Xylella fastidiosa, a vector-borne fastidious plant pathogen infecting multiple crops, GC content ranges between ∼51-52%; however, these values were gathered using limited genomic data. We evaluated GC content variations across X. fastidiosa subspecies fastidiosa (N = 194), subsp. pauca (N = 107), and subsp. multiplex (N = 39). Genomes were classified based on plant host and geographic origin; individual genes within each genome were classified based on gene function, strand, length, ortholog group, core vs accessory, and recombinant vs non-recombinant. GC content was calculated for each gene within each evaluated genome. The effects of genome and gene-level variables were evaluated with a mixed effect ANOVA, and the marginal-GC content was calculated for each gene. Also, the correlation between gene-specific GC content vs natural selection (dN/dS) and recombination/mutation (r/m) was estimated. Our analyses show that intra-genomic changes in nucleotide composition in X. fastidiosa are small and influenced by multiple variables. Higher AT-richness is observed in genes involved in replication and translation, and genes in the leading strand. In addition, we observed a negative correlation between high-AT and dN/dS in subsp. pauca. The relationship between recombination and GC content varied between core and accessory genes. We hypothesize that distinct evolutionary forces and energetic constraints both drive and limit these small variations in nucleotide composition.Nucleotide composition (GC content) varies across bacteria species, genome regions, and specific genes. In  The relevance of nucleotide composition (GC content) in genome evolution has been well established from a genomics , and mule.g. mutation vs selection). As a result, distinct genes, gene/genome regions, genomes, and populations can achieve unique GC content values , X. fastidiosa has undergone substantial genomic and biological changes. The X. fastidiosa genome is half the size of Xanthomonas spp. and characterized by the loss of specific metabolic functions and a slow growth rate , functional (i.e. clusters of orthologous group [COGs]), evolutionary , or genetic variables . Finally, we compared the GC content of X. fastidiosa with four other plant pathogens: Xanthomonas citri, X. campestris, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola (which are closely related to X. fastidiosa), and the more distant Agrobacterium tumefaciens.The available X. fastidiosa whole-genome sequences. Isolates were collected from infected plant material in diverse geographic regions. Isolates belong to subsp. fastidiosa (N = 194), subsp. multiplex (N = 39), and subsp. pauca (N = 107). The number of isolates varied among geographic locations and infected plant hosts. Raw data have been made publicly available (Supplementary Table S1). Most isolates were sequenced using Illumina HiSeq2000; however, five subsp. fastidiosa isolates were sequences using both Illumina HiSeq2000 and PacBio . Samples were sequenced at the University of California, Berkeley Vincent J. Coates Genomics Sequencing Laboratory . The quality of the genome assemblies varied but coverage remained above ∼59×.The following study encompasses 340 https://github.com/lh3/seqtk) and cutadapt v1.14  for subsp. pauca, and M12 (ASM1932v1) for subsp. multiplex). Finally, genomes were annotated using Prokka (fastidiosa isolate XF70 from Costa Rica and removed by mapping FASTQ reads to its closest phylogenetic relative as described by Olea europaea genome assembly (GCA_900603015.1) with bowtie2 v2.3.4.1  and subsequently converted to sorted BAM. Bedtools v2.26.0 (A. tumefaciens (N = 12), X. oryzae pv. oryzae (N = 75), X. oryzae pv. oryzicola (N = 13), X. campestris (N = 14), and X. citri (N = 68) were obtained from NCBI and re-annotated with Prokka. Metadata for these isolates have also been included in Supplementary Table S1.Details on genome assembly and annotation protocols have been provided in previous studies . Individual genome assemblies and annotation files were used to mine gene sequences within each genome. Individual genes were also classified regarding their number of orthologs. Finally, all individual genes were categorized based on their COGs  and by the main categories to which these functions belong . Individual genes without defined COG  were assigned to the Poorly characterized (P) category. Individual genes belonging to two or more functional classes were grouped as Multiple categories (MU). Approximately 30–40% of individual genes were classified within the P category.Roary v3.11.2  was calculated for the core genome alignment of each phytopathogen using the R package “PopGenome”  was also evaluated for individual gene alignments. Briefly, Roary was used to identify individual ortholog groups within each X. fastidiosa subspecies. The detected ortholog groups were programmatically aligned using MACSE v2  was also calculated for each gene within individual X. fastidiosa subspecies. The length of each gene was calculated based on its start and end positions. Variables were assigned as gene functional class, gene size, gene strand, accessory/core gene, number of orthologs, and recombinant/non-recombinant gene (gene-dependent); and geographic location and plant host (genome-dependent). Certain variables known to affect genomic GC content were not studied here. For example, large scope RNAseq data were not available at the time of analysis, precluding any assessment of gene expression levels and their relationship to GC content. It is expected that this type of data will become available in the future. Similarly, most of the whole genome sequences used have been assembled to the contig level, and thus, it was not possible to estimate gene position in a full chromosome. In this regard, gene position was obtained for three finished representative whole-genome assemblies: Temecula1 (GCA_000007245.1) for subsp. fastidiosa, 9a5c (ASM672v1) for subsp. pauca, and M12 (ASM1932v1) for subsp. multiplex. A synteny analysis among the assemblies was conducted using CoGe .A custom python script was used to calculate gene-specific GC content values for each gene within individual A mixed-effect ANOVA was used to evaluate the statistical contributions of gene-dependent and genome-dependent variables in the nucleotide composition of individual genes. The Genome ID and genome GC content were used as a random slope and the gene ortholog group identified by Roary was used as a random effect. The model was used to estimate marginal means for gene-specific GC content . In other words, the marginal-GC content representing the estimated GC content values averaged across all variable levels of the linear regression model.Orthologous genes were identified, aligned, and their ML trees were constructed as described in the “Recombination detection and estimation of global genetic diversity in core alignments” section of the Materials and methods. The relationship between GC content and gene-wide signs of selection was estimated. The rate of non-synonymous over synonymous substitutions (dN/dS) was programmatically calculated for each gene alignment using BUSTED . It should be noted that the shell genome has the widest range in relation to the percentage of strains sharing a given gene (15–95%). Therefore, it can potentially cover a wider range of gene presence/absence variations compared with the soft-core (genes shared by 95–99% of strains) and the cloud genomes (genes shared by <15% of strains).Core and accessory genome sizes varied within examined . The staexamined . Gene-spexamined . Specifit = 14.866, P < 2.2 × 10−16 for subsp. fastidiosa; and t = 7.1555, P = 8.774 × 10−13 for subsp. multiplex) and in shell genes relative to cloud genes . However, this was not seen between the soft-core genes relative to cloud genes in subsp. pauca . Trends in GC content variation based on explanatory variables are discussed below.The nucleotide composition in cloud, shell, and soft-core genes was similar; however, there was a small and statistically significant decrease in GC content in soft-core genes relative to cloud genes (vs GET_HOMOLOGUES (3 variation followed the same trends (Supplementary Table S3). Statistically significant gene- and genome-dependent variables were subsequently plotted to evaluate if biological or ecological trends were present. In addition, the size effect (Cohen’s d) was calculated for the three statistically significant categorical variables: Accessory vs Core, DNA strand, and gene functional class . In addition to marginal-GC, GT was calculated for individual genes in the leading and lagging strand in all X. fastidiosa subspecies . The goal was to establish if G and T nucleotides were enriched in either DNA strand . After removal of ribosomal protein-coding genes, marginal-GC content in the CPS and ISP classes was similar in subsp. fastidiosa and subsp. multiplex core genes . Notably, the marginal GC-content of M class genes was lower in the accessory genome, particularly in the case of subsp. fastidiosa. Genes from the Poorly characterized (P) category were removed from these visualizations.Gene functional class also affected genic GC content. The number of core and accessory genes from different functional groups varied within subspecies. The Metabolism (M) functional class was the most numerous, while the CPS and ISP functions had a similar number of genes . There wi.e. “Translation,” “Amino acid transport and metabolism,” “Defense mechanism,” etc.) (Supplementary Table S5). The relationship between GC content in core vs accessory genes varied across functions, but overall, accessory genes had higher marginal-GC content than core genes of the same function in both subsp. fastidiosa and subsp. multiplex. This was particularly evident within the ISP functional class, where functions associated with “Translation” and “Replication” had lower marginal-GC content. Differences in nucleotide composition across specific functions were less clear in subsp. pauca .Gene function had a significant effect on genic GC content even when genes were classified based on their specific function , X. oryzae pv. oryzae , X. oryzae pv. oryzicola , X. citri , and X. campestris . Neither X. citri, X. campestris, nor A. tumefaciens showed a clear separation in marginal-GC content between functional classes. On the other hand, in both X. oryzae pv. oryzae and X. oryzae pv. oryzicola genes from the ISP class had lower marginal-GC content compared with other functional classes, even after removal of ribosomal protein-coding genes : ng genes . The treX. fastidiosa subspecies showed three chromosomal inversion events between the 9a5c strain (subsp. pauca), and the M12 (subsp. multiplex) and Temecula1 (subsp. fastidiosa) strains .Genes from the ISP function were smaller than those from other functional groups ; howeverpauca , but not for subsp. multiplex  and subsp. fastidiosa . The significant correlation was small. Most individual gene alignments showed signs of gene-wide purifying selection, with only a small proportion having a dN/dS > 1 . Most of the genes under positive selection in each subspecies  were classified in the P group. In subsp. fastidiosa, 12 genes belonged to the CPS class, 15 to the ISP class, and 19 to the M class; in subsp. multiplex, 23 genes belonged to the CPS class, 27 to the ISP class, and 50 to the M class; and in subsp. pauca, 12 genes belonged to the CPS class, 12 to the ISP class, and 38 to the M class (Supplementary Table S7).Genic GC content and the Tukey’s Ladder of Powers transformation of dN/dS were significantly correlated for individual gene alignments of subsp. X. fastidiosa subspecies, intra-subspecific recombination was pervasive across the length of the core genome alignment and equally frequent among functional groups . Core genes from the Poorly characterized (P) category were removed from the visualizations. Genic GC content and the log10 transformation of r/m were significantly correlated for individual gene alignments of subsp. multiplex (r(3869) = −0.1516, P = 2.2 × 10−16) and subsp. fastidiosa (r(3723) = −0.1396, P = 2.2 × 10−16), but not on subsp. pauca (r(1930) = 0.043, P = 0.0588). However, both significant correlations were small.For all analyzed l groups . As a geXanthomonas spp. (∼5 Mb and 65% GC), X. fastidiosa (∼2.5 Mb and ∼51% GC) has undergone significant genomic changes. As in other obligate symbionts  were present in Xanthomonas spp. and the core/soft-core of X. fastidiosa. Repair genes were hypothetically duplicated in the Xanthomonas-Xylella ancestor and later lost in X. fastidiosa ] and, though not directly comparable, smaller than those reported for X. oryzae pv. oryzae [2 × 10−5 mutations per gene per year;   and C. Sulcia muelleri (∼22.7% GC) have low GC genomes.Another explanation for the drop in GC content observed in ronments . During ronments . This, ironments . For X.  limited . In certX. fastidiosa might be the result of selection-driven gene loss eliminating GC-rich accessory genes. This hypothesis should be tested in studies encompassing a larger range of plant-associated bacteria.Alternatively, genome reduction is linked to selection-driven gene-loss on accessory genes . Recent X. fastidiosa. Natural selection can counteract the A/T mutation bias observed in microbial organism . Very few studies have assessed transitions and transversion rates in X. fastidiosa, with most of them using limited genomic data , or if they are masked by other evolutionary forces (i.e. recombination). Future studies should leverage the increasing publicly available genome data for this pathogen to explicitly address this issue.Another point to consider is the unique mutation-selection balance on mic data . While ii.e. lower GC) would be observed by identifying compositional differences between these groups. Such a trend is observed here.GC content was more constrained in the core than the accessory genome. Among accessory genes, the cloud genome (<15% strains) could represent either ancestral gene loss in certain lineages or recent gene gain (X. fastidiosa’s leading strands. This could be explained by differences in the energetic requirements for de novo synthesis of nucleotides and amino acids resulting in strand-biased nucleotide composition (SNC)  also have less significant strand-biased gene distribution , facilitate the opening of the DNA double helix and the initiation of replication  and subsp. multiplex , compared with subsp. pauca . Whether this indicates adaptive changes facilitating rapid replication in some X. fastidiosa subspecies or is the result of architectural chromosome changes (though the cluster is outside three major inversion events) remains to be evaluated.The clustered organization of ribosomal protein genes  matches that previously described in other bacteria. Such an organization facilitates control during transcription and translation   could also be associated with the mechanisms of plant infection. Xylem-limited pathogens move through long distances in a nutrient-limited environment and cause systemic infection while non-vascular pathogens remain restricted to infection sites formed by living parenchyma cells . Most of them belonged to the M class. A less conservative analysis (e.g. a branch-site selection test) might highlight a different pattern, yet, these results indicate that metabolic adaptation is occurring in X. fastidiosa.Purifying selection was predominant in pauca. However, we could not establish if GC content was differently favored between non-synonymous vs synonymous changes from our results. Within bacteria, natural selection favors increased synonymous GC content  = −0.0558, P = 0.0142) was small and future studies should determine its biological relevance, as well as address the potential role of gene expression and transcription in GC content. In addition, it should be noted that in the case of subsp. pauca and subsp. fastidiosa; a significant proportion of our data originates from recently introduced populations. This could limit the action of natural selection due to a recent founder effect.Genes favoring higher dN/dS had lower GC content in subsp. pauca strains , and plays a role in speciation (Nunney ) and host switching . Yet, our results indicate that intra-subspecific recombination does not have a distinct effect on nucleotide composition. Recombinant core genes had lower GC content than non-recombinant core genes, though this was not observed when genes were subdivided according to function. This suggests that the differences in nucleotide composition among functional groups are not caused by recombination, at least in the core genome. In addition, while Recombination occurs between sympatric subsp. eciation  and hosti.e. endosymbionts and intracellular pathogens, opportunistic pathogens, commensal and free-living bacteria, and obligate pathogens), found that nucleotide composition did not differ between recombinant and non-recombinant regions  did not show any differentiating trends. The analysis conducted here confirms these results in X. fastidiosa’s core genome but does not eliminate the possibility nucleotide composition in the accessory genome might be linked to recombination.Previous studies have found that GC-biased gene conversion (gBGC) results in increased GC content in recombinant genes (Xylella fastidiosa has undergone significant biological, ecological, and genomic changes. Thus, it can be a valuable organism to better understand nucleotide compositional changes in bacterial plant symbionts. Our results indicate that GC content has dropped in X. fastidiosa compared with its closest relative. Several hypotheses are presented to explain this drop and should be tested further. Yet, particular focus should be dedicated to better understanding the mutation rate of X. fastidiosa. For this pathogen species, changes in nucleotide composition do exist but are small. It is notable that nucleotide composition of most of the genome of X. fastidiosa is conserved regardless of numerous variables having a statistical effect on it. We hypothesize that distinct evolutionary forces and energetic constraints both drive and limit these small variations. For example, recombination in the core genome and purifying selection would limit the number of nucleotide changes; while recombination in the accessory genome, nutritional limitations in the environment, and mutation/selection biases drive genic GC content drops. Taken together, we show that even in a GC-balanced genome like that of X. fastidiosa, nucleotide changes are observed, and the action of evolutionary forces can be detected.


Schistosoma mekongi is found in the lower Mekong river region and causes schistosomiasis. Low sensitivity of diagnosis and development of drug resistance are problems to eliminate this disease. To develop novel therapies and diagnostics for S. mekongi, the basic molecular biology of this pathogen needs to be explored. Bioactive peptides have been reported in several worms and play important roles in biological functions. Limited information is available on the S. mekongi peptidome. Therefore, this study aimed to identify S. mekongi peptides using in silico transcriptome mining and mass spectrometry approaches. Schistosoma peptide components were identified in adult worms, eggs, and infected mouse sera. Thirteen neuropeptide families were identified using in silico predictions from in-house transcriptomic databases of adult S. mekongi worms. Using mass spectrometry approaches, 118 peptides (from 54 precursor proteins) and 194 peptides (from 86 precursor proteins) were identified from adult worms and eggs, respectively. Importantly, eight unique peptides of the S. mekongi ubiquitin thioesterase, trabid, were identified in infected mouse sera 14, 28, and 56 days after infection. This protein may be a potential target for diagnosis of schistosomiasis. The S. mekongi peptide profiles determined in this study could be used for further drug and diagnostic development. Schistosoma mekongi is a causative agent of schistosomiasis in the Mekong region. The Lao People’s Democratic Republic and Cambodia are endemic areas of this disease [S. mekongi infection generally yields low egg intensity. In some patients, egg shading in feces cannot be observed, and only rectal snipping (a more invasive technique) is able to demonstrate Mekong schistosomiasis [S. mekongi surveillance monitoring, knowledge of the molecular biology of the organism and new interventions for disease control are required. disease . In 2017somiasis . To accoCaenorhabditis elegans [Ascaris suum [S. mansoni neuropeptide—I/Lamide—was identified in S. mansoni larval and adult worms. However, no effects of the peptide on aberrant mobility or morphological phenotypes were detected by knockdown experiments [S. mansoni and S. japonicum [S. mansoni infection from urine samples has been attempted. Ninety-three percent of infected children could be classified correctly based on their urinary peptide profiles [S. haematobium-infected patients even when microhematuria tests were negative [Bioactive peptides have been reported in many worm species such as  elegans  and Ascaris suum . These pris suum . A S. maeriments . Prohormaponicum . In addiprofiles . Hemoglonegative .S. mekongi bioactive peptides is available. In this study, the low molecular weight proteins and peptides of S. mekongi worm, egg, and infected mouse sera were explored. Because the S. mekongi transcriptome is available and was previously published by our group [S. mekongi using data mining and proteomic approach. Bioinformatic analyses were applied for assessing the bioactivity of the S. mekongi peptidome. The worm and egg peptides are essential for understanding the S. mekongi molecular biology, and they also facilitate the schistosome drug development. Moreover, the S. mekongi circulating peptides in the infected sera are useful for diagnosis development. This dataset may lead to the development of interventions for the diagnosis and treatment of Mekong schistosomiasis.Currently, no information on ur group , this daS. mekongi adult worm transcriptome dataset [The  dataset  was used dataset . Non-neu dataset . The sigS. mekongi cercariae were prepared from Neotricula aperta snails without any pre-treatment. ICR mice (N = 6) were anesthetized with Nembutal®  40–60 mg/kg intraperitoneal injection with tuberculin syringe and 26-gauge needle. The mice were exposed with cercariae by abdominal exposure. For a mouse, thirty cercariae were counted under microscope and gently applied to the mouse abdomen by hairpin. Sera were collected pre- and post-infection . After 6–8 weeks, infected mice were sacrificed and dissected. Adult worms were flushed out by vascular perfusion using 0.85% sodium chloride. The perfusion solution was transferred to a sedimentation cone. Adult worms settled at the bottom of the cone were collected. Mouse livers and intestines were homogenized in 0.85% sodium chloride. Homogenates were sequentially passed through 80, 120, 160, and 260 mesh stainless steel sieves to separate S. mekongi eggs from liver and intestinal tissue. The eggs were washed three times with NSS and stored in liquid nitrogen until used.All animal procedures were approved by the Faculty of Tropical Medicine Animal Care and Use Committee (FTM-ACUC), Mahidol University . All experiments were performed in accordance with relevant guidelines and regulations. Eight-week-old female ICR mice (25–35 g) were used in this experiment. S. mekongi adult worms and eggs were dissolved in 8 M urea and sonicated on ice for 10 s. The lysates were centrifuged for 20 min at 20,000 g, 4 °C. The supernatants and mouse sera were individually filtered using Amicon Ultra 0.5 mL centrifugal filters with a molecular weight cutoff of 30 kDa  by centrifugation for 20 min at 15,000× g, 4 °C. The flowthrough peptides were enriched using reverse phase C18 ZipTip chromatography (Millipore). The tips were pre-rinsed with 50% acetonitrile then equilibrated with 0.1% trifluoroacetic acid (TFA). The samples were loaded onto the zips and washed with 0.1% TFA, 80% acetonitrile. The peptides were dried using a speed vacuum .TM 3000 nano-LC system . The column was an Acclaim PepMap RSLC 75 μm × 15 cm nanoviper C18 with a 2 μm particle size and a 100 Å pore size . The LC system was coupled with a MicroToF Q II mass spectrometer . Mass ranges were acquired at 500–3500 m/z. MASCOT search engine 2.3  was used for data analysis. An in-house generated S. mekongi transcriptomic database was used for searching. The search parameters were as follows: one missed cleavage, trypsin digestion, 0.8 Da peptide tolerance, ±0.8 fragment mass tolerance, acetyl (protein N-terminus), amidated (protein C-terminus), Gln→pyro-Glu , Glu-→pyro-Glu , oxidation (Met), and variable modifications. The significance threshold was 0.05.The peptide solution was resuspended in 0.1% formic acid and then injected on an UltiMateS. mekongi ubiquitin thioesterase trabid was retrieved from an in-house transcriptome database. Other ubiquitin thioesterase trabid sequences, including TNN19375.1 (S. japonicum), XP_018651129.1 (S. mansoni), XP_012798773.1 (S. haematobium), TGZ66416.1 (Opisthorchis felineus), GAA33082.2 (Clonorchis sinensis), OON19543.1 (Opisthorchis viverrini), XP_006508032.1 (M. musculus), and XP_006717970.1 (H. sapiens), were retrieved from the nonredundant protein sequence database of the National Center for Biotechnology Information (NCBI). All sequence alignments and calculations of percent identity were performed using Clustal Omega software.The protein sequence of the S. mekongi adult worm transcriptome dataset. A total of 8440 sequences were obtained, and these sequences were further investigated for their biological functions following the workflow in S. mekongi adult worms. FMRFamide related peptide, kisspeptin 1 (KiSS1), opioid peptide, LWamide neuropeptide, arthropod hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone/vitellogenesis-inhibiting hormone (CHH/MIH/GIH/VIH hormone), serpin, egg-laying hormone (ELH), pyrokinin, neurotensin, and neuropeptide Y family peptides were also observed in S. mekongi. Non-neuropeptides were further subjected to gene ontology classification using multilevel analysis. A total of 7013 peptides demonstrated gene ontologies. The top 20 classes according to biological process, molecular function, and cellular component terms are shown in Low molecular weight proteins and peptides with ≤30 kDa molecular weights and ≤280 amino acids in length were retrieved from the S. mekongi adult worms and eggs were separated by filtration (30 kDa), purified using stagetip C18 resin, and then analyzed by mass spectrometry. A total of 118 peptides (from 54 precursor proteins) and 194 peptides (from 86 precursor proteins) were identified from adult worms and eggs, respectively  were identified by mass spectrometry from S. mekongi eggs. The amino acid positions of neuropeptide regions within precursor protein sequences were identified using NeuroPep, a neuropeptide software (http://isyslab.info (accessed on 11 April 2021).). Neuropeptides consisting of TKP, PLG and GGG sequences were predominantly found in S. mekongi adult worms. Neuropeptides containing GGG, YRI and QHL sequences were mainly observed in S. mekongi eggs.Peptides from ectively . Only twectively . The EAAS. mekongi transcriptomic dataset. Any peptides that were identified from uninfected mouse sera were subtracted from those identified from infected mouse sera. A total of 385 peptides (from 120 precursor proteins), 288 peptides (from 90 precursor proteins), and 349 peptides (from 106 precursor proteins) were identified in infected mouse sera 14, 28, and 56 days after infection, respectively  were consistently observed at 14, 28 and 56 days after infection. The identified peptide sequences of S. mekongi ubiquitin thioesterase trabid are presented in S. mekongi compared with a Mus musculus protein sequence database. The protein sequence of the S. mekongi ubiquitin thioesterase trabid was compared with homologs in S. japonicum, S. mansoni, S. haematobium and Homo sapiens. For other helminths, proteins from Opisthorchis felineus, Clonorchis sinensis and Opisthorchis viverrini which showed the highest percent identity to the S. mekongi ubiquitin thioesterase trabid were also used for the comparison. Percent sequence identity is shown in Peptides were identified from uninfected mouse sera (Day 0) and infected mouse sera . Peptide identification was performed by mass spectrometry analysis and by searching against the in-house ectively . The topS. mekongi peptide discovery.In silico transcriptome mining is a powerful tool for peptidome prediction from biological samples. Using homology-based searches and simple bioinformatics workflows, large peptidomes have recently been predicted for a variety of organisms . HoweverS. mekongi neuropeptide family identified using a mining approach. Limited information is available on this peptide family in schistosomes. However, this family has been described in C. elegans. These peptides are expressed in chemosensory neurons, head neurons, spermatheca, hypoderm, intestine, and embryos of C. elegans [GGG peptides belonging to the YGGW-amide related peptide family were identified in S. mekongi adult worms; precursor proteins containing GGG related regions were identified in both S. mekongi adult worms and eggs. In C. elegans, GGG related peptides are also abundant and are localized in chemosensory neurons, sensory neurons, head neurons, tail neurons, the ventral nerve cord, spermatheca, vulval muscles, and intestine [Classical neurotransmitters such as acetylcholine, serotonin and catecholamines have been studied in parasitic platyhelminths and proposed as targets for anthelmintic drugs . However elegans . Using mntestine .S. mekongi FMRFamide-like peptides were identified. Neuropeptides of this family are typically less than 20 amino acids in length and contain an RFamide motif at their C-terminus. FMRFamide-like peptides play a central role in parasite neuromuscular biology. In S. mansoni, FMRFamide-like peptides are widespread throughout the nervous system and produce potent myoexcitation [C. elegans FMRFamide-like peptide signaling impacted locomotory and reproductive behavior. These peptides can bind multiple receptors, making it difficult to indicate the specific receptor being activated by FMRFamide-like peptides [2+ influx through sarcolemmal voltage operated Ca2+ channels (VOCCs). VOCC inhibitors such as nicardipine, verapamil, and methoxyverapamil could inhibit these contractions [Dugesia tigrina [Bdelloura candida [YIRF related peptide was detected in S. mekongi adult worms but was not observed in eggs. This finding supported the possibility that FMRFamide-like peptides play important roles in S. mekongi locomotion. Inhibition of FMRFamide-like peptide receptors might be a practical approach to treat S. mekongi infection.An important neuropeptide class in flatworms is formed by the FMRFamide-like peptides . Using tcitation . The altpeptides . The YIRractions . YIRFami tigrina  and Bdel candida . In our S. mekongi using the transcriptome mining approach. ELH is a member of the neuropeptide F family. Peptides in this family are approximately 40 amino acids in length and are characterized by an RxRFamide sequence at the C-terminus. These peptides are orthologs of the neuropeptide Y family in vertebrates. The neuropeptide F and Y families demonstrate high similarity in terms of structure and function [S. mekongi. In S. mansoni, DRIRFH and HRIRFN related peptides were reported. S. mansoni ELH potently inhibits Forskolin stimulation of cyclic AMP accumulation, leading to downstream signaling pathway regulation [C. elegans, introduction of a defective mutant ELH into wild-type hermaphrodites or females induced an egg-laying defective phenotype [Aplysia californica and Lymnaea stagnalis [S. mekongi ELH may also play an important role in regulation of egg-laying.Seven precursor sequences of molluscan ELH were identified in function . The IRIgulation . In C. ehenotype . Additiotagnalis . The S. S. mekongi using the transcriptome mining approach. The GFVRI amide related peptides are a novel family of bioactive helminth neuropeptides that were first reported in S. mansoni. Treatment of S. mansoni adult worms with this peptide led to significant mobility inhibition [The protein precursor of GFVRI related peptides -family, vitellogenesis-inhibiting hormone (VIH), also known as gonad-inhibiting hormone (GIH), play roles in vitellogenesis [Callinectes sapidus molting and reproduction [S. mekongi arthropod CHH/MIH/GIH/VIH hormone family may have functions in reproduction and development.Eleven precursor sequences of ogenesis . Moreoveoduction . The S. S. mekongi using both transcriptome mining and mass spectrometry approaches. The EAAVVSRQHPVKGEC and QHLSMNPLVESF peptides were identified by mass spectrometry in S. mekongi adult worms and eggs, respectively. TRH influences the release of other hormones, including prolactin, growth hormone, vasopressin, insulin, and the neurotransmitters noradrenaline and adrenaline [Platynereis dumerilii as well as in C. elegans [C. elegans, TRH related peptide is required for growth in body size [S. mekongi growth and development.The EHP, QHL and QHP related peptides are significant TRH domains and were identified in renaline . TRH ort elegans . In C. eody size . SimilarS. mekongi adult worms. A total of 178 sequences were identified by transcriptome mining while five and two sequences were detected in S. mekongi adult worms and eggs, respectively, by mass spectrometry. The TKP related peptides are part of the N/A neuropeptide family. In humans, these peptides could inhibit macrophage/microglial activation via an unknown mechanism [S. mekongi adult worms may involve host immune escape through macrophage inhibition.The precursor sequences of TKP related peptides were dominantly identified in echanism . In miceechanism . The rolS. mekongi. However, there is a lack of confirmation of the functional information on these peptides. The antioxidant, antimicrobial and other properties of these peptides were not evaluated in this study. Further investigations should be performed to confirm the role of each peptide in S. mekongi.Although, the peptides identified in this study may influence several behaviors in S. mekongi infection is currently diagnosed by detection of parasite eggs in stool specimens. It is a low-sensitivity technique, which can underestimate the prevalence and affect mass drug administration. Antibody detection in blood samples is also used to indicate the infection. However, the crude antigen needs to be prepared from the worm or egg which shows variation from batch to batch. Identification of circulating proteins or peptides could improve the diagnosis. The production of monoclonal antibody which binds to these antigens could be one of the strategies for S. mekongi diagnosis development. The S. mekongi peptide profiles in mouse serum 14, 28, and 56 days after infection were dissimilar. This result is consistent with the life cycle of S. mekongi. Approximately 14 days after infection, some S. mekongi worms are in the immature stage. They further develop to the mature adult stage and undergo pairing 28 days after infection. Eggs can be observed 56 days after infection. The SQFQPHFVVDTMSKGA, QWANLMEKIQASVATNPIITPVAQENQ, and NEVHTMLGQSTEEIRA peptides had the highest scores 14, 28, and 56 days after infection, respectively. This result could be useful to detect the early, middle, and late stages of S. mekongi infection. Eight unique peptides of the S. mekongi ubiquitin thioesterase trabid were identified in infected mouse sera starting from 14 days after infection. Comparison of the S. mekongi ubiquitin thioesterase trabid protein sequence with homologs from S. japonicum, S. mansoni, S. haematobium, mouse, and human showed that this protein was conserved among schistosoma species. In Drosophila melanogaster, the ubiquitin thioesterase trabid is a positive regulator of the Wnt signaling pathway. This pathway regulates numerous processes associated with cell development [S. mekongi ubiquitin thioesterase trabid may play roles in growth and development. The protein may be a potential target for early diagnosis of schistosomiasis. However, the individual serum analysis of infected mice needs to be further validated for further diagnosis application.elopment . In humaelopment . The S. S. mekongi worm, egg, and infected mouse sera provided the information insight into the molecular biology of schistosome. Besides, the findings could be useful for further schistosomal drug and diagnostic development.Identification of low molecular weight proteins and peptides from


Amanitaceae, Boletaceae, Russulaceae, and Tricholomataceae were the most diverse family; further, Amanita, Cortinarius, Lactarius, Russula, and Tricholoma were the dominant genera in the area. The macrofungi diversity showed increasing trends from Pinus koraiensis Siebold et Zuccarini forests to Quercus mongolica Fischer ex Ledebour forests. The cumulative species richness was as follows: Q. mongolica forest A > broadleaf mixed forest B > Q. mongolica, P. koraiensis mix forest D (Q. mongolica was the dominant species) > Q. mongolica and P. koraiensis mix forest C (P. koraiensis was the dominant species) > P. koraiensis forest (E). Ectomycorrhizal fungi were the dominant functional group; they were mainly in forest type A and were influenced by soil moisture content and Q. mongolica content (p < 0.05). The wood-rotting fungus showed richer species diversity than other forest types in broadleaf forests A and B. Overall, we concluded that most fungal communities preferred forest types with a relatively high Q. mongolica content. Therefore, the deliberate protection of Q. mongolica forests proves to be a better strategy for maintaining fungal diversity in Wunvfeng National Forest Park.In this paper, we analyze the macrofungi communities of five forest types in Wunvfeng National Forest Park  by collecting fruiting bodies from 2019–2021. Each forest type had three repeats and covered the main habitats of macrofungi. In addition, we evaluate selected environmental variables and macrofungi communities to relate species composition to potential environmental factors. We collected 1235 specimens belonging to 283 species, 116 genera, and 62 families. We found that  Fungal communities are essential for forest ecosystems and have many functions ,2. EctomMany biotic and abiotic factors can affect the diversity and composition of fungal communities ,7. The cQuercus mongolica Fischer ex Ledebour forests and mixed broadleaf forests, which changed when Pinus koraiensis Siebold et Zuccarini was planted in the 1980s [P. koraiensis forest, and P. koraiensis–Q. mongolica mixed forests provide a unique opportunity to investigate the fungal communities of different forest types under the same climatic conditions. Usually, Pinus species are dependent upon macrofungi in symbiotic associations, which are essential for their growth and survival [Pinus forests. Thus, understanding the distribution characteristics of fungal communities in planted and native forests can provide better strategies for fungal diversity conservation, especially for the deliberate conservation of forest types that host a more significant number of fungal species. In this context, we conducted a three-year survey of macrofungi in different vegetation types in Wunvfeng National Forest Park. We study the relationship between selected environmental factors and community composition. We aim to investigate whether fungal communities differ among the five forest types and whether native forests dominated by Q. mongolica are potentially associated with fungal communities.Our study sites are all within the Wunvfeng National Forest Park in the southeastern mountainous region of the Jilin Province, China. The reserve was established in 1992 and is part of the Changbai Mountain system. The original vegetation included he 1980s . Currentsurvival  because survival ,35. Some2 [Q. mongolica. P. koraiensis was planted as a non-native tree species in 1980. At present, it has formed stable forest communities. Dark-brown soil [Our study site, Wunvfeng National Forest Park, covers an area of 6867 hm2  and is s2 . The forown soil  is the mown soil .We designated five following forest types: A, B, C, D, and E. Three 50 m × 50 m sampling plots with at least 500 m distance between them were located within each forest type (pictures of each forest type are provided in Quercus mongolica site (A): Old-growth Q. mongolica forests with the original habitat, and no fallen or standing deadwood has been removed. There are 275 Q. mongolica with a coverage rate of 96.15%.Q. mongolica, Tilia amurensis Rupr., Fraxinus mandshurica Rupr., Acer palmatum Thunb., Juglans regia L., close to nature managed. There are 158 Q. mongolica with a coverage rate of 48.46%.Broad-leaved forest site (B): Mixed broadleaved forest (uneven-aged), with Pinus koraiensis and Quercus mongolica mixed sites (C): 30–50-year-old P. koraiensis forest, close to nature managed, and naturally grown Q. mongolica. P. koraiensis was artificially grown and is the dominant species. There are 42 Q. mongolica with a coverage rate of 18.03%. Quercus mongolica and Pinus koraiensis mixed sites (D): 30–60-year-old Q. mongolica forests, close to nature managed, and artificially grown P. koraiensis. Q. mongolica is the dominant species. There are 183 Q. mongolica with a coverage rate of 74.69%.Pinus. koraiensis sites (E): Single species; 40-year-old P. koraiensis forests planted in 1980, close to nature managed, no fallen trees removed. There are zero Q. mongolica with a coverage rate of 0%.We collected samples 20–25 times per month from June to October 2019–2021. We randomly acquired macrofungi from each plot. We photographed the specimens in the field using a Canon EOS 800D digital camera and recorded fresh morphological characteristics and ecological characteristics . We seleWe collected soil samples four times per month during July–September 2020. After cleaning and removing plant material and debris from the surface, we collected individual soil samples from the center and 4 corners in 15 plots using an auger . We mixed the soil samples from the same plots well and placed them in sealed bags. After removing impurities, we enclosed the fresh samples (20 g) from each clod in an aluminum box. We dried the samples to a constant weight in an oven at 105 °C to measure water content (SWC); we used natural air-dried composite samples (200 g) for each plot to analyze for pH, organic matter (SOM), available phosphorus (P), effective nitrogen (N), and available potassium (K) using the method described by Xing et al. . FinallyWe obtained the temperature and relative humidity of the air and soil temperature from July–September 2020 from meteorological monitoring sites in the forest park. The tested results for the soil are included in We identified the macrofungi using morphological observations methods. We used molecular methods for the species that were morphologically difficult to identify. We measured different microscopic structures of taxonomic importance  . We exam2O, five μL 5 × PCR buffer, and 1 µL dNTP in the PCR reactions that we ran under the following conditions: 95 °C for 3 min, followed by 35 cycles of 94 °C for 40 s, 55 °C for 45 s, 72 °C for 1.5 min, and a final extension step at 72 °C for 6 min before storage at 4 °C We purified the PCR products and sequenced them at Sangon Biotech Co., Ltd. . We performed molecular identification via BLAST comparisons. Species with >98% sequence similarity were also identified with morphological characteristics. GenBank accession numbers obtained are provided in Molecular identification involved sequencing the internal transcribed spacer (ITS). For this, we extracted the DNA of the macrofungi using a NuClean Plant Genomic DNA Kit , following the manufacturer’s instructions. We conducted final elutions in a total volume of 50 μL. We showed a polymerase chain reaction (PCR) with the primer pairs ITS-1F and ITS-4 ; finallyhttp://www.funguild.org (accessed on 18 November 2021) search; these can be found in http://www.indexfungorum.org (accessed on 15 November 2021)). Setting scientific names at all taxonomic ranks in italics facilitates quick recognition in scientific papers [We identified ecological functions  at the genera level using a FUNGuild  reflected the species richness of the community. The Shannon index (D) reflected the diversity of the community species. Pielou’s evenness index (E) reflected the distribution of the number of individuals in each species. The diversity index formulae were as follows:We analyzed the relationships between ectomycorrhizal fungi communities and selected variables using the canonical correlation analysis (CCA) from Canoco 5.0 . We firshttps://hiplot.com.cn/basic/venn (accessed on 20 October 2021)). The pie chart data were derived from the number of macrofungi types. The Venn diagram data included the species in each forest type. The accumulation curve data consisted of the cumulative number of species per collection.We used Origin 9.0 software to construct species stacked histograms at the genera level  to compaRussulaceae was the most diverse family with 36 different species, followed by Tricholomataceae (21 species), Boletaceae (19 species), and Amanitaceae (16 species). Together, these accounted for 32.51% of the total collected species. The most abundant genera were Amanita, Cortinarius, Lactarius, and Russula. The Agaricales were the most prevalent order in the five forest types (59.36%). In terms of the trophic groups, most of the species were ectomycorrhizal fungi (47%), followed by wood-decaying fungi (20.14%) and soil saprotrophs (18.37%).We collected 1235 specimens from 5 forest types, 940 (76.11%) of which we identified at the species level, and we classified these into 283 fungal species. We identified 244 species based on morphology and 39 species using morphology and molecular methods . The uniAgarics accounted for 69.26% of the identified species, followed by boletes, larger ascomytetes, and polyporoid fungi, accounting for 9.89%, 6.36%, and 6.01% of the identified species, respectively. In contrast, hydnaceous and cantharelloid fungi were less abundant, accounting for 1.06% and 0.71%, respectively  of macrofungi . The Rus species .Amanita, Lactarius, and Russula were the most diverse genera. In addition, 34 genera contained 2–4 species, accounting for 29.31% of the genera and 29.68% of the identified species; 67 of the genera contained only 1 species, accounting for 57.76% of the genera and 23.67% of the identified species  of macrofungi . The Ama species .Lactarius (13 species), Amanita (12 species), Coprinellus (10 species), and Russula (7 species) were the most species-rich genera in forest type A. The Russula (6 species) and Gymnopilus (4 species) were the most species-rich genera in forest type B. The Gymnopus (4 species) and Suillus (4 species) were the most species-rich genera in forest type C. The Russula (8 species), Amanita (7 species), and Mycena (4 species) were the most species-rich genera in forest type D. Finally, the Gymnopus (2 species), Helvella (2 species), and Hydnellum (2 species) were the relatively abundant genera in forest type E. More detailed information is shown in The community of macrofungi was different among the five forest types . The speQ. mongolica forest) and B (broad-leaved forest) showed relatively steep upward slopes and produced higher macrofungi abundance values than the other forests. Nevertheless, forest type A (Q. mongolica forest) obtained the highest macrofungi diversity values.The accumulation curves for the species identified in the five forests show a steady increase with more samplings . We reacClitocybe and Tricholoma) were shared in five forest types, but no species were shared. The unique species (found only in 1 forest) increased from E < C < D < B < A and consisted of 13, 22, 31, 50, and 104 fungal species, respectively  Singer only appeared in E and C. The S. luteus (L.) Roussel only appeared in D and C; Helvella crispa (Scop.) Fr. only appeared in B and E. The Amanita oberwinklerana Zhu L. Yang & Yoshim., Amanita orsonii Ash. Kumar & T.N. Lakh., Amanita virosa Bertill., Boletus edulis Bull., Phellinus pomaceus (Pers.) Maire, Russula paludosa Britzelm., and Tricholoma sejunctum (Sowerby) Quél., only appeared in A and D. The Agaricus moelleri Wasser, Gymnopus dryophilus (Bull.) Murrill, Pluteus leoninus (Schaeff.) P. Kumm., and Rhodocollybia butyracea (Bull.) Lennox only appeared in A and C.Two genera  than in forest type C (35 species), indicating that macrofungal species are associated with Q. mongolica. In P. koraiensis forest E, with the smallest species richness index of 2.286, we only found 18 species, accounting for 6.36% of the total species. This indicates that P. koraiensis forests can only provide habitats for a few fungal species.The species richness increased from E < C < D < B < A . Broad-lAmanita, Cortinarius, Lactarius, and Russula. The most common WS were Pleurotus and Polyporus. The highest occurrence of SS was Agaricus, Entoloma, and Hygrocybe. The LS, Clitocybe, Gymnopus, Mycena, and Pluteus, showed the highest occurrence.The main functional groups were ectomycorrhizal fungi (EM), wood-decaying fungi (WS), and soil saprotroph (SS). The EM , WS , SS , and LS  increased from coniferous forest (E) < mixed coniferous forest  < broadleaf forest . EM fungi were most abundant in forest type A . The higQ. mongolica content, effective soil nitrogen, soil available phosphorus, soil available potassium, soil organic matter, soil pH, soil temperature, air temperature, soil water content, effective soil nitrogen, and air relative humidity. The CCA results show that all samples were roughly separated into five groups according to their corresponding locations. Eigenvalue axis 1 (0.8963) is higher than axis 2 (0.7955), with a cumulative contribution of 28.8% and 25.57%, respectively. Of all the variables, the Q. mongolica content and soil moisture content were the most significant factors influencing the EM fungi. Many EM fungi  positively correlate with Q. mongolica and soil water  for the 130 ectomycorrhizal fungi (EM) species recorded in the 5 forest types. The variables included il water .Q. mongolica forests (A), mixed broad-leaved forests (B), artificial P. koraiensis forests (E), and mixed forests . This enabled us to analyze the composition of macrofungi according to the relative content change of Q. mongolica in different forest types. The results show differences in species richness and diversity among forest types with different relative contents of Q. mongolica. The species richness increases with the relative content of Q. mongolica. Forest types with a high cover of Q. mongolica may provide a stable environment for the growth of macrofungi [Quercus is the main host plant of EM fungi [Lactarius [Amanita [Russula [Cortinarius [Q. mongolica forest. Most EM fungi had a significant positive correlation with Q. mongolica content . However, they are not found in the P. koraiensis forest. These species may be associated with Q. mongolica   P. Karst., Hydnellum peckii Banker, and Tricholoma matsutake (S. Ito & S. Imai) Singer). Our results differ from those of Gao [P. koraiensis forests (aged < 150 years). On the one hand, EM fungi may need more time to form a stable symbiosis with the host plants [The species richness of nd Pinus . Howevere of Gao , who fout plants ; on the t plants .Q. mongolica and mixed broad-leaved forests and grows on larger diameter Q. mongolica fallen wood  Della Magg. & Trassin). The wood-dwelling macrofungi may be related to forest type, as they tend to favor specific forest types under similar climatic conditions. In general, these combinations are determined by fungi closely related to the dominant tree, mainly because their enzymes have adapted to wood with different chemical and physical properties [Q. mongolica and mixed broadleaved forests; we found no fallen trees in the P. koraiensis forest, which is another factor that might affect fungal assemblage. The amount of deadwood also affects the macrofungal assemblage, which previous authors highlighted as the most crucial microhabitat in the forest [In our study, wood-dwelling fungi (57 species) are also a critical taxon that is mainly distributed in the operties . Anotheroperties . Furthere forest ,63. The e forest ,65.Q. mongolica and mixed broad-leaved forests, affecting the grass rot fungal assemblage because litter saprotroph fungi strongly depend on deciduous leaves’ presence and volume [Saprophytic soil fungi (52 species) rely mainly on the decomposition of soil organic matter for nutrients, and they tend to prefer specific forest types under similar climatic conditions. Generally, the deciduous leaves of broad-leaved trees are more conducive to soil organic matter accumulation than coniferous forests . The ford volume .Amanita, Cortinarius, Lactarius, and Russula  are from July to September 2020 only. Nevertheless, our results illuminate the potential links between community composition and environmental factors because the July–September 2020 species data include almost all our species.This study with three years of species data is a small contribution that allows us to understand the distribution of fungal species in forest types with the different covers of Q. mongolica forests we analyzed are rich in macrofungal species. Although our data are based on only three years of sampling, we conclude that, as Q. mongolica increases in the forest, the abundance and diversity of macrofungal taxa also increase. We also observed that most EM species favored forest types with high Q. mongolica content , indicating that some EM fungal communities are closely associated with Q. mongolica. We call for further studies to support this claim. In addition, we have only found Tricholoma matsutake (S. Ito & S. Imai) Singer in P. koraiensis forests, which is classified as an endangered species and considered an ectomycorrhizal fungus that is closely associated with Pinus trees. Therefore, according to our research, maintaining P. koraiensis forests is beneficial for conserving endangered species. However, deliberate conservation of Q. mongolica forests would be more useful for maintaining the diversity of macrofungal communities. Whether P. koraiensis affects other fungal species will need to be monitored over 3–5 years.The


Crucihimalaya (Brassicaceae) is an alpine genus occurring in the Qinghai–Tibet Plateau characterized by cold temperatures and strong ultraviolet radiation. Here, we generated a chromosome-level genome for C. lasiocarpa with a total size of 255.8 Mb and a scaffold N50 size of 31.9 Mb. We first examined the karyotype origin of this species and found that the karyotype of five chromosomes resembled the ancestral karyotype of the Brassicaceae family, while the other three showed strong chromosomal structural variations. In combination with the rough genome sequence of another congener , we found that the significantly expanded gene families and positively selected genes involved in alpine adaptation have occurred since the origin of this genus. Our new findings provide valuable information for the chromosomal karyotype evolution of Brassicaceae and investigations of high-altitude environment adaptation of the genus.It remains largely unknown how plants adapt to high-altitude habitats.  Eutrema (Brassiaceae) species distributed in the high or the low altitudeAlpine plants occurring in high-altitude habitats with cold temperatures and strong ultraviolet (UV) radiation have evolved special adaptations to withstand these abiotic stresses.Crucihimalaya (belongs to Brassicaceae) contains 2–4 self-pollination diploid species with chromosome number of 2n = 16, mainly distributed in the high-altitude regions of the Qinghai–Tibet Plateau.,Sisymbrium or Arabidopsis because of their morphological similarity until the recent taxonomic revisions.Sisymbrium or Arabidopsis. The rough genome of one species from this genus, C. himalaica, was reported based on the Second-Generation Sequencer.Capsella and Arabidopsis, gene families related to disease resistance in this species were found to have significantly contracted while those to DNA repair and biquitin-mediated proteolysis expanded. In addition, genes related to reproductive processes were found to have experienced positive selection and/or functional loss in C. himalaica, although it remains unclear whether the identified genomic changes are common for all species of the genus occurring in the high altitudes or especially for C. himalaica. In addition, as the genome sequences of this species had not been assembled into chromosomes, the karyotype origin of the genus Crucihimalaya remains unresolved. Although the karyotype evolution in Brassicaceae is highly diverse, it can be traced to the recombination of two basic ancestral karyotypes.,The genus Crucihimalaya species, C. lasiocarpa, with a total size of 255.8 Mb and a scaffold N50 size of 31.9 Mb. Almost all protein-coding genes (99.13%) are anchored on eight chromosomes. In addition, we first examined the karyotype origin for the genus based on this chromosome-level genome sequence. Two other Crucihimalaya species without genomic resources are very similar to C. lasiocarpa and C. himalaica,Crucihimalaya species. The renewed high-quality genome sequence for this genus will be highly useful for exploring the relative contributions of various genomic mechanisms in driving the adaptive evolution to high-altitude environments.In this study, we report a chromosome-level reference genome for another C. lasiocarpa were collected from Tibet, China  and germinated in the greenhouse .Furthermore, the paired-end Illumina libraries with insert sizes of 400 bp were prepared using the Illumina Genomic DNA Sample Preparation Kit, which were then sequenced on an Illumina NovaSeq platform . The filtered clean data was used for ,For Hi-C sequencing, fresh leaves from one plant were first fixed by formaldehyde. The fixed chromatins were then digested with DpnII restriction endonuclease.k-mer frequency distribution analysis was applied to estimate genome size and heterozygosity (size 21 bp in Illumina DNA short reads). A total of 37.72 Gb raw Nanopore long reads were corrected, assembled using NextDenovo v2.0-beta.1 ) with parameter of seed_cutoff = 38k, reads_cutoff = 1k. The preliminary assembly of C. lasiocarpa was followed by three rounds of polishing using NextPolish,Before genome assembly, C. lasiocarpa genome were identified by a combination method of homology-based and de novo strategies using RepeatModeler v1.0.11T = K/2r using a substitution rate (r) of 7*10−9 substitutions per site per year, where K represented genetic distance.Repetitive elements in C. lasiocarpa genome, an integrated computational approach was adopted based on transcript mapping, homologous protein alignment and ab initio prediction. Illumina RNA-Seq raw reads were first processed using Trimmomatic v0.33Aethionema rabicum, A. lyrata, Arabidopsis thaliana, Boechera stricta, Brassica rapa, Capsella grandiflora, Capsella rubella, Carica papaya, Leavenworthia alabamica, and Tarenaya hassleriana  in ancestral crucifer karyotype (ACK).,A. thaliana gene IDs as start and end coordinates, homologous gene pairs were detected by BLASTP with a cut-off e value of 1e−5 of the predicted protein sequences against the A. thaliana proteome. Next, syntenic relationships were further determined using MCScanXA. thaliana, we determined the corresponding boundaries and intervals of each block in C. lasiocarpa and renamed the pseudochromosomes. The genomic blocks in Capsella rubella were obtained through the same method applied in C. lasiocarpa. In order to eliminate false chromosomal rearrangements caused by assembly errors, the raw Hi-C reads of C. lasiocarpa were used to map to the whole genomic sequence of Capsella rubella using 3D-DNA v180922. Finally, we examined the order, orientation and contiguity of genes across potential adjacent segments, where inter-chromosomal rearrangements were inferred. The most parsimonious scenario of the fusion and fission events during genome evolution of C. lasiocarpa from its ancestral chromosomes was determined. The breakpoint regions were identified by pairwise genome alignment using LAST and MCScanX. By inferring putative homologous genes and collinear genes between Capsella rubella and C. lasiocarpa, we drew a homologous gene dot plot using WGDI.To better understand the evolutionary scenario of the −5) with protein sequences from C. lasiocarpa, Crucihimalaya himalaica, Capsella rubella, A. thaliana, Eutrema heterophyllum, Eutrema yunnanense, Eutrema salsugineum and Aethionema arabicum  was obtained from the TimeTree database.Divergence time estimation was performed by MCMCTree in PAML v4.9 package,P-value and family-wide P-value with the Viterbi method for each orthogroup. Genes in significantly expanded families were then used for Gene Ontology enrichment analysis. The KOBAS softwareWe employed CAFE v4.2C. lasiocarpa and C. himalaica set as foreground branch. For the Branch-Site Model, the χ2 test was conducted for each orthologous to assess statistical significance of Model A and Model A null. Finally, Bayes empirical Bayes (BEB) analysisdN/dS ratio in each orthologs. A likelihood ratio test (LRT) with df = 1 was employed to discriminate between the null model and the alternative model.P-value <0.05 and a higher dN/dS value in the foreground than the background branches were regarded as positively selected genes in the foreground.To search for genes that evolved under positive selection (PSGs), single-copy gene families were extracted by OrthoFinder. Based on the non-synonymous to synonymous substitution ratio, the Branch-Site Model and Branch Model in CODEML from PAML package were used to detect selection with the ancestral branch leading to the C. himalaica and the genus Crucihimalaya is self-compatible,C. lasiocarpa to explore if same mutations could be in C. lasiocarpa. The published S-haplogroups sequences of Crucihimalaya himalaica, Capsella grandiflora, A. thaliana, Arabidopsis halleri, A. lyrata and B. rapa were retrieved from NCBI and used as query in BLASTp searches against the genome assembly of C. lasiocarpa. Via searches, we manually annotated the candidate homologous gene of S locus SCR gene and the flanking genes U-box gene. Intriguingly, there was a candidate protein hit, which matched ARK3 and SRK. Due to the confusing role of candidate ARK3 and SRK protein, the SRK and ARK3 protein of Arabidopsis were downloaded and aligned by MAFFT. Then, a maximum likelihood tree both including SRK and ARK3 was constructed using RaxML. All candidate proteins of S-locus were further confirmed by hmmsearch against the Pfam database.Known that loss of function at the S-locus is the reason for self-fertilization of C. lasiocarpa is estimated at ∼253.6 Mb, with a heterozygosity rate of 0.04% based on k-mer analysis , with a scaffold N50 of just 2.0 Mb (contig N50 = 136.3 Kb), the final genome assembly of C. lasiocarpa exhibited a total size of 255.81 Mb, including 20 scaffolds with a 31.9 Mb scaffold N50 size, and the largest scaffold size was 35.0 Mb. The gap rate of the assembled C. lasiocarpa sequences was estimated to be only 0.003%, while it was 1.63% for the C. himalaica genome assembly  rate of both assemblies as evidenced by BUSCO analysisC. lasiocarpa genome assembly, which was substantially higher than the LAI values obtained for the C. himalaica genome and other relatives under comparison  were anchored to eight chromosomes while only 0.86%  were left on the un-anchored scaffolds. The average gene length, coding sequence length and an average exon number were estimated to be 2,582 base pairs (bp), 236 bp and 5.53 exons, respectively  compared to that in C. himalaica (96%)  , suggestde novo and homology prediction approaches, a total of 134.5 Mb (52.58%) of the assembled C. lasiocarpa genome was identified as repeat regions with LTR elements being identified as the major class (27.01%)  among paralogous genes within the genome (Based on (27.01%) . The pro(27.01%) . Crucihie genome .C. lasiocarpa and other three closely related species. A total of 1,719,930,178 and 1,166 LTR-RTs were identified in C. lasiocarpa, C. himalacia, Capsella rubella and A. lyrata genomes, respectively. LTR retrotransposons in the genome of C. lasiocarpa had recently undergone a rapid proliferation ∼2.5 million years ago  compared with the genomic background  in the genomes of ears ago . As the ckground .n = 8) with 22 conserved genomic blocks  were suggestedCapsella rubella was suggested to be similar to the ACK of the family Brassicaceae,C. lasiocarpa and Capsella rubella using previously reported methods , proto-calepineae karyotype  and translocated PCK . The karyotype of C. lasiocarpa was inferred to evolve from the ACK. The C. lasiocarpa karyotype comprises six relatively conserved chromosomes  and two chromosomes with structural variations occurring   .C. lasiocarpa chromosomes  as the longest inversion across the whole genome . The Hi-omosomes .C. lasiocarpa and closely related species . The C. lasiocarpa–C. himalaica clade diverged from Capsella rubella ∼13.66 MYA.A total 5,993 single-copy gene families were used to construct a maximum-likelihood phylogenetic tree for  species . The phyCrucihimalayam, C. lasiocarpa and C. himalaica , which are enriched for ‘blue light signalling pathway’, ‘response to stimulus’ and ‘abscisic acid-activated signalling pathway’  gene at the S locus and the other pollen-expressed cysteine-rich (SCR/SP11) gene.C. lasiocarpa genome, we identified the SCR gene in our species. The SCR gene of C. himalaica had two mutations across the eight critical conserved cysteines.C. lasiocarpa, which is critical to maintain structural and functional integrity of the SCR proteinSRK gene in C. lasiocarpa. Both SRK and ARK3 belong to the plant receptor-like/Pelle kinase (RLKs) family.SRK and ARK3 sequences from Arabidopsis and other species to search for the corresponding homologous sequences. We constructed the maximum likelihood tree using all homologous sequences from two Crucihimalaya species and other species. We found that all identified homologous sequences from Crucihimalaya were clustered with the ARK3 genes of the other species and none was clustered with the SRK genes (Self-pollination and self-compatibility (SC) in Brassiaceae arise through functional loss or disappearance of the self-incompatibility (SI) genes, including the stigma-expressed receptor kinase  and scaffold N50 (31 Mb). Our scaffolding with Hi-C further facilitated the accurate assignment of all scaffolds to chromosomal positions. The ONT-based assembly also annotated more complete repetitive sequences than the Illumina-based assembly.C. lasiocarpa genome seem to be more accurate and complete than that of C. himalaica .C. lasiocarpa (n = 8) is relatively conserved because five chromosomes  remain stable while the other three show chromosomal structural variations, including two reciprocal translocation, two chromosome fusions and three inversions .Crucihimalaya, C. wallichii, was also found to have a similar pericentric inversion as in C. lasiocarpa for the first chromosome (C. lasiocarpa (Transberingia bursifolia (Brassiaceae).Crucihimalaya and Transberingia is unclear. Therefore, parsimonious karyotype evolution of this species  may involve both fusion and fission events during reciprocal translocation and inversions  for the genus are involved in DNA repair, coldness- and drought-response and reproductive processes.Crucihimalaya might have developed such special genomic characters to adapt to the high-altitude QTP before they diversified into these two species. It remains unclear whether these two species diverged with chromosomal structural variations and what type of genes played a key role during their divergence. In order to address these questions, the chromosomal-scale genome sequence of C. himalaica needs to be assembled and population genomic data of these two species are expected to obtain in the future. In summary, the chromosomal-scale genome sequence of C. lasiocarpa was reported here for the first time. We also clarified the karyotype evolution for the genus of Crucihimalaya. Combined with the previously reported genome sequence for C. himalaica, we found that many genomic changes related to alpine adaptation might have occurred before the divergence of the two species.Self-pollination and SC through functional loss of the SI genes remains a common reproductive assurance for alpine plants when pollinators become scarce.DNARES online.dsac004_Supplementary_DataClick here for additional data file.


Enterococcus faecalis and Enterococcus faecium have been used as probiotics or feed additives. Due to the lack of information in dogs, we aimed (1) to investigate the intra- and inter-host species adhesion of E. faecalis and E. faecium to the duodenal mucosa of six healthy beagles using bacteria derived from dogs and chickens, and (2) to validate a method of quantifying the adhesion of Alexa Fluor stain-labeled bacteria to paraffin-embedded canine duodenal mucosa. Our results show that both canine- and chicken-derived E. faecalis strains adhered better than E. faecium to the duodenal mucosa of beagles. In addition, canine E. faecalis and E. faecium adhere in higher numbers to canine duodenal mucosa, compared to chicken-derived strains of the same species. Our results suggest that both the bacterial strain and the host species may influence the mucosal adhesion properties of E. faecalis and E. faecium.Bacterial adhesion to the intestinal mucosa appears to be an important feature for probiotics. When selecting bacteria for probiotic use, those with high ability to attach to the intestines are preferred. Specific strains of Enterococcus faecalis and E. faecium strains are used as probiotics or feed additives. Adherence to the intestinal mucosa is considered a crucial step for intestinal bacteria to colonize and further interact with the host epithelium and the immune system. In dogs, there are no studies investigating the adhesion of E. faecalis and E. faecium to paraffin-embedded intestinal mucosa. Therefore, we aimed to investigate the adhesion of E. faecalis and E. faecium to the intestinal mucosa of six healthy beagles using bacteria derived from dogs and chickens. In addition, we aimed to validate a method to test the adhesion of Alexa Fluor-labeled bacteria to paraffin-embedded canine intestinal mucosa. The results of our study show that both canine- and chicken-derived E. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa of healthy beagles (p = 0.002). In addition, canine E. faecalis and E. faecium adhered in higher numbers to canine duodenal mucosa, compared to chicken-derived strains of the same species . The determination of the hydrophobicity of bacteria revealed that canine E. faecalis had the highest hydrophobicity level (36.6%), followed by chicken E. faecalis (20.4%), while canine E. faecium (5.7%) and chicken E. faecium (4.5%) had the lowest levels. Our results suggest that both the bacterial species and the host origin of the strain may influence mucosal adhesion.Some  Many commensal and pathogenic bacteria in the gastrointestinal tracts of humans and animals express various adhesin molecules , allowing them to bind to various surfaces in the host ,2,3. BacProbiotics can enhance mucosal health by several proposed mechanisms, including the production of short-chain fatty acids (SCFAs), antimicrobial substances, modulation of the immune response, competitive exclusion of pathogenic bacteria through interference with their adherence to the intestinal mucosa, and enhancement of epithelial barrier functions ,8. ThereFusobacteria, Bacteroidetes, and Firmicutes  × 100, and was expressed as follows: 0–35%, low hydrophobicity; 36–70%, medium hydrophobicity; and 71–100%, high hydrophobicity [The hydrophobicity of the investigated bacteria was determined using an in vitro method to detect the bacterial adhesion to hydrocarbons ,26. Canihobicity . All meaE. faecalis (OD 0.5 and 1) and E. faecium (OD 0.5 and 1) cells and between chicken E. faecalis (OD 0.5 and 1) and E. faecium (OD 0.5 and 1) cells that adhered to the canine duodenal mucosa were determined using the Mann–Whitney U test (intra-host species comparison). The same test was also used to compare the numbers of mucosal-adhered canine- and chicken-derived E. faecalis (OD 0.5 and 1) and E. faecium (OD 0.5 and 1) (inter-host species comparison). The data are presented as median (range). For all analyses, we considered values of p < 0.05 as significant. In addition, we applied the Holm–Bonferroni correction test to deflate type 1 error and adjust the p value for our multiple comparisons. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) software . The normality of data distribution was checked with the Shapiro–Wilk test. The data were found to be non–normally distributed; therefore, nonparametric tests were performed. The differences between the numbers of canine E. faecalis and E. faecium strains were labeled with AF488 and incubated on paraffin-embedded sections from the duodenum of healthy beagle dogs  vs. 62 (59–94) bacteria per field; p = 0.002) and OD600 = 1 (1066 (943–1191) vs. 141 (131–168) bacteria per field; p = 0.002).The adhesion of canine E. faecalis EF368/1 adhered significantly better to the duodenal mucosa of healthy beagles than E. faecium EF369/9 at both OD600 = 0.5 (351 (260–457) vs. 21 (15–23) bacteria per field; p = 0.002) and OD600 = 1 (642.5 (522–1074) vs. 49 (45–52) bacteria per field; p = 0.002) (Chicken = 0.002) B.E. faecalis and E. faecium to the duodenal mucosa of healthy beagles was higher when compared with that of E. faecalis and E. faecium strains of chicken origin  vs. 351 (260–457) bacteria per field; p = 0.015) and OD600 = 1 (1066 (943–1191) vs. 643 (522–1074) bacteria per field; p = 0.015). Similarly, the adhesion of canine E. faecium EF397/1 was significantly higher than that of chicken E. faecium EF369/9 at both OD600 = 0.5 (62 (59–94) vs. 21 (15–23) bacteria per field; p = 0.002) and OD600 = 1 (140.5 (131–168) vs. 49 (45–52) bacteria per field; p = 0.002) (The adhesion of canine n origin . As show= 0.002) B.E. faecalis O-67 was medium (36.6%) and low for chicken E. faecalis EF368/1 (20.4%), canine E. faecium EF397/1 (5.7%), and chicken E. faecium EF369/9 (4.5%) (The hydrophobicity level for canine 9 (4.5%) .E. faecium has been shown to confer benefits to dogs with acute, uncomplicated diarrhea, with a better clinical outcome compared to a placebo [E. faecium SF68® in preventing and treating diarrhea in cats housed in animal shelters. However, the strain did not have significant effects on kennel stress-associated diarrhea in dogs, which may partially be due to the low prevalence of diarrhea in this study [E. faecium strain NCIMB 10,415 4b1707, plus the prebiotics fructooligosaccharides and gum Arabic, did not significantly alter fecal microbiota richness or diversity in dogs with or without food-responsive enteropathy (FRE) [E. faecium (DSM 10663/NCIMB 10415) E1707 as a single-strain treatment had no effect on the clinical activity score, histology scores, or duodenal and colonic gene expressions associated with specific T-helper lymphocyte lines [E. faecalis, which reduced the number of diarrhea episodes and diarrhea severity in five out of six treated dogs with recurrent episodes of self-limiting diarrhea [A few studies have assessed the effects of enterococci on the intestinal health of dogs and cats. Orally administered  placebo . Bybee eis study . In anothy (FRE) . In addite lines . Promisidiarrhea . FurtherE. faecalis and E. faecium strains to the paraffin-embedded duodenal mucosa of healthy dogs, and whether this adhesion might be related to the bacteria’s hydrophobicity. In addition, we aimed to answer the question of whether the adhesion characteristics of these bacteria are related to host specificity.The ability to adhere to and colonize the intestinal mucosa of the host is important for an efficient probiotic bacterium . EspeciaE. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa of healthy beagles. Rinkinen et al. (2000) also reported a relatively low level of adhesion of two E. faecium strains  to immobilized canine intestinal mucus [E. faecium SF 68 and E. faecium M74 of human origin also showed low adherence to canine mucus compared to other lactic acid bacteria [E. faecalis compared to E. faecium. Hydrophobicity is a physicochemical feature related to the capacity of bacteria to adhere to biological surfaces, such as the intestinal mucosa [E. faecalis and E. faecium to the hydrophobic solvent xylene. In our study, canine E. faecalis O-67 showed the highest hydrophobicity percentage. In contrast, chicken E. faecalis EF368/1 and both canine E. faecium EF397/1 and chicken E. faecium EF369/9 strains presented low levels of hydrophobicity, determined as adhesion to xylene. Interestingly, those enterococci strains that had higher hydrophobicity also adhered in higher numbers to the duodenal mucosa in our study. Higher hydrophobicity could therefore be a possible reason why E. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa. Stępień-Pyśniak et al. (2019) also reported a higher level of hydrophobicity for E. faecalis strains than E. faecium strains isolated from wild birds [E. faecium strains isolated from Brazilian cheeses and Bulgarian feta cheese (7.92% to 11.33%) [In our study, both canine- and chicken-derived n et al. 00 also rbacteria . One posl mucosa . We therld birds . A low l 11.33%) . Cell su 11.33%) ,36,37.E. faecalis and E. faecium adhere in higher numbers to canine duodenal mucosa compared to chicken-derived bacteria of the same bacterial species, which suggests the host-specific adhesion of these bacteria. To our knowledge, the most likely reason for the higher binding tendency of canine-derived enterococci to canine duodenal mucosa is the better host adaptation to glycan structures in the epithelium. The glycan structure varies among species, and thus, it seems that enterococci obtained from dogs are better adapted to attaching to the structures on dog epithelium [Pediococcus acidilactici FT28F isolated from piglet feces adheres heavily to pig intestinal epithelial cells without showing any adhesion towards chicken intestinal epithelial cells. In another study, a piglet-derived Lactobacillus acidophilus strain adhered better to intestinal epithelial cells of piglets compared to chicken intestinal epithelial cells, while the opposite was true for a chicken-derived strain [Lactobacillus acidophilus LAB20 adheres better to canine colonic mucus as compared to a bacterial strain isolated from porcine colon. Interestingly, Lauková et al. (2004) compared the adhesion of E. faecalis and E. faecium strains isolated from the feces of various animals  to canine, human, and porcine intestinal mucus, and did not find statistically significant differences between binding ability to any of the tested mucus types [E. faecium SF 68 (human origin) was much higher to the mucus of humans and other species than to dog mucus [The results of our study showed that canine ithelium . The hosithelium ,10,39. Dd strain . Kainulaus types . Rinkineog mucus .E. faecalis and E. faecium strains. Tissue sections give a more physiological context to the adhesion study than cultured cells, as they provide cellular organization and structures that are almost impossible to obtain using in vitro cell culture [In bacterial adhesion tests, the use of various experimental conditions  may influence the results and make inter-assay comparisons difficult ,41. In a culture . Kainula culture ,41.Enterococcus strains. The European Food Safety Authority (EFSA) has developed pioneering guidance for the safety assessment of one of the most common probiotics used in animal feed, E. faecium [Enterococcus strains and assesses each product separately [E. faecium, such as Fortiflora®, Nutrabio®, Cernivet® and Cylactin®, or E. faecalis, such as Symbioflor® 1. Recently, the EFSA approved using Bonvital® (Enterococcus faecium DSM 7134) probiotic for fattening piglets in 2019 [E. faecium demonstrating a resistance to ampicillin greater than 2 mg/L or possessing any of the three virulence marker genes  should not be used as a feed additive [E. faecalis strains proposed for use in the food or pharmaceutical industries.Concerns exist over transferring antibiotic resistance genes and/or potential virulence among  faecium . EFSA taparately . Several in 2019  and chic in 2019 . Based oadditive . SimilarE. faecalis and E. faecium to the duodenal mucosal. Carnoy’s fixative can reportedly be superior to formalin in preserving the mucus layer [In our current study, we used archived formalin-fixed samples from healthy beagles, deparaffinized by xylene and ethanol, to investigate the adhesion properties of us layer , but we us layer . Other sus layer ,48.Despite the increasing number of probiotic products marketed for dogs, there are surprisingly few studies on one of the most important properties of probiotic bacteria, i.e., their ability to attach to the intestinal mucosa. Studies concerning metabolic mechanisms of bacterial crosstalk with canine intestinal mucosa are completely missing. All evidence concerning probiotic adhesion and its possible effects on the intestinal mucosa is based on analogies from other species. Our study is an attempt to overcome these shortcoming by applying, for the first time, a recently published new method  that allT-test), and the results were similarly significant, which indicates their robustness. In addition, we applied the Holm–Bonferroni correction test to deflate type 1 error, and adjusted the p-value for multiple comparisons. Even after adjustment, the p-value remained significant, which proves the accuracy of our results.The main limitation of our study is its small sample size. Basically, this research was more an exploratory than a confirmatory study, and the observed results could be due to chance. In the current study, result variability has been reduced by analyzing the mean of three runs, which is a good estimate of the attached bacteria in a sample. Due to the small sample size and non-normally distributed data, we used the non-parametric Mann–Whitney U test. However, we also analyzed the data with parametric testing (Independent Samples E. faecalis and E. faecium adhere in higher numbers to canine duodenal mucosa compared to chicken-derived bacteria of the same species. In addition, both canine- and chicken-derived E. faecalis strains adhered significantly better than E. faecium to the duodenal mucosa of healthy beagles. The adhesion properties seem to be associated with bacterial hydrophobicity. In this study, we used, for the first time, paraffin-embedded dog intestinal tissue sections to investigate the adhesion properties of Alexa Fluor-labeled E. faecalis and E. faecium. Our results suggest that both the bacterial strain and the host may influence the mucosal adhesion properties of E. faecalis and E. faecium. Host gut-derived bacteria might be preferable as probiotics, as they have adapted to the gastrointestinal environment of the host and can proliferate and express biological activity in a more competitive way compared to microbes from other host species.The results of this study showed that canine


Introduction: Approximately 10–20% of patients WITH myasthenia gravis (MG) are refractory to conventional immunotherapies. The purpose of this study was to conduct a systematic review and meta-analysis to explore the optimal therapies for refractory MG.Method: Correlative studies were performed through a search in PubMed, Cochrane Library, and Embase databases. The primary outcome was defined by changes in the quantitative myasthenia gravis score (QMG). Secondary outcomes were defined by the Myasthenia Gravis Activities of Daily Living Scale (MG-ADL), Myasthenia Gravis Foundation of America (MGFA) post intervention status, adverse events, and disease exacerbation after treatment.Result: A total of 16 studies were included with 403 patients with refractory MG on therapies with rituximab, eculizumab, tacrolimus, and cladribine. Therapeutic efficacy of rituximab and eculizumab was identified with an estimated reduction in QMG score (4.158 vs. 6.928) and MG-ADL (4.400 vs. 4.344), respectively. No significant changes were revealed in efficacy or exacerbation density between the two independent therapeutic cohorts. The estimated adverse event density of eculizumab was more significant than that in the rituximab group (1.195 vs. 0.134 per patient-year), while the estimated serious event density was similar.Conclusion: The efficacy and safety of rituximab and eculizumab have been approved in patients with refractory MG. Rituximab had a superior safety profile than eculizumab with a lower incidence of adverse events.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021236818, identifier CRD42021236818. Myasthenia gravis (MG) is an autoimmune disorder due to a transmission defect in the neuromuscular junction. These patients clinically manifest with fluctuating muscle weakness in an ocular, limb, and axial muscles. The majority of patients with MG have excellent responses to acetylcholinesterase inhibitors, rescue therapies (such as intravenous immunoglobulin and plasma exchange), immunosuppressants including glucocorticoid, azathioprine, mycophenolate mofetil, and thymectomy . HoweverThe meta-analysis protocol has been registered in PROSPERO  under ID: CRD42021236818.One author (FX) performed an article search in PubMed, Cochrane Library, and Embase. Medical subject headings (MeSH) and free-text words were used for PubMed and Cochrane: Myasthenia Gravis/therapy (MeSH) and refractory. In Embase, Emtree, and free-text words were used: MG (Emtree), therapy (Emtree), and refractory. The primary search was completed on December 16, 2020 and the final search was completed on March 15, 2021. Only English language papers were included.Randomized controlled trials and observational studies with at least 10 patients with MG with each intervention were included. Refractory MG was defined according to criteria of Mantegazza : 1. PatiNewcastle-Ottawa Scale (NOS) was applied for quality evaluation for observational studies . AccordiTwo authors (FX and WM) independently extracted data. The discussion aimed to resolve any disagreements until a consensus was reached, or by consulting a third author (ZW). The following data were extracted: author, year of publication, country, original inclusion criteria, the total number of patients included in the study, intervention, related quantitative outcomes, and adverse events. Serious adverse events include adverse events that are life-threatening or result in death, hospitalization, or persistent or significant disability or incapacity, are congenital anomalies or birth defects, or are essential medical events . WorseniThe primary outcome was the changes in the quantitative myasthenia gravis score (QMG) . SecondaQ-test and I2-statistics were used for heterogeneity analysis. Fixed effect model or random effect model will be used based on heterogeneity (random effect model will be used if I2 > 50%). Publication bias was explored with Egger's test.Statistical analysis was based on STATA (version 14 for Windows). If some of the included studies failed to provide the SD of the change of reported outcomes, we would calculate it based on the methods described by The Cochrane Collaboration in the Cochrane Handbook for Systematic Reviews of Interventions   and tacrolimus group . There were 339 cases with the antiacetylcholine receptor (AChR) antibody positive and 43 cases with muscle-specific tyrosine kinase (MuSK) antibody positive. All the cases with positive anti-MuSK antibodies were recruited in studies about rituximab. The remaining 21 patients were seronegative for MG-related antibodies.In the included studies, eight studies were on rituximab and five studies were on eculizumab, while two studies were about tacrolimus and cladribine, respectively . In thesp = 0.281).In the included studies, eight studies provided the primary outcome data in the QMG score . The stup < 0.001).Two of the included studies provided an MGC scale , 27, andp = 0.706).Six of the included studies had reported MG-ADL results . The stup = 0.570). In addition, after the removal of Musk antibody-positive patients, the estimated MM rate of rituximab became 61% (95% CI: 0.28–0.90).Five of the included studies had provided the percentage of patients with MGFA-PIS as minimal manifestations (MM) . A randop = 0.822). The estimated incidence density of crisis for rituximab was 0.059 per patient-year (95% CI: 0.049–0.072). The estimated incidence density of crisis for eculizumab was 0.026 per patient-year (95% CI: 0.011–0.062) (p = 0.390).The incidence density of MG exacerbation and crisis was estimated with a random effect model. The estimated incidence density of MG exacerbation for rituximab was 0.178 per patient-year (95% CI: 0.099–0.319). The estimated incidence density of MG exacerbation for eculizumab was 0.218 per patient-year (95% CI: 0.182–0.262) . No smal1–0.062) . No smalp = 0.009).All included studies except for Brauner et al. reported adverse therapy events on patients with refractory MG. The incident density of adverse events and serious adverse events were calculated. A random-effect model was used for quantitative synthesis. The estimated adverse event density of rituximab was 0.134 per patient-year (95% CI: 0.064–0.281), while the estimated adverse event density of eculizumab was 1.195 per patient-year (95% CI: 0.635–2.248) . TacroliCommon adverse events of eculizumab include headache , nausea , myalgia , nasopharyngitis , upper respiratory tract infection , and diarrhea . Common adverse events of rituximab include upper respiratory tract infection , infusion reaction , herpes zoster infection , and enteritis  .p = 0.058).The estimated serious adverse event density of rituximab was 0.082 per patient-year (95% CI: 0.035–0.190), while the estimated adverse event rate of eculizumab was 0.281 per patient-year (95% CI: 0.146–0.540) . No smal2 twice a month. Studies of low-dose rituximab included Choi et al. . However, no significant difference in adverse severe event density was found between them. The most common adverse events of eculizumab were headache (18%), nausea (14%), and myalgia (11%). The most common adverse events of rituximab were infusion reaction (2%), upper respiratory tract infection (1%), herpes zoster infection (1%), and enteritis (1%). Preventive non-steroid anti-inflammatory drugs may be considered for eculizumab treatment, and management of mild adverse events were important for adherence.2 two times a month. High-dose rituximab had a higher MM rate than low-dose rituximab , the Southern China International Cooperation Base for the Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003), Guangdong Provincial Engineering Center for Major Neurological Disease Treatment, Guangdong Provincial Translational Medicine Innovation Platform for Diagnosis and Treatment of Major Neurological Disease, and Guangdong Provincial Clinical Research Center for Neurological Diseases. The sponsor or funding organization had no role in the design or conduct of this research.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.