Hunting and logging, ubiquitous human disturbances in tropical forests, have the potential to alter the ecological processes that govern population recruitment and community composition. Hunting-induced declines in populations of seed-dispersing animals are expected to reduce dispersal of the tree species that rely on them, resulting in potentially greater distance- and density-dependent mortality. At the same time, selective logging may alter competitive interactions among tree species, releasing remaining trees from light, nutrient or space limitations. Taken together, these disturbances may alter the community composition of tropical forests, with implications for carbon storage, biodiversity conservation and ecosystem function. To evaluate the effects of hunting and logging on tree fecundity and seed dispersal, we use 3 years of seed rain data from a large-scale observational experiment in previously logged, hunted and protected forests in northern Republic of Congo (Brazzaville). We find that low-intensity logging had a meaningful long-term effect on species-specific seed dispersal distances, though the direction and magnitude varied and was not congruent within dispersal vector. Tree fecundity increased with tree diameter, but did not differ appreciably across disturbance regimes. The species-specific dispersal responses to logging in this study point towards the long-lasting toll of disturbance on ecological function and highlight the necessity of conserving intact forest. Hunting and logging can alter ecological communities, but their long-term effects are seldom studied in Afrotropical forests. Hunting-related declines in seed-dispersing animal populations are expected to reduce dispersal of the tree species that rely on them, resulting in fewer successful offspring. At the same time, selective logging may alter competitive interactions among species, releasing remaining trees from light, nutrient or space limitations. We find that low-intensity logging affected seed dispersal two decades after the logging event. The difficult-to-detect effects on a key ecological process could have direct consequences for the diversity and function of forest ecosystems. Logging concessions now cover almost 56 million ha of forest in West and Central Africa . Most coLogging directly disturbs tropical forest communities through the extraction of large trees  and disrStudies investigating how hunting and logging affect seed dispersal have yielded mixed results  in part Clark To evaluate the separate and combined effects of hunting and logging on both fecundity and dispersal for animal and abiotically dispersed trees, we collected 3 years of seed rain data from a large-scale observational experiment in previously logged, hunted and protected forests in northern Republic of Congo (Brazzaville). By controlling for logging and hunting in our sampling design, we offer a first opportunity to test their relative effects. We hypothesized that the fecundity and dispersal distances of tropical trees will be sensitive to both hunting and logging. Specifically, we expected that: (i) tree fecundity is greater in logged forests relative to protected forests, regardless of whether trees species are abiotically or animal dispersed; and (ii) hunting reduces dispersal distances of animal-dispersed species, but not the dispersal distances of abiotically  dispersed species. Understanding the separate and combined effects of disturbances on seed dispersal is critical to predict changes in forest species composition and diversity.Meliaceae, Euphorbiaceae and Annonaceae  report a range of natural stand mortality from 0.86 to 2.02 %, with a best estimate of adjusted stem turnover rate of 1.81 ± 0.16 %  and the Kabo logging concession (267 000 ha) in northern Republic of Congo . The fornonaceae . Rainfalt volume . Althoug± 0.16 % .2 in area were centred along three transects at 25, 50 and 75 m from a plot border, with 10 m separating each trap. All traps were at least 20 m from the nearest plot border. Seeds and fruits were collected every 2 weeks and identified to species or genus level. Previous evidence demonstrates that parameter estimates are dominated by the relatively abundant seeds falling from within these distances  dispersal mode. In addition to dispersal mode, the mean tree DBH (cm) and tree density (stems per hectare) for each species were also calculated by forest type to relate dispersal parameters to species characteristics.The dispersal mode for each tree species was assigned based on fruit morphology and observations of fruit consumption  . Variable selection was based on Deviance Information Criterion (DIC). Model estimates reported in the text are posterior means and 95 % credible intervals (CIs) based on the Gibbs sampler realizations.Gibbs sampling was used for posterior simulation. For each tree species Hunting and logging influenced the mean distances of dispersal kernels , with the greatest effects on animal-dispersed species, though the direction and magnitude varied. Two-thirds of all species (22/33) in disturbed forests had 95 % CIs for dispersal parameters that did not overlap with estimates from protected plots, indicating a role of disturbance. This trend held true whether a species relied on animals for dispersal entirely (13/18), in part (5/8) or not at all (4/7).Celtis mildbraedii, Diospyros canaliculata, Erythrophleum suaveolens, Greenwayodendron suaveolens, Lannea welwitschii, Pausinystalia macroceras, Rinorea oblongifolia, Staudtia kamerunensis, Strombosia nigropunctata), and eight species had lower dispersal estimates .Of the 22 species affected by disturbance, 17 species showed an effect of logging alone: nine species had higher dispersal estimates in logged compared to protected forest  or less than  logging alone. Notably, three species exhibited divergent effects of disturbance regime on dispersal estimates: logging positively affected D. canaliculata and E. suaveolens, whereas the combination of hunting and logging negatively affected dispersal estimates relative to protected plots. Strombosiopsis tetrandra displayed the opposite pattern . Predictions were congruent within each dispersal vector, regardless of disturbance type . AbioticTo evaluate the group-level effects of disturbance, we clustered dispersal estimates of all species by disturbance type, including protected , hunted and logged  and logged forests . The large overlap in dispersal estimates among forest types indicates a lack of consistent effects of disturbance on dispersal distance.Radlkofera calodendron, Lepidobotrys staudtii, S. kamerunensis, R. oblongifolia, Xylopia chrysophylla, Diospyros bipindensis, Camptostylus mannii and D. canaliculata. Logging only influenced fecundity estimates of three species .Estimated tree fecundity increased with tree diameter , but wasWe find that low-intensity logging affected seed dispersal two decades after the logging event. Guidelines aimed at reducing the ecological damage stemming from logging can substantially reduce short-term impacts  and compContrary to our expectations, the dispersal vector of a seed type, abiotic or animal, was not a reliable indicator of the magnitude or direction of the responses of tree species to disturbance. Our results do not support the argument that dispersal decreases for animal-dispersed species following perturbation of the disperser community  (GRF-1106401) and a Neil Williams Presidential fellowship; J.R.P. and C.J.C. were supported by a University of Florida Presidential fellowship (J.R.P.), a School of Natural Resources and Environment alumni fellowship (C.J.C.) and Environmental Protection Agency Science to Achieve Results (STAR) fellowships (91630801-0 to J.R.P. and 91643301-0 to C.J.C.); J.S.C. was supported by NSF-EF-1137364, NSF-EF-1550911 and NASA’s AIST programme.C.L.N. posed the central questions, wrote the original manuscript, and analyzed the data; J.S.C. wrote the the R and C++ code for the MASTIF model with testing and feedback by C.L.N. through development; J.R.P. and C.J.C. collected data with help from those in Acknowledgements section; J.R.P. and J.S.C. edited for content and provided guidance on structure and style.None declared.The following additional information is available in the online version of this article—Figure S1. Boxplots comparing the distribution of tree diameters within each plot type show no systematic difference across plot types.Figure S2. Boxplots comparing the distributions of total stems per plot show significant overlap across plot type.Figure S3. Stacked bar plots comparing community composition show a consistent distribution of 33 focal species across plots.Figure S4. Comparison of standardized root mean squared prediction error  with size of circle indicating relative number of seeds from that species present in the study.Figure S5. (A–D) Example of individual results (Nesogordonia kabingaensis) that were amalgamated across species for in-text summary figures.Figure S6. (A–C) Examples of model diagnostics for Nesogordonia kabingaensis.Table S1. Table of species information and dispersal vectors.Supplementary MaterialClick here for additional data file.


Polygonum cuspidatum , a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance. Polygonum cuspidatum Sieb. et Zucc., commonly known as Huzhang in Chinese and Japanese knotweed in English, is a medicinal plant that is widely distributed in eastern Asia whose roots have served for centuries as an important traditional Chinese medicine for dispelling wind-evil, damp elimination, analgesic therapy, relieving coughs, and reducing sputum , Fagopyrum cymosum , and Polygonum multiflorum, as well as the pseudocereal crop Fagopyrum tataricum (Tartary buckwheat). In contrast to its medicinal uses, P. cuspidatum is regarded as an invasive plant in Europe and North America due to its aggressive growth, allelopathic effects, and extremely strong abiotic stress tolerance  (Arachis hypogaea) (Vaccinium spp.) (Theobroma cacao) (Sorghum bicolor)  , peanutsypogaea) , blueberum spp.) , cocoa (a cacao) , and sorbicolor) . Resverabicolor) . Resveradiseases . Althougdiseases . The larspidatum .P. cuspidatum and have potential antimicrobe and anticancer applications , cinnamic acid 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), and stilbene synthase (STS) . PAL, C4ompounds . While Compounds .Rubia and related species, such as Rubia cordifolia  events. Our study paves the way for the genetic analysis of P. cuspidatum. The draft genome produced in this study should facilitate the improvement of P. cuspidatum, provide a better understanding of the metabolic pathways of its natural products, give insight on its remarkable stress tolerance, and facilitate the development of methods for the biocontrol of this plant for weed management.The lack of genomic information represents a major obstacle to exploring the biological features of P. cuspidatum plants were cultivated in a field in Hubei Province, China . The fresh leaves of 1-year-old plants were collected for DNA extraction. To construct sequencing libraries, 5 μg of DNA was used. Libraries were constructed using an Illumina TruSeq DNA Preparation Kit following the manufacturer’s recommendations. The four sequencing libraries, with insert sizes of 550 bp, 2 to 3 kb, 5 to 7 kb, and 10 to 15 kb, were sequenced on the Illumina HiSeq platform . In addition, 250-bp paired-end reads were generated on the Illumina HiSeq 2500 platform.http://qb.cshl.edu/genomescope/) .A genome survey was performed using 67 Gb of clean Illumina sequencing data (PE150) using Jellyfish software following the instructions from the GenomeScope website (escope/) . The genescope/) . For SOAescope/) . For plaescope/) . For scaP. cuspidatum based on the principle of structural prediction and de novo prediction. Sequences in this database were classified using PASTEClassifier  was utilized with a transposable element (TE)-masked genome (http://www.mbkbase.org/Pinku1/), Arabidopsis thaliana (https://www.arabidopsis.org/) and grape (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/), and the monocot species rice (Oryza sativa) (http://rice.plantbiology.msu.edu/). For transcriptome-based prediction, the HISAT (version 2.0.4) and StringTie (version 1.2.3) programs were used for transcript assembly (https://github.com/TransDecoder/TransDecoder/) was used to predict gene models. All gene models obtained using these approaches were integrated with EVM software , Arabidopsis (https://www.arabidopsis.org/), grape (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/), and tomato (Solanum lycopersicum) (ftp://ftp.solgenomics.net/tomato_genome/annotation/ITAG3.2_release/). All data were subjected to BLASTp analysis , and the resulting data were grouped into gene families with OrthoMCL. To predict the expansion and contraction of the gene families and to infer lineage-specific gene gains and losses, the five genomes were analyzed using OrthoFinder v.2.2.6 . Only genes with no homologs in the 79 other species were retained; these genes were defined as species-specific genes.To explore how many genes were unique to the P. cuspidatum genome using HISAT, and gene expression levels were estimated using StringTie software . The results were manually checked based on gene annotation information. Partial genes with incomplete assembly or representing pseudogenes were excluded from further analysis. The protein sequences were aligned with ClustalX software  detector. An InertSustain C18 column  with a suitable guard column  was used. The mobile phase consisted of deionized water and acetonitrile using the following gradient program: 28% for 0–7 min, 72% for 7–9 min, and 28% for 9–20 min, with a flow rate of 1.2 ml/min and column temperature of 25°C.P. cuspidatum was not available, we sequenced 102 Gb of Illumina data to estimate the size and heterozygosity of the P. cuspidatum genome via genome survey analysis . We also sequenced PE250 data for genome assembly. In total, we generated 377 Gb of short-read data . Of these sequences, 55% were classified as class I TEs, including 30.68% gypsy-type long terminal repeats (LTRs), 9.88% copia LTRs, and 10.67% large retrotransposon derivatives (LARD)  of these genes among species examined, indicating that P. cuspidatum and B. vulgaris share many homologous genes  of the genes could be annotated in at least one database  > 0.3] in at least one tissue, including 52 TF genes specifically expressed in roots (FPKM of roots >2 and FPKM of aboveground tissues <0.3) and 132 specifically expressed in aboveground tissues (FPKM of aboveground tissue >2 and FPKM of roots <0.3) (Transcription factors (TFs) play important roles in regulating secondary metabolism, development, and environmental responses in plants. Through a comparison with P. cuspidatum, we compared all genes with those of four other species, including F. tataricum, S. lycopersicum, Arabidopsis, and V. vinifera. Among these, F. tataricum (Tartary buckwheat) is the most closely related species to P. cuspidatum, which also belongs to the Polygonaceae family. The reference genome of Tartary buckwheat was recently released . By integrating our RNA-Seq data, we determined that 509 genes were upregulated in roots compared to aboveground tissues  between single-copy collinear paralogous genes of P. cuspidatum and the closely related species F. tataricum at the whole-genome level. The analysis of Ks values revealed two peaks of distribution in P. cuspidatum , little is known about the regulation of the biosynthetic pathways of these compounds. We therefore investigated genes that are preferentially expressed in P. cuspidatum roots on a genome-wide scale by conducting comparative transcriptome analysis between roots and aboveground tissues based on genome assembly and gene annotation information.Although crude RNA-Seq revealed 21,415 genes that were expressed in roots, 23,100 that were expressed in aboveground tissues (FPKM > 0.1), and 20,492 that were expressed in both tissues. Of these, 923 genes were expressed only in roots and 2,608 genes were expressed only in aboveground tissues , we performed GO enrichment analysis. DEGs that were upregulated in roots were highly enriched in several important biological processes, such as in glycolysis and gluconeogenesis and in the monosaccharide biosynthetic process, the fructose 6-phosphate metabolic process, and glucose catabolic process , 4CL (EVM0052610), and STS/CHS (EVM0039564) , the Cultivating Project for Young Scholar at Hubei University of Medicine , the Natural Science Foundation of Hubei Provincial Department of Education (Q20182104), the Fundamental Research Funds for the Central Universities (GK201702016), Hubei Provincial Natural Science Foundation of China (2017CFB674), the Foundation of Health Commission of Hubei Province (ZY2019Q004), Open Research Fund of Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry Ministry of Education (2019005), Fund for Key Laboratory Construction of Hubei Province , Hubei Provincial Outstanding Young and Middle-aged Science and Technology Innovation Team Project (Grant No. T201813), the Scientific and Technological Project of Shiyan City of Hubei Province (18Y06) and the Hubei Provincial Technology Innovation Project (2017ACA176).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.


Toxoplasma gondii infection in immune-competent individuals is usually asymptomatic or causes a mild flu-like illness, it may become severe and can occasionally be fatal in immune-compromised people, such as AIDS patients or pregnant women.  Although Toxoplasma gondii, Toxoplasmosis, pregnant women, HIV/AIDS, and Ethiopia.  Electronic English databases , parasitology congresses, and theses of Ethiopian medical universities, were systematically searched (published or unpublished data). Full-length articles and abstracts were collected using keywords such as T. gondii in HIV/AIDS patients and pregnant women. In this review, a total of 4,030 individuals were included and analyzed. The pooled prevalence of T. gondii in this review was 81.00% (95% CI = 69.10–89.78). Sub-group analysis showed that 2,557 pregnant women were evaluated. In pregnant women, the pooled sero-prevalence was 71.2 .  Analysis of seroprevalence estimates was pooled using a random effects meta-analysis. Seventeen studies were included in the present systematic review and meta-analysis. One of these studies reported seroprevalence of Toxoplasma infection of 81% among immunocompromised patients. Scaling up prevention and control methods mainly strengthening educational efforts are necessary to avoid reactivation and to stop the spread of T. gondii infection. This systematic review and meta-analysis identified a high seroprevalence of  Toxoplasma gondii is one of the most prevalent obligate intracellular protozoan parasites of man and livestock [Toxoplasma gondii and a wide range of animals serve as intermediate hosts. Globally, it is estimated that about one-third of the population is infected with T. gondii [T. gondii infection in immune-competent individuals is usually asymptomatic or causes a mild flu-like illness [Toxoplasma gondii infection in AIDS patients and pregnant women causes severe encephalitis, and neurologic diseases, and can affect the heart, liver, inner ears, and eyes (chorioretinitis) [T. gondii during pregnancy can result in spontaneous abortion, still-birth, fetal death, neonatal death, or various congenital defects, such as hydrocephalus, central nervous system abnormalities, and chorioretinitis [T. gondii. It also causes cervical lymphadenopathy or ocular disease in HIV infected immune competent individuals.ivestock –5. Felin. gondii . Althoug illness , it may  illness , 6. Toxotinitis) . Infectietinitis . HIV infT. gondii after ingesting raw or undercooked meat, by ingesting cat-shed oocysts via contaminated soil, food or water; or trans-placental transmission [Humans get infections with smission –5, 7.T. gondii diagnosis such as enzyme-linked immunesorbent assay (ELISA) and indirect immunofluorescence assay (IFA) are considered as gold standard for the detection of Toxoplasma-specific antibodies (IgG or IgM) [Serological methods used for  or IgM) .T. gondii infection in Ethiopia has been reported in some populations; mainly in HIV infected individuals, pregnant women, child bearing age women, and in general population [In Ethiopia, human toxoplasmosis infection is a neglected disease . The prepulation .T. gondii infections, information about the seroprevalence of toxoplasmosis in different special populations is very important. So, this systematic review of the literature aimed to evaluate the sero-prevalence of T. gondii among pregnant women and HIV/AIDS patients in Ethiopia.For appropriate diagnosis, treatment, and control of Toxoplasma gondii, Toxoplasmosis, pregnant women, HIV/AIDS, and Ethiopia. These key words were also used in combination using boolean operators. Overlapped articles found in more than one databases were excluded. Annual research conferences and preceding of Ethiopian medical laboratory association (EMLA), and Ethiopian public health laboratory associations' (EPHLAs) records were reviewed to identify relevant unpublished articles.Relevant literatures were searched on Pubmed, Pubmed central, Google scholar, science direct databases, Scopus, theses of Ethiopian medical universities, and Ethiopian journals such as Ethiopia journal of health development and proceedings of professional associations from May, 2018 to June, 2018. Full-length articles and abstracts were collected using keywords such as The reference lists of published studies included in this meta-analysis were searched for additional articles. Two authors independently reviewed all collected studies for eligibility and they extracted the required information based on the objective this meta-analysis.T. gondii infection in pregnant women and HIV/AIDS patients were included. All collected studies were assessed by two authors for eligibility to be included in this review. In case of disagreement on the eligibility of identified studies, authors held discussion to resolve the issue by considering the quality of identified study and the aim of the present review. Studies performed in study groups other than pregnant women, and HIV/AIDS and those studies that did not use random sampling methods as a sampling technique were excluded .Point estimates and 95% confidence intervals of seroprevalence of all included studies were calculated. An overall seroprevalence and group-specific sero-prevalences were calculated among HIV/AIDS patients and pregnant women. Heterogeneity among studies was visualized using a forest plot chart. The T. gondii in pregnant women, whereas 8 studies reported T. gondii infection in HIV/AIDS patients. There was also one study which reported seroprevalence of T. gondii in HIV/AIDS patients and pregnant women. This study was considered in both pregnant women and HIV/AIDS patient groups during analysis. The study designs of all eligible and included studies were cross-sectional. In this review, Enzyme-linked immunosorbent assay (ELISA) and latex agglutination slide tests were the commonly used methods for the detection of T. gondii antibodies. The higher sero-prevalence of T. gondii (96.7% and 93.6%) was reported in HIV/AIDS patients from Mizan Aman Hospital and selected areas of Ethiopia, respectively. On the other hand the lower seroprevalence (18.5% and 34.9%) reported in this study was in pregnant women from Felege Hiwot Hospital from Jigjiga Hospital  were Cochran Q = 1,233.145293  and I2 (inconsistency) = 98.6% (95% CI = 98.5%–98.8%)  while it was 76% (95% CI = 61%–89%) in recent studies (2014–2017). Majority of the studies were conducted in recent years (2014–2017). Sub group analysis showed the sero-prevalence of T. gondi is decreased in recent years.In general, in this review a total of 4,030 individuals were included and analyzed. The pooled prevalence of toxoplasmosis in this review was 81.00% (95% CI = 69.10–89.78). The %–98.8%) . In thisQ statistic and the pooled sero-prevalence were Cochran Q = 845.808548  and 71.2  = 99.1%  and 88.45 (95% CI = 80.87%–94.31%) respectively. I2 (inconsistency) = 94.1% (95% CI = 91.5%–95.7%) (In HIV/AIDS %–95.7%) .P-value >0.05). There was significant bias among studies included about HIV/AIDS patients .Begg and Egger tests were used for evaluating publication bias. Significant publication bias was not revealed in studies included about pregnant women  methods for diagnosis. The highest prevalence of toxoplasmosis was observed in individuals diagnosed with ELISA. For instance, in HIV/AIDS patients, 96.70% sero-prevalence of T. gondii was observed using ELISA. The incidence and prevalence of T. gondii infection varies depending on geographic areas, weather conditions, and age groups in Ethiopia.In Ethiopia, a review studied on the sero-prevalence of T. gondii in general population revealed the overall pooled prevalence was 74.73% [In Ethiopia, s 74.73% . The serT. gondii in pregnant women and HIV/AIDS patients in Ethiopia. Our finding indicates that researchers must give attention to the seroprevalence of toxoplasmosis in immunocompromised patients especially in HIV/AIDS patients, and pregnant women. Scaling up prevention and control methods and mainly strengthening educational efforts are necessary to avoid reactivation and to stop the spread of T. gondii infection.As far as we know and our literature search, this is the first systematic review and meta-analysis that provides information about the seroprevalence of


Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood.Pine trees challenged by B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus.Here, we described anti-phytoalexin genes in Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.The results indicated that the anti-phytoalexin gene  Bursaphelenchus xylophilus, the causative agent of a devastating epidemic of pine wilt disease, is a migratory endoparasite that causes severe damage to pine forests in East Asia and Europe  [dT] 3′) was selected.Fluorescent siRNAs corresponding to Bx-cathepsin W with the primers BxCWqF (TTG CAT TCT ACG GCC AGT CC) and BxCWqR (ACT GAC TTT CGA TGG CTC CG). Bx-Actin RNA was used as an internal control with the primers BxACqF (TTC AGG TGT TAC CCA CAC CG) and BxACqR (GCG GTG GTG GTG AAA GAG TA). Each treatment was performed on three separate biological replicates, and each replicate was measured three times. The quantification and normalization of the data followed the log2fold method according to the Promega instructions. Student’s t-test was used to analyze differences between group grades. The second group was used to assess survival differences. The survival assay of B. xylophilus was performed in 24-well polystyrene culture plates containing 500 μl ddH2O or carvone (800 mg/L) per well in the dark at 25 °C. One thousand nematodes were then added to each well. Moving and immobilized nematodes were counted every 2 days. The third group was used to assess the pathogenicity difference of P. massoniana. RNAi-treated nematodes were inoculated into 3-year-old P. massoniana seedlings  to test the pathogenicity of P. massoniana. The inoculations of M9 buffer-treated nematodes and ddH2O were set as the control. Thirty days after inoculation, nematodes were isolated and counted from P. massoniana seedlings. Three biological replicates were performed for the inoculation.RNAi was performed using mixed stages of nematodes soaked in M9 buffer with 10 mM octopamine and siRNAs (2 μg/μl) or M9 buffer with 10 mM octopamine as a control overnight in the dark at 25 °C, as outlined in Wang, et al. . The RNAB. xylophilus genes. The module to which Bx-cathepsin W belonged and the association between its expression level and the susceptibility of different species of pines were identified. The survival rate of B. xylophilus and the disease susceptibility index of different species of pines  were selected as two traits for WGCNA.Weighted gene coexpression network analysis (WGCNA) is a method frequently used to explore the complex relationships between genes and phenotypes . The disP. massoniana for 1 day and 15 days, P. thunbergii for 1 day and 15 days, P. sylvestris var. mongolica for 15 days, and CK2, a control of nematodes cultured on B. cinerea) were sequenced and analyzed according to the same method described above and were designated as the second sequencing. Two transcriptomes from the first sequencing (Treatment 1 and CK1) and all 6 transcriptomes from the second sequencing were analyzed by weighted gene WGCNA to find clusters (modules) of highly correlated anti-phytoalexin genes .To characterize the anti-phytoalexin gene transcript patterns, 4 cDNA libraries  of An average of 79.69 Mb raw reads was obtained for each sample. After removing the low-quality reads, more than 66.74 Mb clean reads (6.62 Gb clean bases) for each sample were obtained. Clean reads were mapped to the reference genome, the result showed that more than 93% of the transcripts could mapping to the reference genome  but had no homolog in the genome of C. elegans. The 3rd group included 54 genes that had a homolog in the genome of C. elegans. The 4th group included 20 xenobiotic-metabolizing enzyme (XME) genes  database or the genome of Among those 187 anti-phytoalexin genes, 95 genes could be enriched as 66 KEGG orthologs , was cloned from the Treatment 2 cDNA library. Bx-cathepsin W contained a coding sequence of 1179 bp, encoding a polypeptide of 392 amino acids composed of a putative 16-residue signal peptide, 141-residue propeptide, and a 235-residue mature protein. Protein sequence comparisons revealed 77% homology with human cathepsin W  [C. elegans cathepsin F , and approximately 53~75% homology with human cathepsins F, O, X, K, H, and S . The deduced amino acid sequence contained the highly conserved residues of the catalytic triad of papain-like cysteine proteases  , 76% homBx-cathepsin W silencing was found in B. xylophilus after the nematode soaked in siRNAs overnight. The patterns of fluorescein observed for the nematodes indicated that FAM/Cy3 labeled siRNA was taken up by B. xylophilus effectively  and morphology . This result indicated that the resistance of B. xylophilus to carvone decreased after the knockdown of Bx-cathepsin W.However, the survival rate of the Bx-cathepsin RNAi-treated nematodes was found  and pine susceptibility , which indicated that genes in this module were related to the interaction between nematodes and pines. The GS of Bx-cathepsin W with survival rate was 0.79 , the GS of Bx-cathepsin W with pine susceptibility was 0.77 , and the MM of Bx-cathepsin W with yellow module was 0.79 . This result indicated that Bx-cathepsin W had a strong positive correlation with the susceptibility of pines; in other words, Bx-cathepsin W could improve the anti-phytotoxin ability of B. xylophilus, which was consistent with the Bx-cathepsin W RNAi analysis and suggested that Bx-cathepsin W functions as an anti-phytotoxin via an additional method of analysis.The survival rate of ait Fig. a. The moBx-cathepsin W-correlated genes were identified from the yellow module; among them, the connections of the corresponding topological overlap of 439 genes were above a threshold of 0.08  in the pine or the vector Monochamus alternatus. In particular, B. xylophilus must cope with phytoalexins, which are induced by pine in response to nematode infection. Anti-phytoalexin is a crucial process for B. xylophilus to cope with its pine hosts.Furthermore, in order to find a common core among different phytoalexins induced  studies , 32. HowP. massoniana and carvone. However, the detailed functions of these anti-phytoalexin genes require further study. The roles of nematode cathepsins, one proteinase of cysteine proteinases (EC 3.4.22), may include invasion of host tissues, parasite nutrition and evasion of host defense responses [Heterodera glycines [Meloidogyne incognita [Necator americanus [Teladorsagia circumcincta [H. glycines [M. incognita [M. incognita preparasitic infective J2 s is directly related to the parasitic aspects of the plant-nematode interaction [In this study, we identified 187 anti-phytoalexin genes that showed the same promoted expression trends upon induction by both esponses , 34. Catglycines  and Meloncognita . Cysteinericanus  and Telaumcincta . Previouglycines  and M. incognita  females.eraction .Bx-cathepsin W functioned in the anti-phytoalexin process of B. xylophilus in response to P. massoniana and carvone stresses. Carvone-treated B. xylophilus was not strictly correlated with feeding in Treatment 2, and the promotion of Bx-cathepsin W expression indicates that some function of Bx-cathepsin W is indeed more directly related to the plant-nematode interaction, especially to the anti-phytoalexin process. The results of the RNAi assessment indicated that Bx-cathepsin W supported the survival of B. xylophilus under carvone or P. massoniana stress. From the relationship between the expression level of Bx-cathepsin W and the survival rate of B. xylophilus in different pines, we found that the gene expression was positively related to the survival rate of B. xylophilus. Therefore, the correlation of the expression level of Bx-cathepsin W with the susceptibility of pines was examined. These results showed that Bx-cathepsin W played an important role in improving the anti-phytotoxin ability of B. xylophilus. The survival rate of B. xylophilus was positively correlated with the susceptibility of pines; that is, the nematodes that infect more susceptible pines are more likely to survive and therefore are greater in number than nematodes that infect less susceptible pines. Therefore, based on the above results, Bx-cathepsin W is important in the pine wood nematode infection process. According to the correlation between genes in the yellow module, genes that shared the same expression pattern and had a high correlation with Bx-cathepsin W might have a strong regulatory relationship with Bx-cathepsin W, which could indicate a direction for further research on Bx-cathepsin W and a new target for the control of the nematode.Therefore, we were particularly interested in determining whether Bx-cathepsin W, support the survival of the nematode B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification and WGCNA algorithm provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.The results indicated that the anti-phytoalexin genes, particularly Additional file 1: Table S1. Statistical analysis of the RNA sequencing data. Table S2. Xenobiotic-metabolizing enzyme genes. Table S3. Results of KEGG enrichment. Table S4. Anti-phytoalexin genes related to the Lysosome pathway. Table S5. The number of genes in the 25 modules. Table S6.Bx-cathepsin W-correlated anti-phytoalexin genes. Table S7. Identification of B. xylophilus xenobiotic metabolism genes. Text S1. Transcriptome library preparation and sequencing. Text S2. Data analysis of reads. Text S3. Transcriptome assembly. Text S4. The differential gene expression analysis. Text S5. The WGCNA algorithm.


Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function.BRAFV600E mutation was analyzed by next generation sequencing.NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins.In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14–29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. V600E protein is degraded in NI needs further investigation.NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAF The existence of intranuclear inclusions (NI) in many normal and neoplastic tissues has been known for a long time, in diabetic patients  in hepatIn thyroid carcinoma (TC), however, NI were seen as invaginations of the cytoplasm, i.e. bordered by the nuclear membrane with a considerable variety of size and shape even in the same tumor . Also, oAlthough there are many observations regarding NI, there is no study that addresses the issue of whether these NI have a biological function. Therefore, we have recently investigated NI in hepatocellular carcinoma and have shown that they contain autophagy-associated proteins and correlate with prolonged survival . These iBRAF Mutation is an important diagnostic tool for papillary thyroid cancer Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I commend the authors of the study for their findings. Their correlation of autophagy genesI think the authors samples maybe slightly skewed however and they may need to address the prevalence of BRAF in the literature that occurs in classical, poorly differentiated and FVTC. Rates in most studies of BRAF positive at in the 60-70% range. Can the authors explain their sample selection and why they were unable to see BRAF mutations in these other subgroups. There is a need to increase your sample size to explain the rational for not observing these changes as it would mean the clinical sample size is too low.I note the authors correlate the autophagy genes in patients with the BRAF mutation. There are 14 patients with the BRAF mutation and a significantly higher number of tumor samples with at least upregulation of one or more of the autophagy genes. Can the authors comment on the total cumulative number of patients with at least one of the autophagy genes unregulated. Can the authors also explain if the BRAF mutation is only present in 14% of patients and more than 29% of patients have a change in at least one autophagy gene the effect is only at best 50% of patients with changes in the autophagy genes. And what is different about these tumors and the tumor biology versus the patients without these features. Potentially a correlation with recurrence and disease free survival and outcomes would be important.It would be also important to see the labeling of BRAF wt in the inclusions. I think the authors need to see if there is clustering of wt in nuclear inclusions.Reviewer #2: This manuscript has surprising, convincing, important new data on the structural features of intranuclear cytoplasmic inclusions: Intranuclear cytoplasmic inclusions are (at least in the carefully studied examples) completely bound by nuclear lamina and nuclear membranes.There are minor editorial corrections/suggestions:1. Line 79 take out word "briefly".2. Line 204 Did you really homogenize the cells in 70% ethanol (more likely aquaeous)? Also, cells should not have needed permeabilization since they went through xylene.3. Line 318-19. The writing is not clear. I think you mean "No other genes were mutated in more than one case".4. Line 324. Not clear. I think you can delete the beginning of the sentence to say "Lamin A/C positive inclusions occur most frequently..."5. Line 389-390. I don't think you have any basis to speculate about the temporal aspects of the fate of inclusions. I would say something like "as if it could become ejected soon"6. The authors may want to include this reference on intranuclear cytoplasmic inclusions:Rezk S, Brynes RK, Nelson V, Thein M, Patwardhan N, Fischer A, Khan A.2004. beta-Catenin expression in thyroid follicular lesions: Potential role innuclear envelope changes in papillary carcinomas. Endocr Pathol 15:329–337.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Oct 2019Editor Comments: First of all, we want to thank you for taking the time to review our manuscript. We have highlighted the changes in our manuscript within the document by using coloured (blue) text; deleted text is marked in red and crossed out. The information regarding page numbers refers to the previous version of the manuscript. Journal Requirements (JR) and the responds of the author (AC) are listed below. Journal Requirements:JR.1. When submitting your revision, we need you to address these additional requirements.http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfPlease ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at AC.1. We have checked PLOS ONE's style requirements and we have incorporated corresponding author’s initials in parentheses after the email address. Additionally, we have corrected the formatting of the headings.JR.2. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed:doi: 10.1002/cjp2.129In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.AC.2. Thank you for your comment regarding minor instances of text overlap with our own previous publication. We have checked this and found some parts mainly concerning background information and some idioms and we apologize for this. We rephrased or quoted any duplicated text outside the methods section in the revised manuscript.Response to the Reviewer: First of all, we want to thank you for taking the time to review our manuscript. We have highlighted the changes in our manuscript within the document by using colored (blue) text; deleted text is marked in red and crossed out. The information regarding page numbers refers to the previous version of the manuscript. The comments of the reviewer (RC) and the responds of the author (AC) are listed below. Response to Reviewer 1 RC: I think the authors samples maybe slightly skewed however and they may need to address the prevalence of BRAF in the literature that occurs in classical, poorly differentiated and FVTC. Rates in most studies of BRAF positive at in the 60-70% range. Can the authors explain their sample selection and why they were unable to see BRAF mutations in these other subgroups. There is a need to increase your sample size to explain the rational for not observing these changes as it would mean the clinical sample size is too low.AC: Thank you for pointing out the prevalence of BRAF in the literature regarding the different entities. We incorporated additional references into the discussion section (line 612 of the previous manuscript) of the revised manuscript. It is documented that the occurrence of BRAF V600E mutation depends, among others, on the region, where the study is conducted and the available diet there [1]. Guan et al demonstrated the association of high iodine intake with BRAF V600E mutation [2]. The study cohort consisted of patients with classical PTC and revealed that in regions with high iodine contents in natural drinking water the prevalence of BRAF V600E mutation was significantly higher (69%) than in regions with normal iodine content (53%) [2]. A study of Jin et al revealed that 63.7% of the Chinese patients with PTC harbored the BRAF V600E mutation and they pointed out that high dietary iodine intake from rich sea foods may contribute to this high prevalence of BRAF V600E mutation; however they did not elaborate on the variants of studied PTCs. The prevalence of BRAF V600E mutation is also depended on the studied variant of PTC. It is documented that BRAF V600E mutation occurs significantly more frequently in the conventional variant of PTC than in the follicular variant with 58% versus 31% [3] and 75% versus 40% [4]. Only classical PTC cases were analyzed in the above mentioned study by Guan et al. with the detection of 53% BRAF V600E mutation in regions with normal iodine content. There are also studies reporting a lower prevalence of BRAF V600E mutation in about 45% (30 to 70%) of PTCs [5–9], with 12% of follicular variant PTC and 77% of tall cell variant PTC harboring this mutation [5]. In our study cohort 14 of 43 PTCs (32%) harbored the BRAF V600E mutation. The reason for this relatively low occurrence of the BRAF V600E mutation could be that first of all, our study group consisted of patients from the European region, who do not consume iodine-rich seafood so often. Secondly, in our study group were also 9 follicular variant PTCs, 1 hobnail variant PTC, 1 columnar cell variant PTC and 4 solid variant PTCs, whereby PTC solid variant rarely harbors BRAF V600E mutation [10] but more frequently BRAF VK600-1E mutation  and BRAF V600delinsAL . Our study cohort consisted of 10 ATCs, 10 PDTCs and 9 FTCs, which were all BRAF V600E negative. It is documented that PDTCs and ATCs were more likely to have BRAF V600E mutation when they arise from papillary carcinoma . The BRAF V600E mutation was found in about 5-20% [14–16] of PDTCs and in 11-45% of ATCs [17–19]. In the study of Landa et al, in which 45% of ATCs harbored the BRAF V600E mutation, they used NGS technique and adopted an ultra-deep sequencing strategy, enabling an average depth of coverage . In our current study, we used also NGS but we did not perform ultra-deep sequencing described by Landa et al. Another reason why we detected no BRAF mutation in our PDTC and ATC cases might be that they developed de novo rather than arised from papillary carcinomas . Nevertheless, our current study did not focus on the prevalence of BRAF V600E mutation in thyroid cancer. Our aim was to show that NI so often observed in daily microscopic observations, are completely surrounded by the nuclear membrane. In addition, we wanted to show that these inclusions, which have so far been considered of less important, might have a function because they contain accumulations of proteins associated with autophagy and proteolysis. Therefore, although we appreciate your comment, we believe that increasing the number of ATC and PDTC cases would not be useful, as the message regarding the characteristics of intranuclear inclusions remains the same.RC: I note the authors correlate the autophagy genes in patients with the BRAF mutation. There are 14 patients with the BRAF mutation and a significantly higher number of tumor samples with at least upregulation of one or more of the autophagy genes. Can the authors comment on the total cumulative number of patients with at least one of the autophagy genes unregulated. Can the authors also explain if the BRAF mutation is only present in 14% of patients and more than 29% of patients have a change in at least one autophagy gene the effect is only at best 50% of patients with changes in the autophagy genes. AC: We apologize that the manuscript was not clear at this point, which led to misunderstandings, as we did not investigate the expression of genes. We only found out that in 14-29% of all cases there was an accumulation of autophagy-associated proteins within the inclusions. Further, the number of NI, which showed immunopositivity for autophagy-associated proteins within the inclusions was higher in BRAF V600E positive cases than in cases with BRAF wild-type. We agree that about 50% of the cases harbored NI with an accumulation of autophagy-associated proteins within them although they have no BRAF mutation. But the number of such filled NI was lower in BRAF wild-type cases. In our opinion the BRAF V600E mutation is only one factor, which may regulate the accumulation of the autophagy-associated proteins within NI and the formation of NI (line 635-637 of the previous manuscript). Nevertheless, we assume that there are also other factors that play a role in these biological processes. RC: And what is different about these tumors and the tumor biology versus the patients without these features. Potentially a correlation with recurrence and disease free survival and outcomes would be important.AC: Unfortunately, we don’t have survival data of the patients. RC: It would be also important to see the labeling of BRAF wt in the inclusions. I think the authors need to see if there is clustering of wt in nuclear inclusions.https://www.novusbio.com/PDFs/NBP1-47668.pdf). AC: Thank you for your suggestion. We provided the antibody anti-BRAF , which detects BRAF wild-type . Further, we observed varying degrees of immunoreactivity for BRAF wild-type within the NI (arrows) whereby some NI showed relatively weaker immunostaining (arrows). We found BRAF wild-type immunopositivity also in the case in which formerly BRAF V600E mutation was proved by NGS. This result is unexpected and can have several reasons. One explanation may be heterogeneity in this case with both BRAF wild-type and BRAF V600E cells coexisting in the same tumor. Also other studies have observed the existence of BRAF heterogeneity in some tumors . Another explanation can be that the mutated BRAF protein also reacts to some degree with this antibody. We contacted Novus Biologicals, from which the antibody anti-BRAF (#NBP1-47668) was purchased, and asked how specific this antibody is. The answer was that since this antibody is made from a full length recombinant, which is based on the wild type sequence, NP_004324 its possible that it may or may not pick up the mutated form. Additionally, they said that as long as the epitope of NBP1-47668 is not directly over the mutation in BRAF V600E then this antibody should pick up the wild-type and mutant form. In our manuscript we documented the accumulation of mutant-BRAF protein within NI and now we also found BRAF wild-type protein within NI. It is documented that high BRAF expression was significantly correlated with poor patient survival in melanoma . As we also found clustering of BRAF wild-type in NI this might due to a biological mechanism to deal with excess BRAF protein. Additional Figure for ReviewImmunohistochemistry: BRAF Antibody (OTI5A9) [NBP1-47668] The image is displayed in the attached Word document.The images depict immunostaining of representative PTC cases harboring BRAF wild-type  and BRAF V600E mutation  proven by NGS; varying degrees of immunoreactivity for BRAF  is shown both in the intranuclear inclusions (arrows) and in the cytoplasm. Original magnifications: 1,000 X. Response to the Reviewer: First of all, we want to thank you for taking the time to review our manuscript. We have highlighted the changes in our manuscript within the document by using colored (blue) text; deleted text is marked in red and crossed out. The information regarding page numbers refers to the previous version of the manuscript. The comments of the reviewer (RC) and the responds of the author (AC) are listed below. Response to Reviewer 2RC: 1. Line 79 take out word "briefly".AC: 1. Thank you for this point; we have deleted it. RC: 2a. Line 204 Did you really homogenize the cells in 70% ethanol (more likely aquaeous)? http://dx.doi.org/10.17504/protocols.io.78phrvn). AC: 2a. Thank you very much for your comment. We agree with your assessment and apologize for this mistake. We missed to incorporate that after the rehydration-step with 70% ethanol, we homogenized the cells in Target Retrievel Solution pH9 . We rewrote the section “3D imaging of immunofluorescence-labeled isolated nuclei” and added it as a separate laboratory protocol with its own identifier (DOI) to our manuscript . We permeabilized the cells with saponin according to a modified flow cytometry and cell sorting protocol [27]. We incubated the antibodies in the presence of saponin because it is documented that in contrast to many other detergents cell permeabilization with saponin is a reversible process and once saponin is removed from the sample, the pores of the membrane will be closed [28]. Saponin intercalates in the membrane structure, replacing cholesterol and leaves much of the membrane structure intact [29–31].AC. 2b. This is a very interesting point. We agree with you that permeabilization is not always needed as xylene interacts with the cell membrane and changes the permeability . Nevertheless, it is documented that the exact mechanism is not fully understood [25]. We are of the opinion that it also depends on the thickness of the tissue sections. To isolate complete nuclei, paraffin-embedded tissue sections were cut relatively thick (60 μm) to keep the nucleus intact as effectively as possible. In this case addition of xylene was performed to remove paraffin from the paraffin-embedded tissues. Since the mechanism of how xylene interacts with the cell membrane is not fully understood, we think it is better to add saponin to the Dako REAL Antibody Diluent  to ensure that the antibody penetrates the nucleus. However, regarding our double-labeling immunofluorescence studies of FFPE tissue sections cut at 1 μm, experiments were carried out according to standard protocols and we did not add saponin to the antibody dilution buffer . In the troubleshooting data of abcam it is recommended if the antibody cannot penetrate the nucleus, where the protein is located (nuclear protein) to add a strong permeabilizing agent like Triton™ X-100 to the blocking buffer and antibody dilution buffer  cases (supporting information S6 Table). These are mutations, which we did not want to discuss in our manuscript in more detail. For more openness and transparency, however, we have replaced the former text (line 318-19 of the previous manuscript) as follows: “We detected with a maximum of 3/107 (2.8%) cases a rather low incidence of mutations in the genes of the Wnt pathway and we did not find any associations between these mutations and the occurrence of NI.” RC: 4. Line 324. Not clear. I think you can delete the beginning of the sentence to say "Lamin A/C positive inclusions occur most frequently..."AC: 4. We have deleted the beginning of the sentence.RC: 5. Line 389-390. I don't think you have any basis to speculate about the temporal aspects of the fate of inclusions. I would say something like "as if it could become ejected soon"AC: 5. We agree with you and we have replaced the section “which seems to be pushed to the edge of the nucleus for being ejected soon.” by “as if it could become ejected soon."RC: 6. The authors may want to include this reference on intranuclear cytoplasmic inclusions:Rezk S, Brynes RK, Nelson V, Thein M, Patwardhan N, Fischer A, Khan A. 2004. beta-Catenin expression in thyroid follicular lesions: Potential role in nuclear envelope changes in papillary carcinomas. Endocr Pathol 15:329– 337.AC: 6. We have added to the introduction section (line 99 of the previous manuscript): “In addition, it is also documented that strong immunopositivity for ß-catenin was detected within NI in 83% of PTCs [32]. ”References1. Jin L, Chen E, Dong S, Cai Y, Zhang X, Zhou Y et al. BRAF and TERT promoter mutations in the aggressiveness of papillary thyroid carcinoma: a study of 653 patients. Oncotarget. 2016;7:18346-18355.2. Guan H, Ji M, Bao R, Yu H, Wang Y, Hou P et al. Association of high iodine intake with the T1799A BRAF mutation in papillary thyroid cancer. J Clin Endocrinol Metab. 2009;94:1612-1617.3. Smith RA, Salajegheh A, Weinstein S, Nassiri M, Lam AK-y. Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant. Hum Pathol. 2011;42:500-506.4. Lim JY, Hong SW, Lee YS, Kim B-W, Park CS, Chang H-S et al. 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Dettmer M, Schmitt A, Komminoth P, Perren A. Poorly differentiated thyroid carcinoma. Pathologe. 2019;15. Eloy C, Ferreira L, Salgado C, Soares P, Sobrinho-Simoes M. Poorly differentiated and undifferentiated thyroid carcinomas. Turkish Journal of Pathology. 2015;3116. Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nature Reviews Endocrinology. 2011;7:569-580.17. Kunstman JW, Juhlin CC, Goh G, Brown TC, Stenman A, Healy JM et al. Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing. Hum Mol Genet. 2015;24:2318-2329.18. Landa I, Ibrahimpasic T, Boucai L, Sinha R, Knauf JA, Shah RH et al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. The Journal of clinical investigation. 2016;126:1052-1066.19. Tiedje V, Ting S, Herold T, Synoracki S, Latteyer S, Moeller LC et al. NGS based identification of mutational hotspots for targeted therapy in anaplastic thyroid carcinoma. Oncotarget. 2017;8:42613-42620.20. Xu B, Ghossein R. Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma. Endocr Pathol. 2016;27:205-212.21. Kurtulmus N, Ertas B, Saglican Y, Kaya H, Ince U, Duren M. BRAFV600E mutation: has it a role in cervical lymph node metastasis of papillary thyroid cancer. European thyroid journal. 2016;5:195-200.22. Takata M. Identifying BRAF and KIT mutations in melanoma. Expert Review of Dermatology. 2013;8:171-176.23. Bhandaru M, Safaee Ardekani G, Zhang G, Martinka M, McElwee KJ, Li G et al. A combination of p300 and Braf expression in the diagnosis and prognosis of melanoma. BMC cancer. 2014;14:398.24. Safaee Ardekani G, Jafarnejad SM, Khosravi S, Martinka M, Ho V, Li G. Disease progression and patient survival are significantly influenced by BRAF protein expression in primary melanoma. Br J Dermatol. 2013;169:320-328.25. 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Beta-Catenin expression in thyroid follicular lesions: Potential role in nuclear envelope changes in papillary carcinomas. Endocr Pathol. 2004;15:329-337.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 22 Nov 2019New insights into intranuclear inclusions in thyroid carcinoma: association with autophagy and with BRAF V600E mutationPONE-D-19-21366R1Dear Dr. Schwertheim,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Colchicum genus, C. szovitsii subsp. szovitsii is also of medicinal importance in Turkish traditional medicine. However, its biological properties have not been fully investigated. Herein, we focused on the evaluation of the in vitro antioxidant and enzyme inhibitory effects of flower, root and leaf extracts, obtained using different extraction methods. In addition, a comprehensive (poly)-phenolic and alkaloid profiling of the different extracts was undertaken. In this regard, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) allowed us to putatively annotate 195 polyphenols and 87 alkaloids. The most abundant polyphenols were flavonoids (83 compounds), whilst colchicine and 2-demethylcolchicine were some of the most widespread alkaloids in each extract analyzed. However, our findings showed that C. szovitsii leaf extracts were a superior source of both total polyphenols and total alkaloids . Overall, methanolic leaf extracts showed the highest (p < 0.05) ferric reducing antioxidant power (FRAP) reducing power (on average 109.52 mgTE/g) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging (on average 90.98 mgTE/g). Interestingly, each C. szovitsii methanolic extract was more active than the water extracts when considering enzymatic inhibition such as against tyrosinase, glucosidase, and acetylcholinesterase (AChE). Strong correlations (p < 0.01) were also observed between polyphenols/alkaloids and the biological activities determined. Multivariate statistics based on supervised orthogonal projections to latent structures discriminant analysis (OPLS-DA) allowed for the detection of those compounds most affected by the different extraction methods. Therefore, this is the first detailed evidence showing that C. szovitsii subsp. szovitsii might provide beneficial effects against oxidative stress and the associated chronic diseases. Nevertheless, the detailed mechanisms of action need to be further investigated.Like other members of the  Medicinal plants have historically proven their value as sources of molecules with therapeutic potential, and even in this new era of highly allopathic medicine, herbal medicines are an area of focus for researchers around the world to complement modern drugs and as sources of novel drug leads . The estOne of the important medicinal plants are the Colchicum species, which have been traditionally used more than 3000 years ago to treat various ailments by different ethnic groups. Colchicum is a valuable genus (family: Colchicaceae) with approximately 100 species, mainly distributed in Mediterranean countries, South Africa, and the Caucasus . The namThe medicinal value of the Colchicum species is ascribed to the presence of tropolonic alkaloids, colchicinoids, mainly colchicine . AncientColchicum szovitsii subsp. szovitsii, a spring flowering species from Turkey  (ABTS) scavenging capacity, ferric and cupric reduction activity, and metal chelating activity  and aqueous macerated (52.76 mgTE/g) extracts were found to be the best DPPH scavengers. For the root extracts, the methanolic extracts obtained using the Soxhlet and maceration methods had the highest free scavenging potentials with the DPPH assay . For the ABTS assay, a similar trend was observed for flowers and roots, whilst for the leaf extracts, the highest DDPH quenching ability was observed with the infused extract (66.63 mgTE/g).In the current study, we investigated the antioxidant capacities of activity . Based oPH assay . SimilarC. szovitsii subsp. szovitsii were the most potent reducing agents.Reducing power is considered as one of the most important antioxidant mechanisms and this corresponded to the electron-donating capacity of antioxidant ability . In the C. szovitsii subsp. szovitsii. Transition metal ions such as Fe2+ accelerate ROS production in the body. Therefore, the Fe chelating activity of a compound may be related to its antioxidant capacity. Interestingly, for all the plant parts studied, the infused extracts were the most effective metal chelator , tyrosinase, α-amylase, and α-glucosidase were selected to determine the enzyme inhibitory potentials of the C. szovitsii subsp. szovitsii flowers, roots, and leaves showed remarkable inhibition capacity against the tested enzyme.Approximately 15% of the world population invest in skin whitening agents, with Asia dominating the market . AccordiC. szovitsii subsp. szovitsii, except the macerated roots extract, were very potent.New α-amylase and α-glucosidase inhibitors are also being sought to regulate post-prandial glucose levels because of the adverse side-effects linked to commercially available anti-diabetic medications. The findings  revealedC. szovitsii extracts. A summarizing table reporting all of the correlation coefficients for each part analyzed  is reported in the C. szovitsii roots were found to be highly (p < 0.01) correlated to ABTS (0.97), AChE inhibition (0.95), tyrosinase inhibition (0.96), and amylase inhibition (0.95) values. These findings were also supported by several researchers, who reported that alkaloids were the main players against cholinesterase [p < 0.01) were observed between anthocyanins and CUPRAC values (0.86), flavanols and tyrosinase inhibition (−0.99), flavones and CUPRAC values (0.85), phenolic acids and FRAP values (−0.87), tyrosols and tyrosinase inhibition (0.97), and stilbenes and DPPH values (0.93). Regarding C. szovitsii leaf extracts, we found no significant correlations between the total alkaloid content and the biological activities inspected (p < 0.01) to the DPPH, FRAP, CUPRAC, and ABTS assays. In accordance with our results, polyphenols were reported as the main contributors to antioxidant properties in earlier studies [p < 0.01) recorded for flavanols . Regarding polyphenols characterizing flower extracts, significant correlations (p < 0.01) were obtained between anthocyanins and enzymatic inhibition assays, with the highest coefficient recorded for amylase inhibition . These findings could be valuable for designing functional food ingredients and anthocyanins have exhibited promising antidiabetic effects in several studies [Pearson’s correlation coefficients were then calculated in order to check the contribution of polyphenols and alkaloids to the biological properties observed when considering the different esterase ,38 and testerase ,40. Looknspected . However studies ,42. On tlavanols . These r studies ,47. In t studies . InteresC. szovitsii. This fact may be explained by the different exposure of abiotic and biotic stress factors for each plant part and thus these parts could produce different levels of secondary metabolites. These findings were also supported by scientific evidence [C. szovitsii could be of interest for food, pharmaceutical, and cosmetic areas.Clearly, the present correlation values depended on the parts of evidence ,50. To sColchicum genus, there have been very few studies including C. szovitsii subsp. szovitsii. This study reports the biochemical characterization of Colchicum szovitsii subsp. szovitsii, according to their phenolic and alkaloid compositions, together with antioxidant and enzyme inhibitory activities. Of the plant parts studied, the extracts from leaves exhibited the highest phenolic levels associated with potent antioxidant and key enzyme inhibition associated with chronic pathologies, namely neurodegenerative complications (acetyl and butyrylcholinesterase), hyperpigmentation (tyrosinase), and diabetes mellitus (α-amylase and α-glucosidase). Detailed chemical profiling using UHPLC-QTOF mass spectrometry allowed us to confirm the wide phenolic and alkaloid distribution in the different extracts tested.When considering previous studies regarding the pharmacological attributes of the C. szovitsii, the presented findings advocate this species as a potential source for obtaining natural bioactive products for medical and pharmaceutical applications. However, further studies  on C. szovitsii should be developed, aiming at replacing synthetic antioxidants and enzyme inhibitors.This work reinforces the notion that (poly)-phenols and alkaloids largely contribute to antioxidant and other pharmacological attributes, in the case of enzyme inhibition, making the studied species a promising source of drugs and whitening agents for medicine and cosmetics applications. In addition to fill the scientific gap on


Apis mellifera (the Western honey bee) is the most important commercial pollinator worldwide. The Gram-positive bacterium Melissococcus plutonius is the causative agent of European foulbrood (EFB), a global honey bee brood disease. In order to detect putative virulence factors, we sequenced and analyzed the genomes of 14 M. plutonius strains, including two reference isolates. The isolates do not show a high diversity in genome size or number of predicted protein-encoding genes, ranging from 2.021 to 2.101 Mbp and 1589 to 1686, respectively. Comparative genomics detected genes that might play a role in EFB pathogenesis and ultimately in the death of the honey bee larvae. These include bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an epsilon toxin, proteolytic enzymes, and capsule-associated proteins. In vivo expression of three putative virulence factors  was verified using naturally infected larvae. With our strain collection, we show for the first time that genomic differences exist between non-virulent and virulent typical strains, as well as a highly virulent atypical strain, that may contribute to the virulence of M. plutonius. Finally, we also detected a high number of conserved pseudogenes (75 to 156) per genome, which indicates genomic reduction during evolutionary host adaptation.In Europe, approximately 84% of cultivated crop species depend on insect pollinators, mainly bees.  Apis mellifera) is the most important commercial pollinator worldwide [Melissococcus plutonius [M. plutonius. Subsequently, M. plutonius colonizes the larval gut. Dying larvae are displaced to the wall of the comb cell, often turn yellow and finally take on a brownish color, as they decompose after death [M. plutonius typical strains, atypical strains , has recently been shown [M. plutonius were not described, and therefore the pathogenicity mechanism remains unclear.The Western honey bee  displayd Japan, . Furtherd Japan, ,9, whichen shown . The auten shown . Field pen shown . Until nM. plutonius strains and a comparative genome analysis with type strain ATCC 35311 [M. plutonius that might play an important role in EFB pathogenesis. Additionally, we investigated the expression profiles of three putative virulence factors in naturally EFB-infected larvae. In this way, the study contributes to the prospective development of a conceptual pathogenicity model and the underlying molecular mechanisms of EFB disease.In this study, we investigate if phylogeny-related virulence is a consequence of variance in the gene content. For this purpose, we present the whole genome sequences of 12 M. plutonius ATCC 35311 (type strain) was originally isolated in United Kingdom and represents a typical strain according to Bailey and Collins [M. plutonius S1 is a derivative of strain 49.3 and was isolated after five cultivation steps. Collins . Strain  Collins . The typM. plutonius using species-specific 16S rRNA primers  as recommended by the manufacturer.Honey bee larvae with clinical EFB symptoms were collected from the aforementioned EFB outbreaks in Switzerland and Norway, and dissected under sterile conditions. Isolates were prepared from diseased larvae mixed with Bailey medium . Larval  primers . GenomicM. plutonius strain S1 was performed with a combined sequencing approach using a Genome Analyzer IIx  and a 454 GS-FLX Titanium XL+ system . 454-shotgun and Illumina Nextera XT shotgun libraries were prepared as recommended by the manufacturers. Sequencing resulted in 149,969 total 454 shotgun reads and 869,292 Illumina 112 bp paired-end reads, which were used for a hybrid assembly with MIRA v3.4.0.1 [M. plutonius isolates B5, H6, L9, 21.1, 60, 82, 764-5B, and 765-6B was carried out by generating paired-end libraries (2 × 112 bp) with the Nextera XT library preparation kit and employing the Genome Analyzer IIx as recommended by the manufacturer . Strains 49.3, 90.0 and 119 were sequenced at the Beijing Genomics Institute  with an Illumina Hiseq2000  by employing 90 bp paired-end reads. After read trimming and quality check using Trim_Galore v0.3.7  and FastQC v0.11.2 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), respectively, a de novo assembly of the paired-end Illumina reads was done with the SPAdes software 3.7.0 [Whole-genome shotgun sequencing of v3.4.0.1 . An averv3.4.0.1 . Misassere 3.7.0 . BacteriProkka v1.9  was usedM. plutonius ATCC 35311 (BioProject: PRJDA61383) and DAT561 (BioProject: PRJDA73165) were obtained from NCBI and re-annotated as described above for comparative genome analyses , JSBA00000000 (M. plutonius 49.3), JSBE00000000 (M. plutonius 60), JSBF00000000 (M. plutonius 82), JSAZ00000000 (M. plutonius 90.0), JSBB00000000 (M. plutonius 119), JSAW00000000 (M. plutonius B5), JSBC00000000 (M. plutonius H6), JSBD00000000 (M. plutonius L9), CP006683-CP006684 (M. plutonius S1), JSAV00000000 (M. plutonius 764-5B), JSAX00000000 (M. plutonius 765-6B).DNA sequences obtained and GenBank submissions: The genome sequences reported in this paper have been deposited in the GenBank database under accession numbers: JSAY00000000 (M. plutonius strains were determined by multilocus sequence typing (MLST) [in silico using the public available ST data from the PubMLST database . The goeBURST algorithm as implemented in the program PHYLOViZ v2.0 [−5) [Melissococcus genomes as input. For this purpose, cat_seq v0.1 and cds_extractor v0.6 were used [M. plutonius 49.3 was used as a reference strain. Gubbins was used to identify loci with high densities of SNPs [The sequence types (ST) of g (MLST)  according (MLST) . The seqViZ v2.0  was used2.0 [−5)  by usingere used . Based oere used  with 100ere used  was usedere used  were use of SNPs  that may of SNPs . Mugsy ( of SNPs  was used of SNPs .http://www.bioinformatics.org/ftp/pub/gipsy/) was used to detect genomic islands. Additionally, a blastp  search of the deduced protein sequences against the virulence factor database (VFDB) [The Phage Search Tool (PHAST)  was usede (VFDB)  was perfe (VFDB) , Bactibae (VFDB) , and IMGe (VFDB) . The MERe (VFDB)  was usede (VFDB)  and easye (VFDB)  were useApis mellifera) displaying EFB symptoms  were sampled from the same colony in Lützelflüh, Switzerland, in June 2013. Furthermore, healthy honey bee 5th instar larvae  were used as a negative control. Sampled larvae were frozen in liquid nitrogen immediately after collection. Honey bee larvae were individually homogenized in 200 µL sterile TE buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA) supplemented with 3 mg/mL lysozyme. The homogenate (50 µL) was used for a fast EFB confirmation test using the EFB Diagnostic Test kit  and 10 µL was used for colony PCR with specific primer pairs targeting the 16S rRNA gene of M. plutonius [M. plutonius genes. Transcription of M. plutonius specific genes in EFB-infected larvae was tested using RT-PCR , and rho (transcription termination factor) were used as positive controls. Genomic DNA of strain 49.3 was used as positive control and strain S1 as negative control as the toxin gene is not present in this strain  was defined within CC13. No strain could be allocated to the known CC12 to which the atypical strains (including DAT561) from Japan belong. The type strain M. plutonius ATCC 35311 from England was assigned to ST1 (CC13) according to Haynes et al. [In order to analyze the molecular epidemiology and population structure of ceptions . Four isceptions . The rem of CC13 . Interess et al. .M. plutonius 21.1, 49.3, 60, 82, 90.0, 119, B5, H6, L9, S1, 764-5B, and 765-6B range from 2.021 to 2.101 Mbp ) and comprise between 1589 and 1686 predicted protein-encoding genes , and strain heterogeneity and contamination were estimated as 0%.  in some strains, but detected in others, can be assigned as absent. As mentioned previously  only sequences below 0.5 kbp have not been included in estimating genome sizes and for virulence gene screening. Hence, with such high values for genome completeness, it is very unlikely that genes have not been detected, caused by missing genome sequences. Comparing the different Melissococcus genomes with respect to their coding density revealed a high density with neglectable variance for the 14 different strains , suggesting no substantial assembly or gene calling errors  , which wM. plutonius ATCC 35311 [All strains harbor a plasmid with high DNA sequence similarity to the recently published plasmid pMP1 (NC_015517) of M. plutonius strains as 1304 proteins, which represents on average approx. 71% of the proteins encoded by a M. plutonius genome. The pan-genome of our strain panel comprises 1846 proteins.Based on the identification of orthologous proteins we calculated the core genome of this set of M. plutonius evolutionary diversity, rather than the result of independent infection events.Comparing the different genome properties, this is the first study providing evidence for multiple strain infections in a single honey bee larva. B5, H6 and L9 were isolated from the same larva but have different genome sizes, number of coding sequences and pseudogenes, and the pMP19 plasmid is only present in H6. All three strains cluster together closely , which mArai et al.  and NakaM. plutonius strains have 275 orthologues in common, which were not identified in the atypical strain DAT561  [Enterococcus faecalis has highly toxic effects on honey bee larvae leading to classical EFB symptoms [We identified an and CO2) . The genand CO2)  showed tsymptoms . M. plutonius genomes, a potential chitin-binding domain-containing protein, consisting of a signal peptide and a type 3 chitin-binding domain, was identified (PlCBP49 (JX185746) of Paenibacillus larvae. PlCBP49 is able to degrade the peritrophic matrix of the honey bee larva [Bacillus thuringiensis serovar kurstaki str. (P. larvae DSM 25719 (22% identity with ERIC1_1c29890) [P. larvae DSM 25719/25430, which are fragmented by transposase insertions or mutations , whereas the enhancin of atypical strain DAT561 is slightly truncated (728 amino acids). However, analyzing the most recent version of the DAT561 genome [N-acetylgalactosaminidase (EC 3.2.1.97) that catalyzes the release of oligosaccharides via hydrolysis of the O-glycosidic bond between alpha-acetylgalactosamine at the reducing end of mucin-type sugar chains (O-glycan) and serine/threonine residues of proteins, which is putatively non-functional in the M. plutonius strains 82, 90.0, 119 and ATCC 35311 due to nonsense mutations. The peritrophic matrix lines the midgut of invertebrates and is comprised of secreted chitin and (glyco)proteins, mainly peritrophins . In the entified . It beloee larva . Additioee larva ,57,58,59aki str. . The pep1c29890)  and it iutations  and are 1 genome  revealedN-acetylgalactosaminidase were transcribed in vivo during EFB pathogenesis .A fibronectin/fibrinogen-binding domain (DUF (Domain of unknown function) 814)-containing protein was discovered in all strains examined in this study . The corStaphylococcus aureus, it was shown that some DUF1542-containing proteins are involved in cell cluster formation, cellular adhesion and antibiotic resistance [A putative extracellular matrix-binding protein , which is a common secondary invader in EFB disease [Only the genomes of the typical ding ORF , while a disease . Most imE. faecium strains by Palmer et al. [E. faecium 504 and E. caccae ATCC BAA-1240 share high sequence similarity to this cluster, although all Melissococcus strains contain nonsense mutations in genes involved in capsule formation -locus of E. faecalis [Enterococcus haemoperoxidus and E. caccae  cycle and the electron transport system for oxidative phosphorylation. Enzymes for a glycolysis system were found in all strains, but the genes encoding pyruvate kinase and transketolase of the atypical strain DAT561 are interrupted by frameshift mutations in the original genome sequence of this strains [M. plutonius is shown in Competition for resources with the host results in an evolutionary pressure on bacteria. Therefore, we studied in more detail the potential pathways for energy and sugar metabolism of  strains . A recen strains . EnzymesM. plutonius. We used the obtained bioinformatical results to discuss critical steps for the pathogenesis and infection of M. plutonius; including proliferation of M. plutonius in the gut of the honey bee larva, competition for host resources and putative encapsulation of M. plutonius. However, nearly all of the pathogenesis mechanisms are completely unknown and consequently purely speculative. M. plutonius may have a different lifestyle as genome data showed, with genes which might also be relevant for an invasive lifestyle and actively killing honey bee larvae. The following discussion is based on the pure presence of the identified genes in the M. plutonius genomes and experimental evidence is needed to develop a fully functional infection model. We did not include the potential function of the secondary invaders ) that are usually found in the remains of EFB-diseased honey bee larvae. Our genome analyses revealed that all M. plutonius strains are putatively able to degrade the pectin backbone of the pollen cell wall using a large variety of enzymes. Interestingly, the strains differ in their genetic equipment of these enzymes, as different enzymes are putatively non-functional due to mutations in the corresponding genes of all typical strains from Norway and Switzerland  of the honey bee pathogen  biosynthesis and only the genome of the atypical strain M. plutonius DAT561 lacks the respective genes , proteins involved in adhesion to host extracellular matrix , proteases and proteolytic enzymes; however, functionality and relevance for pathogenicity of all these proteins have to be verified in vivo.Virulence of pathogenic bacteria of insects is mostly determined by the ability to degrade host glycoproteins and metabolizing resulting carbohydrates. The honey bee larval gut is coated by a chitin-containing peritrophic matrix, which is degraded during nfection . This abu et al.  showed fc matrix . NeverthM. plutonius 49.3 with highly similar contigs in strains 21.1, 60, and H6. The plasmid comprises 20 ORFs of which two ORFs encode for an extracellular matrix-binding protein and melissotoxin A. Interestingly, the virulence plasmid pMP19 is not stably maintained during in vitro propagation. The five-time laboratory-passage of M. plutonius 49.3 cured the strain from the plasmid and resulted in strain M. plutonius S1. Furthermore, M. plutonius B5, H6 and L9 were all isolated from the same EFB-infected honey bee larva and exhibited a close phylogenetic relationship  was found in tionship , but onl was founboratory  and lackboratory  and mighboratory  might beboratory .M. plutonius forms a capsule [M. plutonius produces capsules in vivo in infected larvae, and at least for DAT561, capsule-formation has not been detected using light and electron microcopy [M. plutonius strains analyzed in this study  lack putatively important virulence factors  in comparison to CC3 (ST3/7). Additionally, strains harboring the virulence plasmid pMP19 may have additional advantages versus strains lacking the plasmid. Typical and atypical strains most likely have different virulence mechanisms, with atypical strain DAT561 encoding for different bacteria cell envelope-associated and host cell adhesion-associated proteins. However, the atypical strain might compensate this with faster growth in the larval gut by increased metabolic capabilities with respect to usage of different nutrient sources. Faster nutrient consumption of atypical strains lead to starvation of the honey bee larvae. We assume that in case of atypical strains the combination of fast nutrient consumption and establishment of virulence factors leads to an accelerated death of the honey bee larvae. Nevertheless, additional infection studies are needed to predict a difference in virulence between strains and clonal complexes. Such studies also have to be tested with different host genetic backgrounds, as host genotype contributes to the course of the disease [Based on the identification of putative virulence genes from different  strains . Typical disease . Therefo