Acinetobacter baumannii, CR Pseudomonas aeruginosa, CR Enterobacterales, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. are recognized by the World Health Organization as global priority pathogens of critical or high priority [Antimicrobial resistance and multidrug-resistant organisms currently constitute a severe public health problem. Multidrug-resistant organisms are resistant to multiple antibiotic classes, resulting in limited therapeutic options and difficult-to-treat health-care-associated and community infections, with high morbidity and mortality rates. In particular, carbapenem-resistant (CR) priority .A. baumannii-calcoaceticus complex. The epidemics of A. baumannii are characterized by the spread of multidrug-resistant clonal lineages [A. baumannii isolates responsible for wound infections in Ghana. Chopjitt et al. [Acinetobacter pittii isolates from Taiwan and China.This Special Issue was dedicated to updates on the “Molecular Epidemiology of Antimicrobial Resistance”. Two manuscripts investigated new patterns in the epidemiology of infections caused by isolates belonging to the lineages . Monnheilineages  analyzedt et al.  investigEnterobacterales, have been the subject of four studies. The research paper by Bilal et al. [NDM-1bla bearing IncX3 plasmid in clinically isolated ST11 Klebsiella pneumoniae from Pakistan. A new resistance-mediating plasmid chimera was detected in OXA-48bla-positive Klebsiella pneumoniae strain at a German university hospital [IMP-4bla were identified both in Escherichia coli Australian clinical samples and in multiple sublineages of E. coli ST216 colonising silver gulls [E. coli resistant to the antibiotic nitrofurantoin, which is used as first-line treatment of urinary tract infections. Lin et al. [Enterococcus and ST59 S. aureus was mediated by insertion sequence IS1216V.The molecular determinants, genetic and genomic elements of antimicrobial resistance in l et al.  reportedhospital . HI2 plaer gulls . Edowik n et al.  showed tOne manuscript focussed on diagnostics of antimicrobial resistances. Vasilakopoulou et al.  reportedEnterobacteriaceae isolates collected in Taiwan. Siméon et al. [Enterobacter hormaechei. Karruli et al. [Therapeutics and management for the prevention and control of infections caused by multidrug-resistant organisms were investigated in three manuscripts. Yang et al.  analyzedn et al.  reportedi et al.  identifiIn conclusion, the genomic epidemiology of multidrug-resistant organisms, the analysis of molecular determinants of antimicrobial resistance, the identification of innovative diagnostic and therapeutic approaches are important to prevent and control antimicrobial resistance.


Solidago canadensis L.) remains elusive. In the present study, we explored the sources and the number of introduction events of this species from its native areas into China. Using the genotyping-by-sequencing approach, we identified 34,035 selectively neutral single-nucleotide polymorphism (SNP) markers to infer the evolutionary trajectories of 77 S. canadensis individuals. Both the principal component analysis and the ADMIXTURE analysis revealed two genetic groups that are sympatric to each other in China and suggested the absence of genetic admixtures. The phylogenetic analysis indicated three feasible introduction routes and multiple introduction events of Canada goldenrod into China. Specifically, the one from the USA directly into China, the other from the USA into China through Japan, and the third from the USA into China through Europe. Based on the site frequency spectrum of these identified SNPs, we inferred strong bottleneck events for both genetic groups, and that the multiple introductions did not rescue the decline of genetic diversity. To conclude, multiple introduction events, genetic bottlenecks, and potential human-mediated spread characterize the introduction history of Canada goldenrod in China. The present study harnesses the power of SNP data in deciphering the evolutionary trajectory of invasive plants and paves the way for future studies concerning the invasion mechanism of Canada goldenrod.Despite numerous studies reported in the context of ecology, the introduction history of the infamous invasive plant Canada goldenrod ( As a distinguishing feature of the Anthropocene, biological invasions pose severe threats to local communities and ecosystems ,2,3. ForHowever, there is a genetic paradox that falls out of the scope of direct interactions between invasive and native species. How do alien species persist and subsequently become invasive when they suffer from strong founder effects (bottlenecks) in the meantime ,11,12,13Solidago canadensis L.) is now a notorious invasive weed globally .,42.S. canadensis . Long-ranadensis  43]. It. ItS. cae nature . Recentlerstated ,46. Nevenadensis . Some donadensis ,48. SpecS. canadensis on introduction to different areas. Alexander et al. .,33.S. caogically  and rescequently . The typcrossing ,51,52. Geriments ,28,43. At et al. .Reynoutria japonica Houtt., the Japanese knotweed; Taraxacum officinale G. H. Weber ex Wiggers, the dandelion), the low genetic diversity together with the phenotypic plasticity of S. canadensis in China strongly implies that epigenetic variations could play critical roles in its invasion success [Sporobolus alterniflorus (Loisel.) P.M.Peterson and Saarela) were related to non-coding RNAs’ activities [S. canadensis [S. canadensis. Therefore, we strongly recommend in-depth explorations of epigenetic variations and their underlying molecular mechanisms in future works that aim at decoding the invasion mechanisms of Canada goldenrod in China.Similar to many poster-child invasive plants  to explore the invasion routes of this species in China, we collected one European  and two Japanese  samples as representative individuals . Later, the DNA libraries were pooled, size-selected (300–400 bp) on 1% agarose gels, column-cleaned using PCR purification kits, and amplified for 12 cycles using Phusion™ DNA polymerase . The average fragment size was estimated on a 2100 Bioanalyzer Instrument , and the library quantification was performed using the PicoGreen™ kit . Finally, the pooled libraries were sequenced with the NovaSeq™ 6000 platform to generate paired-end reads of 150 bp . All the sequencing data have been deposited at GenBank under the accession number PRJNA937432.The GBS procedure was conducted following the original protocol ,65. Firsde novo assembly of clean reads and the clustering of tags were performed using the Stacks (version 1.43) pipeline [We conducted the SNP calling with the following steps. First, the raw sequencing data were processed using fastp (version 0.18.0)  to removpipeline . Basicalpipeline . Next, wpipeline . Later, pipeline .S. canadensis individuals with PCA using PLINK (version 1.9) [K) with 5-fold cross-validations.Prior to the subsequent analyses, we tested whether these retained SNPs were selectively neutral using BayeScan (version 2.1) with the default settings . Based oion 1.9) . Meanwhiion 1.9) . Five boS. canadensis individuals with different geographical origins, we reconstructed the individual phylogeny based on the putatively neutral SNPs with both the NJ method and the ML method [To explore the evolutionary affinities among L method . The conL method ,76. The L method ,78. As iL method . Both thS. canadensis (see Results) as follows. First, we obtained the folded site frequency spectrum (SFS) using the SNPs identified above and easySFS “https://github.com/isaacovercast/easySFS” (accessed on 4 September 2022). Then, the effective population size (eN) of the two genetic groups over time was simulated using Stairway Plot 2 (version 2.1.1) [S. canadensis was assumed to be one year [−9 base substitutions per site per generation following Arabidopsis thaliana [eN dynamics were inferred with 100 estimates.We inferred the demographic history of the two distinct genetic groups of n 2.1.1) . The genone year . The mutthaliana . For eacS. canadensis.By exploring genomic SNPs of both the native and invasive Canada goldenrod individuals, our results indicate that multiple introduction events, genetic bottlenecks, potential human-mediated dispersal events, and clonal reproduction all play roles in the invasion history of Canada goldenrod into China. The present study highlights the power of genome-wide SNPs in inferring the evolutionary history of invasive plants and paves the way for future studies regarding the invasion mechanism of


Picromerus lewisi, an important predatory stink bug and an effective biological agent, were comprehensively identified by transcriptome analysis, with a total of 59 neuropeptide precursor genes and 58 potential neuropeptide receptor genes found. In addition, several neuropeptides and their receptors enriched in salivary glands of P. lewisi were identified. The present study and subsequent functional research contribute to an in-depth understanding of the biology and behavior of the predatory bugs and can provide basic information for the development of better pest management strategies, possibly including neuropeptide receptors as insecticide targets and salivary gland derived venom toxins as novel killing moleculars.Predatory stink bugs derive from phytophagous stink bugs and evolved enhanced predation skills. Neuropeptides are a diverse class of ancient signaling molecules that regulate physiological processes and behavior in animals, including stink bugs. Neuropeptide evolution might be important for the development of predation because neuropeptides can be converted to venoms that impact prey. However, information on neuropeptide signaling genes in predatory stink bugs is lacking. In the present study, neuropeptide signaling genes of  Neuropeptides are a large diverse class of signaling molecules with key roles in insect physiology and behavior . NeuropeLygus hesperus (Miridae) (Nezara viridula (Pentatomidae) and Halyomorpha halys (Pentatomidae)  are an ideal group to explore the evolution of trophic strategies . HeteropMiridae) , Nezara tomidae) , and foudipennis . Comprehnfestans , and Cimtularius , and twoviridula  and Apol lucorum . To datePicromerus lewisi Scott (Hemiptera: Pentatomidae) is widely distributed in China and other Asian regions  Insect neuropeptides might be repurposed into novel insecticides; 2) finding a neuropeptide pathway that is different or absent in beneficial predators, could lead to insecticides that do not hurt beneficial predators; 3) natural enemy evolution might repurpose neuropeptides as venom toxins, causing them to be expressed in salivary glands. To begin testing these hypotheses, we identified neuropeptides and their receptors in P. lewisi were jointly performed , Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot , KEGG (Kyoto Encyclopedia of Genes and Genomes), and GO (Gene Ontology) databases.In our previous study, full-length transcriptome and RNA-seq transcriptome analysis of erformed . Howeverus study . Brieflyus study . All libus study . All unip values (P_adj) to control the false discovery rate. Genes with P_adj values <0.05 and | log2 (fold change) | > 1 were designated as differentially expressed genes (DEGs). Heat maps showing gene expression profiles were constructed based on Log10 (FPKM +1) values.Differential expression analysis of transcripts from SG, G and H with three biological replicates each was performed using the DESeq2 R package (1.20.0). The Fragments per kilobase of transcript per million fragments mapped (FPKM) value was calculated to estimate the expression level of each transcript from each library. The Benjamini-Hochberg approach was used to adjust To validate the transcriptome data, qRT-PCR analysis of eighteen genes had been performed . BrieflyP. lewisi transcriptome. Amino acid sequences of known neuropeptide precursors from H. halys (L. hesperus (Nilaparvata lugens (R. prolixus (N. lugens (N. viridula (A. lucorum (R. prolixus (Drosophila melanogaster and Aedes aegypti were obtained from Carausius neuropeptide-like precursor (CNP), parathyroid hormone (PTH), PaOGS36577, and RFLamide (RFLa), were identified based on their homologous genes from Periplaneta americana  database (SRR10134979) via the NCBI tBLASTn program.Local tBLASTn searches were performed to predict the genes of neuropeptide precursors and their receptors from the reassembled H. halys , L. hesphesperus , Nilapara lugens , and R. prolixus  were use. lugens , N. viriviridula , A. luco lucorum , and R. prolixus  were colmericana  or Triboastaneum . In addiastaneum , CNMamidastaneum  and elevastaneum , were alP. lewisi. Briefly, the presence of signal peptides was predicted using the online program SignalP 5.0 . Prohormone cleavage sites and post-translational modifications were identified based on the information presented in Veenstra . For the alignments of neuropeptides, the putative active peptides or the amino acid sequences removing the putative signal peptides were adopted. For the alignments of neuropeptide receptors, full-length sequences were used when available, or partial sequences were used. Amino acid identity was subsequently determined and the alignment result was visualized using GeneDoc version 2 (http://weblogo.berkeley.edu/logo.cgi)  .https://itol.embl.de/) (Maximum likelihood (ML) phylogenetic analysis was conducted using IQ-TREE . In a fimbl.de/) .P. lewisi used for cloning were supplied by the Fenggang County Natural Enemy Breeding Center of the Guizhou Tobacco Company Zunyi Branch, Zunyi, Guizhou Province, China. Total RNA was isolated using TRIzol reagent . RNA quality and quantity were determined using the RNA Nano 6000 Assay Kit in the Bioanalyzer 2,100 system . Total RNA was treated with DNase I  to remove any residual genomic DNA. cDNA were synthesized from 1 μg of the total RNA using a SMARTer PCR cDNA Synthesis Kit . Neuropeptide precursor transcripts were amplified in a 50-uL reaction system using 2 × Phanta Flash Master Mix . The PCR procedure was set as follows: pre-denaturation at 98°C for 30 s, 35 cycles of 98°C for 10 s, 55°C for 5 s and 72°C for 5 s, and termination at 72°C for 1 min. The PCR products were cut from the gel, purified and sequenced by Beijing Tsingke Biotech Co., Ltd.Two species-specific neuropeptide transcripts, Crustacean cardioactive peptide (CCAP) and Orcokinin B (OKB), were amplified by PCR and sequenced to verify their reliability. Primers were designed to amplify the coding sequences . Five fide novo assembly procedure, resulting in a total of 62,183 unigenes with a mean length of 1,203 bp, and an N50 length of 2,040 bp  with an H. halys OKB peptide, whereas other P. lewisi OKB peptides had 45%∼90% identity with their H. halys orthologues .The sequence . The H. P. lewisi (SFCNSFTGC) has two variant residues and lacks a C-terminal glycine residue .CCAP is known as the most conserved arthropod neuropeptide, with an identical amino acid sequence, PFCNAFTGCamide, found in all insects examined. Unexpectedly, the mature CCAP peptide predicted from  residue . The samP. lewisi PTTH transcript has features typical of other insect PTTHs, encoding a putative active peptide containing seven cystine residues that form inter- and intra-chain disulfide bonds. PTTH has also been found in another heteropteran species, L. hesperus, but it is absent in a number of heteropteran species such as H. halys, N. viridula, and R. prolixus  in heads, salivary glands, and guts of P. lewisi  were significantly more highly expressed in salivary glands, with log2 (fold) >1 and P_adj <0.05 . OKB had. lewisi .P. lewisi can be classified into 27 groups based on their putative ligands: Receptors for AKH, AKH/corazonin-related peptide (ACP), AST-C, allatotropin (AT), CCAP, CNMa, corazonin (Crz), Ele, myosuppressin (MS), NTL, proctolin (Pro), SIFamide (SIFa), Short neuropeptide F (sNPF), SK, capability/cardioacceleratory peptide 2b (CAPA), ETH, pyrokinin (PK), AST-A, FMRFamide (FMRFa), tachykinins (TKs), AST-B, leucokinin (LK), bursicon (Bur), GPA2/GPB5, neuropeptide F (NPF), insulin-like peptide (ILP), and orphan  and PTH  presented the FPKM value higher than 10 in one repeat  in heads, guts and salivary glands is shown in e repeat . Comparee repeat . Receptoe repeat .P. lewisi, with a total of 59 neuropeptide precursor genes and 58 potential neuropeptide receptor genes found. The number of neuropeptide precursors and their receptors identified in P. lewisi is similar to that found in H. halys and higher than those in other heteropterans such as N. viridula, L. hesperus, and R. prolixus  and five receptor genes were significantly upregulated in the guts when compared to heads, suggesting that they may have functions in feeding regulation.Tissue-specific expression profiles of neuropeptides and their receptors in xcretion . DH31, Cxcretion . In P. lhttps://motif.mvls.gla.ac.uk/FlyAtlas2/), ion transport peptide (ITP), ILP6, NPLP2, NPLP4, and receptors for Crz, GPA2/GPB5, ILPs and NPLP1 were expressed in Drosophila salivary glands  and eight receptors were enriched in salivary glands. In all these cases, the physiological functions of only a few neuropeptides and their receptors in insect salivary glands have been determined. More research is needed on this topic to describe specific functions.Multiple neuropeptides and their receptors were identified as genes expressed in insect salivary glands. In the flyAtlas database  and the caterpillar Acharia stimulea (RF-amide peptides)  . NeverthP. lewisi. To our knowledge, CCAP is the identical neuropeptide (PFCNAFTGC-NH2) in all examined insect species. CCAP from P. lewisi (SFCNSFTGC) identified in the present study has two variant amino acid residues and no amidation at the C-terminus. Thus our findings indicate a highly novel primary structure for this peptide which we will seek to confirm through further studies utilizing liquid chromatography tandem mass spectrometry analysis. CCAP is mainly expressed in the central nervous system and/or in the intestine in most of the determined insects, such as D. melanogaster and R. prolixus  and highly expressed in heads, indicating that salivary gland-derived CCAP of P. lewisi could be considered as an endogenous endocrine factor. It is worth mentioning that the presence of the atypical CCAP in P. lewisi venom as a toxin cannot be ruled out given an intriguing example of a CCAP-related peptide discovered in the venom of Conus villepinii and having the activity of decreasing the heart frequency in Drosophila larvae (The most unexpected result in the present study was finding a very atypical CCAP in prolixus . In R. py glands . However feeding , but itsa larvae .Chrysoperla carnea, based on the transcriptome analysis information that insect kinins were not found in C. carnea (P. lewisi and other heteropterans, P. lewisi harbors almost all kinds of neuropeptide signaling system identified in other heteropteran species. Because of the conservation of this signaling system, it seems difficult to develop green pest control strategies based on neuropeptide systems putatively lost in benefical predatory bugs. Although sequence alignment of mature peptides between P. lewisi and its close herbivorous bug species showed several neuropeptides like OKB, CCAP, and PTTH, which are highly diverse in bugs, these neuropeptide signaling classes could be potential targets for the development of highly selective insecticidal agents needs to be further determined.The insect neuropeptidergic system has been considered as an ideal target for the development of greener pest control strategies. Comparative genomics and transcriptomics provide useful information for appropriate design strategies to develop target-specific insecticidal molecules that could successfully control pests while protecting beneficial species. To date, insecticidal activity and biosafety have been demonstrated for a few insect neuropeptides and their analogues, such as kinins, proctolin, CAPA, and TKs . For exa. carnea . Based oP. lewisi, providing basic information for in-depth study on repurposing neuropeptides and their receptors into insecticides and targets.In the present study, a total of 59 neuropeptide precursors and 58 potential neuropeptide receptor genes were identified through transcriptomic analysis. The present study also revealed a set of neuropeptides and their receptors that were enriched in the salivary glands of


Escherichia coli and characterized, and one of these showed remarkable activity, being very promising for the synthesis of value-added products, such as lactose hydrolysates or galactooligosaccharides, from dairy industry effluents. Kamli et al. [Aneurinibacillus. Antimicrobial and antiviral activities, carbohydrate-active enzymes and genes involved in heavy metal resistance were identified. The potential of this bacterial genus for several industrial applications was revealed. Guo et al. [Pseudomonas, and was named P. marinensis. Two genes encoding for cold-active lipases were identified in its genome, although one lipase showed much higher activity and stability than the other. This difference was explained in the function of the aminoacidic composition. Through mutagenesis experiments, the Ser/Asp/His catalytic triads of both lipases were proved essential for enzyme activity. These enzymes are promising for several industries due to the reduced energy consumption required for the catalysis. Silva-Salinas et al. [Bacillus licheniformis strain LB04, isolated from hot springs in Mexico. The enzyme was immobilized by entrapment on agar-agarose beads showing a higher hydrolytic activity than the free enzyme. It was stable at pH 3.0 and up to 80 °C, and was found to be efficient for starch hydrolysis at temperatures above 65 °C and in acidic conditions, making this α-amylase interesting for the refined syrup and bakery industries. Almahasheer et al. [Bacillus cereus group) from poultry farm waste in Saudi Arabia. Keratinase activity was enhanced through random mutagenesis. Mutations did not affect the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad. Due to their thermostability, activity at alkaline pH and binding affinity, the keratinases are potentially useful for industrial feather processing. Wang et al. [Acetivibrio thermocellus and Thermoclostridium stercorarium using several kinds of hemicellulose and cellulose as substrates. A. thermocellus mainly performed cellulose hydrolysis, whereas T. stercorarium mainly performed hemicellulose hydrolysis. The two cellulase systems proved to have a synergistic effect. Ethanol yields of the co-culture of A. thermocellus and T. stercorarium nearly doubled those of the monocultures, which indicates a novel method of ethanol production from cellulosic biomass through consolidated bioprocessing. González-González et al. [Thermus thermophilus HB27 cloned and expressed in Kluyveromyces lactis) as a biocatalyst in aqueous and organic media. The recombinant enzyme showed high thermal stability and activity in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S provided high yields for the acetylation of alcohols, and catalyzed the stereoselective hydrolysis of -ibuprofen methyl ester (87% ee). It is potentially applicable for industrial bioconversions.Eberhardt et al.  achievedi et al.  performeo et al.  isolateds et al.  studied z et al.  tested KThese published articles represent new and exciting contributions to upgrade enzyme biotechnology-based industries.


However, whether similar evolutionary processes exist in invertebrates remains poorly understood. Derived from the second-largest animal phylum, scallops are a special group of bivalve molluscs and acquire the evolutionary novelty of the swimming lifestyle, providing excellent models for investigating the coordinated genome and lifestyle evolution. Here, we show for the first time that genome sizes of scallops exhibit a generally negative correlation with locomotion activity. To elucidate the co-evolution of genome size and swimming lifestyle, we focus on the Asian moon scallop (Amusium pleuronectes) that possesses the smallest known scallop genome while being among scallops with the highest swimming activity. Whole-genome sequencing of A. pleuronectes reveals highly conserved chromosomal macrosynteny and microsynteny, suggestive of a highly contracted but not degenerated genome. Genome reduction of A. pleuronectes is facilitated by significant inactivation of transposable elements, leading to reduced gene length, elevated expression of genes involved in energy-producing pathways, and decreased copy numbers and expression levels of biomineralization-related genes. Similar evolutionary changes of relevant pathways are also observed for bird genome reduction with flight evolution. The striking mimicry of genome miniaturization underlying the evolution of bird flight and scallop swimming unveils the potentially common, pivotal role of genome size fluctuation in the evolution of novel lifestyles in the animal kingdom.Genome miniaturization drives key evolutionary innovations of adaptive traits in vertebrates, such as the flight evolution of  Eukaryotic genomes vary greatly in size by more than five orders of magnitude Neomenia permagna in solenogastres to 7.68 Gb for Diplommatina kiiensis in gastropoda. Swimming is one of the special lifestyles among molluscs with calcified shells. Different from most bivalves with sessile and buried lifestyles, jet propulsion is produced by rapid and successive contraction of adductor muscle Amusium pleuronectes; Linnaeus, 1758), a tropical Indo-West Pacific bivalve, is naturally distributed along the coast from Myanmar to Dampier Archipelago, north to southern Japan, and east to Papua New Guinea Amusium genus the most active scallops (belonging to the ecomorph E) With more than 100,000 extant species, mollusca is the second-largest animal phylum, which globally distributes among diverse aquatic and terrestrial environments A. pleuronectes and comparative genomic analyses reveal genome miniaturization and relevant gene pathways that contribute to the evolution of the swimming lifestyle, which strikingly mimics genomic changes underlying bird flight evolution and provides insights into the pivotal role of genome size fluctuation in the evolution of novel lifestyles in the animal kingdom.Here, we show for the first time that the genome sizes of scallops exhibit a generally negative correlation with locomotion activity. Genome sequencing of http://www.genomesize.com) and lifestyle classification (r = −0.78) between genome size and swimming distance during one free swimming burst  and the Queen scallop (Aequipecten opercularis), reside at higher latitude oceans (A. pleuronectes) for genome sequencing and comparative analysis, due to its exhibition of the smallest genome with strikingly high swimming activity.The energy metabolism level and shell morphology are two important influencing factors on scallop locomotion activity e oceans . The sheA. pleuronectes, resulting in a total of 275.3 Gb of genomic data   were supported by at least one known protein sequence . We alsogo (MYA) B.A. pleuronectes was further evaluated by gene synteny analysis. For macrosynteny analysis, we found that A. pleuronectes showed equivalent macrosynteny conservation levels with similar karyotypes and conservation index values in comparison with other scallops, including the P. yessoensis genome that was recognized with ancestral bilaterian karyotype Hox and ParaHox gene clusters, which play crucial roles in the anterior-posterior body plan patterning during animal development ZMPSTE24-Taxilin-HECA-SYAP1-PSMG1) conserved in protostomes A. pleuronectes genome , the most dominant gene families with large gene numbers were contracted in A. pleuronectes genome  and tandem repeats] among scallop species, and found that TEs but not tandem repeats showed a significant reduction in s genome . Compare 150 MYA B. We fous genome , includis genome . Howeverronectes . The TE A. pleuronectes when compared with the other five scallop species  and basal metabolic pathways , which is one of the fastest flying birds and possesses the highest metabolic rates normalized by body mass among vertebrates C. anna genome Chelydra serpentina, Xenopus laevis, and Mus musculus) and found that these genes were also significantly enriched in relevant pathways, including basal metabolic pathways and oxidative phosphorylation pathway , the recessing scallop P. yessoensis (ecomorph D), and the byssal-attaching scallop Chlamys farreri (ecomorph B). We compared the gene expression profiles in the striated muscles of three scallop species for the three gene pathways that were most enriched with size-reducing genes in A. pleuronectes , P. yessoensis (ecomorph D), and C. farreri (ecomorph B). We found that most of the biomineralization-related genes have fewer copy numbers in A. pleuronectes than those in the other scallops  were gathered from the Tung Ping Chau Bay  for whole-genome sequencing. The DNA samples were stored at the key laboratory of marine genetics and breeding, Ministry of Education, Ocean University of China (specimen code: OUC-MGB-2019-Apl-08). Dissected tissues were immediately frozen in liquid nitrogen and preserved at −80 °C for DNA extraction. The conventional hexadecyltrimethylammonium bromide (CTAB) procedure was used to extract high molecular-weight genomic DNA (gDNA) from the adductor muscle The Asian moon scallops , the sticky ends of the treated fragments were repaired with biotinylated residues to form blunt-end fragments. End repair, adaptor ligation, and polymerase chain reaction (PCR) amplification were performed before sequencing. Finally, the library was sequenced with the Illumina Xten 2500 (PE150) platform.The Hi-C library was constructed using adductor muscle tissues obtained from the same individual of A. pleuronectes was estimated using k-mer frequency distribution analysis based on Illumina sequencing reads. The Trimmomatic software k-mer number/k-mer depth.The genome size of de novo assemble the genome using the PacBio long reads. Based on the fuzzy-Bruijn graph (FBG) algorithm, the raw reads were assembled by the wtdbg2 module, and the consensus calling of the preceding assembly was conducted with wtdbg-cns, which was also used for reducing sequencing errors. The Illumina clean reads were mapped to the assembly using Burrows-Wheeler aligner (BWA) software The wtdbg2 software A. pleuronectes. The clean reads were aligned to the assembly by using BWA, and uniquely aligned reads with high mapping quality were retained for further analysis. Invalid interaction pairs were filtered by HiC-Pro software For chromosome-level scaffolding, the Hi-C sequencing technique was applied for chromosome construction for de novo predictions and homology-based methods. De novo repetitive sequence database was constructed by RepeatModeler v1.0.11 A. pleuronectes genome, the tRNAscan-SE software Tandem repeats were identified by Tandem Repeats Finder v4.09 de novo prediction, and RNA sequencing (RNA-seq) supporting alignment. The de novo prediction was conducted using Augustus P. yessoensis, C. farreri, A. purpuratus, Crassostrea gigas, Pinctada fucata, and Modiolus philippinarum) to the A. pleuronectes genome assembly using TBLASTN. The accuracy of spliced alignments was then assessed by aligning homologous genome sequences to the matched proteins using GeneWise The gene structure was predicted by integrating three methods, including homology-based search, The mantle, striated/smooth muscle, kidney, gill, gonad, and foot tissues/organs from three adult individuals were chosen for RNA-seq. Tissues/organs were dissected and immediately frozen in liquid nitrogen, followed by preservation at −80 °C until RNA extraction. Total RNA was isolated from each tissue/organ according to previously described protocols A. pleuronectes, P. yessoensis, C. farreri, and P. maximus), the conservation of gene macrosynteny compared to the presumed bilaterian ancestral linkage groups (ALGs) was assessed according to the method described by Wang and colleagues A. purpuratus and Argopecten irradians), we employed a heuristic hierarchical method to cluster the scaffolds from the draft genome and set the tree-cutting threshold as 0.25.For species with chromosome-level assemblies .For detecting conserved gene clusters among the six scallop species, we conducted A. pleuronectes and other nine scallop species , as well as one outgroup species (Delectopecten randolphi). The phylogenetic tree was built using BEAST2 v2.6 The phylogenetic tree of scallops with different lifestyles was constructed based on the available rRNA genes , including A. pleuronectes, P. yessoensis, C. farreri, A. purpuratus, A. irradians, and P. maximus. The sequences of protein-coding genes of the six scallop species were retrieved from the MolluscDB database. Orthologous groups from the six genomes were identified using OrthoFinder v2.3.12 E value of 1E−5 for BLASTP. Then, 4159 single-copy orthologs were used for multiple alignment analysis by using MAFFT software Lottia and Crassostrea; > 330 MYA between Patinopecten and Crassostrea) retrieved from the TimeTree database Six scallop species were employed to construct the genome-based phylogeny, including K of TEs was calculated according to the Jukes-Cantor formula K = 3/4Ln(1 − 4d/3), in which K is the genetic distance and d represents the number of observed substitutions normalized by the sequence length. We employed the formula T = K/2rr represents the nucleotide substitution rate for each scallop.To investigate the dynamics of TE activities during the evolution of scallops, we employed the free-ratio model of PAML software to calculate the nucleotide substitution rates A. pleuronectes was conducted by comparing the average domain counts in the other five scallop species using the Fisher test in R. And the gene families with P < 0.05 were identified as significantly contracted or expanded ones.To categorize the gene families, six scallop species were scanned using the HMM approach on known Pfam functional domains. A protein containing multiple copies of a domain was only counted once. The gene family expansion/contraction analysis for E value of 1E−5 for BLASTP. To identify size-reducing genes in the scallop A. pleuronectes, the length of paired single-copy orthologous genes in six scallop species were standardized using gene length divided by the genic region of the genome. Genes with the relative length value in A. pleuronectes smaller than the average value of the other five less-active scallop species were defined as size-reducing genes. Similarly, single-copy orthologous genes from the four tetrapods were identified using OrthoFinder with an E value of 1E−5. To identify size-reducing genes in the bird C. anna, the length of paired single-copy orthologous genes in four tetrapods were standardized using gene length divided by the genic region of the genome. Genes with a relative length value in C. anna smaller than the average value of the other three non-flying tetrapods were defined as size-reducing genes. Genome assemblies of four tetrapods were downloaded from the NCBI genome database, including C. anna , C. serpentina (NCBI genome: ASM1885937v1), X. laevis (NCBI genome: Xenopus_laevis_v10.1), and M. musculus (NCBI genome: GRCm39).Single-copy orthologous genes among six scallop species were identified using OrthoFinder v2.3.12 A. pleuronectes and C. anna were employed for KEGG enrichment analysis by EnrichPipeline P < 0.05) was shown by bubble plot. We compared the expression levels (TPM) of size-reducing genes enriched in the oxidative phosphorylation pathway in striated muscles between A. pleuronectes and the other two scallop species (P. yessoensis and C. farreri). The complex colors of the oxidative phosphorylation pathway plots represent the up-regulation degree for genes in A. pleuronectes according to the ratio of the TPM of a gene in A. pleuronectes divided by the average TPM of the gene in the other two less-active scallop species. In addition, we downloaded the adductor muscle transcriptome data of A. purpuratus from NCBI (SRA: SRR6849474), and compared it with mixed transcriptome data of striated and smooth muscles of A. pleuronectes, P. yessoensis, and C. farreri. Transcriptome analyses of basal transcription factors and aminoacyl-tRNA biosynthesis pathways were performed among four scallop species . Transcriptome data of C. farreri and P. yessoensis were retrieved from studies of Wang et al. A. purpuratus were obtained from NCBI .Size-reducing genes in A. pleuronectes, P. yessoensis, and C. farreri) was compared by ggboxplot package in R, and a comparison of expression levels of biomineralization-related genes in mantle was shown in the bar plot. To construct the co-expression gene network, the R package of WGCNA A. pleuronectes, P. yessoensis, and C. farreri. The modules that enriched genes specifically highly expressed in the mantle tissue was considered a biomineralization-related module. The connection strength for genes in each module was represented by the intramodular connectivity value (Kwithin), which is an important factor for determining hub genes. Then, we used Cytoscape software to visualize the top 40 hub genes in the biomineralization-related modules of the three scallop species. OrthoFinder was used for detecting single-copy orthologous genes in the three scallop species, and relative gene lengths were employed to determine length-increasing genes in A. pleuronectes in the biomineralization-related module by comparing to the other two scallop species. And the results were shown by a heatmap plot in R.The identification of putative biomineralization-related genes was conducted by aligning to well-known Pfam protein domains according to the bivalve biomineralization toolbox G. gallus. RNA-seq data of G. gallus were downloaded from NCBI .For the analysis of bone formation-related genes, we collected marker genes of immature cartilage, mature cartilage, and bone according to the previous study https://ngdc.cncb.ac.cn/biocode/tools/BT007309.The code developed for the study has been submitted to BioCode at the National Genomics Data Center (NGDC), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences (CAS) / China National Center for Bioinformation (CNCB) (BioCode: BT007309), which is publicly accessible at A. pleuronectes scallop genome project has been deposited in NCBI (BioProject: PRJNA797172). The genome sequencing data have been uploaded to Sequence Read Archive . The Illumina sequencing data of 20 transcriptomes for adult tissues/organs have also been uploaded to SRA (SRA: SRR17752968–SRR17752977 and SRR17752979–SRR17752988). The assembled data of A. pleuronectes have also been submitted to Genome Warehouse GWHBJCZ00000000), and are publicly accessible at https://ngdc.cncb.ac.cn/gwh. The raw sequencing data have also been deposited in the Genome Sequence Archive CRA006995), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa.The Yuli Li: Data curation, Methodology, Formal analysis, Writing - review & editing. Yaran Liu: Software, Formal analysis, Data curation, Methodology, Writing - original draft. Hongwei Yu: Formal analysis, Methodology. Fuyun Liu: Software, Data curation. Wentao Han: Formal analysis. Qifan Zeng: Data curation. Yuehuan Zhang: Data curation. Lingling Zhang: Writing - review & editing. Jingjie Hu: Writing - review & editing. Zhenmin Bao: Conceptualization, Project administration, Writing - review & editing. Shi Wang: Conceptualization, Project administration, Writing - review & editing. All authors have read and approved the final manuscript.The authors declare that they have no competing interests.


C. minima), a Chinese medicinal herb, showed anti-cancer effects in lung cancer. However, the active components and underlying mechanisms remain unclear. In this study, we used network pharmacology to evaluate C. minima active compounds and molecular mechanisms in lung cancer.Traditional Chinese medicine (TCM) has been extensively used for neoplasm treatment and has provided many promising therapeutic candidates. We previously found that Centipeda minima  network and a protein–protein interaction (PPI) network using Cytoscape software, and we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using R software. To verify the network pharmacology results, we then performed survival analysis, molecular docking analysis, as well as in vitro and in vivo experiments.C. minima bioactive compounds and 179 corresponding targets. We screened 804 targets related to lung cancer, 60 of which overlapped with C. minima. The top three candidate ingredients identified by D-I-G-D network analysis were quercetin, nobiletin, and beta-sitosterol. PPI network and core target analyses suggested that TP53, AKT1, and MYC are potential therapeutic targets. Moreover, molecular docking analysis confirmed that quercetin, nobiletin, and beta-sitosterol, combined well with TP53, AKT1, and MYC respectively. In vitro experiments verified that quercetin induced non-small cell lung cancer (NSCLC) cell death in a dose-dependent manner. GO and KEGG analyses found 1771 enriched GO terms and 144 enriched KEGG pathways, including a variety of cancer related pathways, the IL-17 signaling pathway, the platinum drug resistance pathway, and apoptosis pathways. Our in vivo experimental results confirmed that a C. minima ethanol extract (ECM) enhanced cisplatin (CDDP) induced cell apoptosis in NSCLC xenografts.We identified a total of 21 C. minima active ingredients and molecular mechanisms in the treatment of lung cancer, providing a molecular basis for further C. minima therapeutic investigation.This study revealed the key The online version contains supplementary material available at 10.1186/s12906-023-03915-y. Lung cancer is one of the most common malignancies and the leading cause of all cancer-related deaths worldwide , 2. Non-C. minima), also called Ebushicao in TCM, belongs to the daisy family Asteraceae -3,5-dimethoxybenzene). Our results indicated that these 11 compounds may be related to C. minima’s anti-cancer effects. According to the 11 compounds’ D-I-G-D network degrees and molecular docking analysis, quercetin emerged as a key C. minima active compound against lung cancer. Quercetin is a member of the flavonoid subclass of flavonols and has been found to exhibit significant anti-tumor properties [We screened 21 potential compounds from operties . Quercetoperties –41, and operties . Anotheroperties . Our in C. minima active compound anti-lung cancer activity. TP53 encodes p53 tumor suppressor protein and acts as a critical failsafe mechanism for cellular anti-cancer defenses [Further, our results suggest that TP53, AKT1, and MYC may be key targets for defenses –46. TP53defenses –50. A TPdefenses . Alpha sdefenses –55. AKT1defenses . Wu et adefenses . Thus, Adefenses . A previdefenses . In our CDDP has been widely used in first-line lung cancer treatment . HoweverC. minima clearly has the potential for use in developing new NSCLC treatments. Additionally, our study demonstrated that network pharmacology predictions can be used to characterize TCM pharmacological mechanisms in detail. Evidence suggests that C. minima functions as an anti-cancer drug through multiple targets, amongst which inducing apoptosis and influencing platinum drug resistance were pathways identified by both in vivo and in vitro experiments. However, the current work has several limitations. First, more comprehensive TCM target gene databases are needed to obtain more reliable results. Furthermore, it is necessary to explore how the three hub genes change in response to single-drug therapy or two-drug combination therapy. Finally, although we determined that quercetin was the most important bioactive compound in C. minima, it may not be completely interchangeable for C. minima. Thus, further studies investigating C. minima’s anti-lung cancer molecular mechanisms are needed.Based on our network pharmacology results and experimental evidence from previous work, C. minima’s anti-lung cancer material basis and mechanism of action. We found that quercetin was the key active ingredient and that TP53, AKT1, and MYC may be three potential C. minima targets in lung cancer treatment. Additionally, we found that C. minima may exert anti-cancer effects by inducing apoptosis and influencing platinum drug resistance. In conclusion, our research provides a reference for further study of C. minima’s potential clinical application for lung cancer treatment.Here we used network pharmacology along with molecular docking and experimental validation to explore Additional file 1. Western blotting of GAPDH. Western blotting of Bcl2.


Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families  had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three , but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech ( Thrombosis refers to a variety of disorders characterized by aberrant blood clots in the blood that restrict or occlude the lumen of blood vessels, resulting in the ischemia and infarction of organs and malfunction . ThromboAlthough antithrombotic drugs have slowed the occurrence of thrombotic events in patients, their success in reducing the lethality of thrombotic disease is limited. The primary reason for this failure is that these drugs generally rely on a single target of action, and it is difficult to account for individual differences in dosing, which ultimately leads to frequent drug resistance, internal bleeding, liver and kidney damage, and other serious side effects that put patients’ lives at risk. For instance, warfarin, an anticoagulant, may lead to adverse events, including cerebral microhemorrhage, hemorrhagic stroke, subarachnoid hemorrhage, and leukocyte rupture vasculitis ,5. The uHirudo medicinalis) has been a prevalent tool in the treatment of various ailments in European countries since the 17th century. These conditions include, but are not limited to, osteoarthritis [Hirudo nipponia, is a common practice in traditional Chinese medicine for treating various diseases, particularly those related to thrombosis, including stroke and coronary heart disease. These leeches are often administered orally and have been widely studied by researchers [Leeches  are well-known annelids due to their obligate blood-feeding habits and are found on every continent except Antarctica . Over 80rthritis , chronicrthritis , cutaneorthritis , and posrthritis ,17. The earchers ,18,19.There are many antithrombotic active components in leeches ,20, but There are significant variations in the genomes of different leech species which lead to considerable disparities in the composition, function, and evolution of genes related to thrombosis prevention . NotablyHirudinaria manillensis , homotypic synonym: Poecilobdella manillensis) is classified under the family Hirudinidae, just like the European medicinal leech H. medicinalis. However, it is highly specialized in parasitizing mammals, as opposed to its counterpart. Sawyer [Hirudo spp.). Furthermore, it has been shown that the antithrombotic properties of the Asian buffalo leech are superior to those of commonly used Hirudo species, including H. medicinalis and H. nipponia [The Asian buffalo leech . Following the removal of their digestive tracts, genomic DNA was collected from fresh tissue using the DNeasy Blood and Tissue Kit . The quality and integrity of the DNA samples were evaluated through agarose gel electrophoresis, NanoDrop spectrophotometry  and Qubit fluorometry . Subsequently, the genomic DNA, which met the standards of quality and quantity, was utilized for building the PacBio and Illumina libraries.https://github.com/PacificBiosciences/ccs, accessed on 19 April 2022) with default settings.HiFi SMRTbell libraries were prepared with the SMRTbell Express Template Prep Kit 2.0 . The genome DNA was fragmented to 15–18 kb using a g-TUBE , and damage and fragment ends were repaired with reagents provided in the Template Prep Kit. The repaired ends were then ligated with SMRTbell hairpin adapters, and AMPure PB beads  were employed to concentrate and purify the library. To acquire SMRTbell libraries with large inserts, templates larger than 15 kb were selected using the BluePippin system . The sequencing procedure was carried out by BioMarker  utilizing the PacBio Sequel II platform. Thereafter, high-precision HiFi reads were generated using the CCS software . The overhangs were blunted with bio-14-dCTP  and Klenow enzyme (NEB). After dilution and religation using T4 DNA ligase (NEB), the genomic DNA was extracted and sheared to 350–500 bp with a Bioruptor . The biotin-labeled DNA fragments were enriched with streptavidin beads (Invitrogen) and were sequenced using the Illumina HiSeq 2000 sequencing platform  with both directions of 150 bp reads. We also generated ~100× short reads  for polishing the genome assembly. DNA libraries with ~350 bp insertions were constructed and were then sequenced with both directions of 150 bp reads using the Illumina HiSeq 2000 sequencing platform. Quality control for raw reads of Hi-C and Survey was performed using fastp 0.20.0 with default settings and parameters .For RNASeq, total RNA was extracted from head tissue using TRIzol reagent (Invitrogen) and purified using the RNeasy Mini Kit . Oligo(dT)-loaded magnetic beads were used to purify poly(A) mRNA from total RNA. The mRNA was then fragmented into small pieces (300~500 bp) at 94 °C for exactly 5 min. The cleaved RNA fragments were reverse-transcribed into first-strand cDNA using SuperScript II reverse transcriptase and random primers, and second-strand cDNA was generated using GEX second-strand buffer, DNA polymerase, RNase H, and dNTPs. These cDNA fragments were subjected to end repair and 3′ adenylation. Paired-end adapters were ligated to the 3′-adenylated cDNA fragments. cDNA fragments of ~300 bp were selected and enriched through 15 cycles of PCR amplification using Phusion DNA Polymerase. Finally, the constructed cDNA libraries were sequenced bidirectionally (150 bp in each direction) using the Illumina HiSeq 2000 sequencing platform. Quality control of raw RNASeq reads was also performed using fastp 0.20.0 with default settings and parameters.https://github.com/Nextomics/NextDenovo, accessed on 3 November 2021). The initial HiFi assembly contigs underwent polishing with Illumina PCR-free data utilizing NextPolish v1.4.0 [The genome was assembled using PacBio HiFi reads through NextDenovo V2.5.0  v.4.1.4  with theBoth ab initio and RNASeq-based prediction strategies were used to predict protein-coding genes in the masked genomes. For the ab initio prediction strategy, GlimmerHMM v3.0.4  and SNAPH. manillensis genome. It should be mentioned that the above automated gene prediction approaches varied in their predictive sensitivity to antithrombotic genes. Even the relatively best BRAKER approach could predict only three out of five hirudins (see below). Fortunately, there are many RNASeq reads of H. manillensis (accession SRR15881191~SRR15881251) in the sequence reads archive of GenBank, which greatly facilitated our manual prediction on antithrombotic genes.To the best of our knowledge, we collected all the available literature on leech antithrombotic genes and/or proteins. Sequence information was obtained directly from the literature or retrieved from genetic databases  according to the accession number in the literature. All coding sequences were translated into protein sequences using MEGA software. These reported leech antithrombotic proteins (RAPs) were used as query sequences to identify homologous proteins in the H. manillensis genome using the est2genome model in Exonerate v2.2.0 [To obtain as many antithrombotic genes as possible, we used a so-called BRAKER-plus strategy to improve the prediction of potential antithrombotic genes. First, we assembled the RNA-Seq data sequenced in this study and those from GenBank using Trinity. The generated unigenes were used for CDS prediction using GeneMarkS-T v5.1 . Second,e v2.2.0 . The hige v2.2.0 . After chttps://dtu.biolib.com/SignalP-6, accessed on 5 September 2023). For each gene family, the proteins predicted in this study and the corresponding archetypal protein were combined and aligned using Clustal W in MEGA v11.0.13 [The CDS of all antithrombotic genes were translated into protein sequences using Seqkit v0.10.2 . The sigv11.0.13 . The prov11.0.13 . The lonThe relationships among members of the hirustasin superfamily and the HMEI family are too complex to be directly observed. We reconstructed phylogenetic trees of the two protein families using IQ-TREE v1.6.12 . The besPichia pastoris as expression vector to produce recombinant hirudin variants of H. manillensis according to Hu et al. [P. pastoris strain GS115. The cells were plated on yeast extract peptone-dextrose medium containing 0, 0.25, 0.50, 1.0, and 2.0 mg/mL G418. The positive transformants harboring recombinant hirudin variants were identified through PCR and grown overnight in 5 mL buffered glycerol-complex medium at 30 °C with shaking. After 2 days of culture, hirudin protein expression was induced by daily addition of 1.0% v/v methanol. The harvested supernatant was then centrifuged, and the protein of interest was extracted with 70% ammonium sulfate.Hirudin was the first identified and most studied antithrombotic protein. The antithrombotic (mainly anticoagulant) activities of hirudin variants from different leech species have been repeatedly analyzed . We usedu et al.  with minThe antithrombin activity of the recombinant protein was evaluated using the antithrombin titration method as previously described , with miH. manillensis. A total of 23.80 Gb of highly accurate HiFi reads were obtained, with an average length of 18.73 Kb. The de novo assembly generated 34 contigs with a total length of 149.59 Mb and an N50 of 9.58 Mb. We then used the Illumina HiSeq platform for the NGS sequencing of Hi-C libraries, and based on the obtained 21.09 Gb Hi-C reads, we consolidated the contigs into 22 scaffolds with a total length and an N50 of 148.59 Mb and 11.46 Mb, respectively. The first 13 longest scaffolds were 14.76~8.30 Mb in length, while the remaining 9 scaffolds were less than 0.3 Mb each. The highly discontinuous scaffold length distribution and the well-resolved Hi-C maps . The genome assembly was deposited in the Figshare online repository: https://doi.org/10.6084/m9.figshare.24187377.v1, accessed on 24 September 2023  with the eukaryota_odb10 database. The results showed that 98.0% of the BUSCOs were captured, including 92.5% complete and single-copy BUSCOs, 3.1% complete and duplicated BUSCOs, and 2.4% fragmented BUSCOs. We also used Merqury to estimate the quality of the assembly and obtained a quality score of 33.53. The BUSCO and Merqury results indicated that we had obtained a high-completeness and high-quality genome of this species. We also searched for repeat sequences in the genomes using RepeatModeler and RepeatMasker. A total of 18.75% of the genome was identified as repeats, including retroelements (6.59%), DNA transposons (1.76%), rolling circles (0.20%), unclassified elements (8.41%), satellites (0.2%), and simple repeats (1.8%). No small RNA or low-complexity repeat elements were found. The masked genome was used for gene prediction.We used two ab initio prediction approaches (GlimmerHMM and SNAP) and three RNASeq-based prediction approaches  to predict protein-coding genes in the masked genomes. The ab initio approaches detected more genes than the RNASeq-based approaches, but with much lower N50 values of the CDS (<1 kb). Of the three RNASeq-based prediction approaches, BRAKER detected the smallest number of genes  but had the largest N50 values of the CDS (2151 bp) . After cTo test the prediction sensitivity, we used the well-known hirudin (HV1) as a query to blast the CDS predicted by each approach. The results showed that two hirudin-coding genes were predicted by each of the two ab initio prediction approaches, and three were predicted by the BRAKER approach. Unexpectedly, no such gene was detected by PASA, Stringtie, or the EVidenceModeler integrated sequences . ConsideWe aligned the protein sequences predicted by the BRAKER approach against the NCBI NR, Uniprot TrEMBL, eggNOG, and Pfam databases to perform functional annotations. About 2/3 of the proteins were functionally assigned to the NR, TrEmbl, EggNOG, and Pfam databases, respectively . After sH. manillensis elastase inhibitor (HMEI)  thrombin inhibitors, including hirudin and progranulin; (2) Factor Xa inhibitors, including antistasin, lefaxin, and therostasin; and (3) serine protease inhibitors, including hirustasin, guamerin, piguamerin, bdellastasin, poecistasin, eglin, bdellin, leech-derived tryptase inhibitor (LDTI), and r (HMEI) . We usedillensis ,42. A toillensis   were successfully identified from the 21 gene families, outperforming previous studies. Of the 72 corresponding antithrombotic proteins, 59 were encoded by 12 multi-gene families  that contained 2 or more closely related members. The remaining nine proteins  were encoded by single gene families. Nine of the twelve multi-gene families were clustered in a single chromosome, while three families  were involved in two or three chromosomes. The GFF file and predicted CDS of the BRAKER-plus gene prediction approach, and all CDS of the 72 antithrombotic genes were also deposited in the Figshare online repository: ber 2023 .H. medicinalis. Subsequently, additional subtypes or variants were sporadically reported from H. medicinalis [H. manillensis [Haemadipsa sylvestris [H. nipponia [Whitmania pigra [H. manillensis genome. The corresponding five proteins of these genes were 66~85 amino acid residues in length. Online blasting showed that these proteins had a 57.63~77.83% identity with the hirudins deposited in GenBank  [Haementeria [Antistasin belongs to a class of serine protease inhibitors characterized by a well-conserved pattern of cysteine residues . Antistaicinalis  and H. gilanten) , but no menteria ,92, therH. depressa. Inhibition of Factor Xa by lefaxin was demonstrated by inhibition of its amidolytic activity and its ability to inhibit thrombin generation in the prothrombinase complex [H. manillensis genome. In contrast to the other Factor Xa inhibitors, no signal peptide or conserved cysteines were found in the corresponding proteins. The archetypal lefaxin from H. depressa corresponded to the N-terminus of the six lefaxins (Lefaxin (leech Factor Xa inhibitor), as its name suggests, was also an effective inhibitor of Factor Xa. Lefaxin was first purified from dissected salivary complexes of a leech  complex . Three glefaxins . The seqT. tessulatum. Like the other known inhibitors of Factor Xa, Therostasin is expressed and stored in the cells of the salivary glands of the leech. Therostasin is a cysteine-rich protein with 16 cysteine residues [H. manillensis. The corresponding protein sequences contain a signal peptide region that is much longer than that of T. tessulatum. In contrast, the functional region was much more conserved between the two leeches, especially the 16 cysteine residues  is a small protein with potent inhibitory activity against chymotrypsin and subtilisin-like serine proteinases acting on non-cationic substrates . This mohepsin G . Two eglhepsin G , suggestonserved . The sigH. medicinalis [H. manillensis genome. The corresponding protein sequences were relatively conserved, including a signal peptide region of 19 amino acids, an N-terminal region of about 40 amino acids and six cysteines, and a C-terminal region with a highly charged repetitive sequence  is another Kazal-type serine protease inhibitor that inhibits mast cell tryptase in particular, but also trypsin and chymotrypsin . LDTI waicinalis . Recombiicinalis ,109. Herysteines . Sequencproteins . The seqH. manillensis elastase inhibitor) is a newly discovered elastase inhibitor from H. manillensis. The protein belongs to a multigene family because at least two members, HMEI-A and HMEI-B, have been detected. The HMEI-A showed potent abilities to inhibit elastase and as a result inhibited the formation of neutrophil extracellular trap (NET). Although the authors did not test the activity of HMEI-B, a similar function was expected based on the high-sequence identity, especially the highly conserved cysteine residues [H. manillensis genome, indicating that they belong to a large gene family. Of the 18 corresponding proteins, three (HMEI_Hman03 ~ HMEI_Hman05) were identical to each other. Phylogenetic analysis showed that HMEI_Hman16 and HMEI_Hman15 were closely related to HMEI-A and HMEI-B, respectively . Here, we identified five apyrase proteins from the H. manillensis genome. One signal peptide region and two conserved cysteine sites were detected (H. robusta apyrase (XP_009028854.1) and the six newly obtained proteins were 58.64~81.71%.Apyrase (adenosine 5′-diphosphate diphosphohydrolase) is a nonspecific inhibitor of platelet aggregation by acting on adenosine 5′-diphosphate, arachidonic acid, platelet-activating factor, and epinephrine. Apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent release of apyrase from salivary glands into the host during blood feeding . The bionilotica , but seqdetected . The seqL. rubellus [Eisenia fetida [H. manillensis genome. These proteins had high sequence similarity to the archetypal lumbrokinase from L. rubellus (GenBank No. AAN28692.1). All leech lumbrokinases and the archetypal lumbrokinase from L. rubellus were relatively conserved in the signal peptide region and 14 cysteine residues -Lys isopeptidase that cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine c-carboxamide and the e-amino group of lysine. The molecule can disrupt covalent bonds formed between fibrin monomers under the influence of Factor XIIIa in blood plasma . Destabiicinalis , and thricinalis . Similarresidues . Surprisresidues . The seqH. medicinalis. This leech GGT was observed to have proteolytic activities on factor XIIIa cross-linked fibrin [H. manillensis. Six cysteines but no signal peptide region was detected in this protein (γ-glutamyl transpeptidase (GGT) is a cell surface enzyme that hydrolyzes the γ-glutamyl bond of extracellular reduced and oxidized glutathione, initiating its cleavage into glutamate, cysteine (cystine), and glycine. It is a critical enzyme in maintaining cellular redox homeostasis and is used as a marker of liver disease and cancer . A homold fibrin . A GGT g protein . The seqH. medicinalis. LCI binds tightly to pancreatic carboxypeptidases A1, A2, B and to plasma carboxypeptidases B. Assuming that leeches secrete LCI during feeding, the inhibitor could help maintain the fluid state of blood by inhibiting thrombin-activatable fibrinolysis inhibitors [H. manillensis genome. The corresponding protein had a signal peptide region and eight cysteines  is a metallo-carboxypeptidase inhibitor isolated from the medicinal leech hibitors . Here, aysteines , which aypal LCI  and thatH. medicinalis, was the most specific enzyme known for the identification of hyaluronic acid [H. manillensis. One corresponding protein , but not the other two, had a signal peptide region. Two conserved cysteines were detected in these proteins  and the newly obtained six proteins were 62.39~97.55%.Hyaluronidase is a spreading or diffusing substance that modifies the permeability of connective tissue through the hydrolysis of endoglucoronidic bonds of hyaluronic acid . Leech hnic acid . After tnic acid . It has nic acid , althougproteins . The simP. pastoris as a eukaryotic expression vector to produce recombinant hirudins. The results showed that hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5 exhibited obvious anticoagulant activities with mean ± SD of 960.1 ± 142.4, 508.8 ± 12.4, and 803.0 ± 101.3 U/mg, respectively. In contrast, hirudin_Hman3 and hirudin_Hman4 and the control group showed no anticoagulant activity.Hirudin is the first identified and most concerned antithrombotic protein. Here, we use H. medicinalis (HV1), H. manillensis , H. nipponia (hirudinHN), and W. pigra (Wpig_V1). The active hirudin_Hman2 was distributed in another monograph (group B1), which included two active hirudins from H. manillensis (HLF5 and HLF8). In contrast, the inactive hirudin_Hman3 and hirudin_Hman4 were distributed in another monograph (group B2) with inactive hirudins from H. manillensis (HLF7a and HLF6) , which included seven active hirudins from nd HLF6) .H. manillensis. A total of 13 chromosomes were identified, consistent with the recent study [H. manillensis genome. The Merqury analyses yielded a quality score of 33.5, which is also higher than that of Zheng et al. [H. manillensis to date.A high-quality genome is essential for effective identification and analysis of functional proteins. Recent high-throughput sequencing and assembly technologies make it easier to obtain a chromosome-scale genome. Here, we used state-of-the-art third-generation sequencing (PacBio HiFi) and next-generation sequencing  to obtain a nearly complete chromosome-scale genome of nt study . Howevernt study ). The BUnt study , only 88g et al.  (32.1). In contrast to the high quality of the genome assemblies, the results of the genetic prediction of antithrombotic genes were not very satisfactory. To test the prediction quality of different methods, we used the finally identified hirudins as query sequences to BLAST (tblastn) the CDS data sets predicted using these methods. Among the five hirudins, 2~3 hirudins were successfully predicted by GlimmerHMM, SNAP, and Braker, while none were predicted by PASA or Stringtie. Unexpectedly, no hirudin gene was found in the consensus prediction results generated by EVidenceModeler. It should be noted that the prediction of antithrombotic genes (at least for hirudins) does not necessarily seem to be better with higher completeness of genomes than in previous studies ,42,86, iUsing a so-called BRAKER-plus strategy, combining BRAKER prediction and manual prediction, we finally identified 72 genes from 21 gene families involved in antithrombotic functions. All protein products of these genes had more than 50% sequence similarity to their corresponding archetypal proteins, indicating that our identification methods are reliable. Except for lefaxin, eglin, apyrase, and hyaluronidase, most of the antithrombotic proteins were cysteine-rich, forming three or more disulfide bonds. By increasing protein stability, disulfide bonds were thought to make protein structures relatively independent of their amino acid sequences, thus acting as buffers against deleterious mutations and allowing for accelerated sequence evolution . InteresHirudo spp., Hirudinaria spp., Haemadipsa spp., Haementeria spp., Theromyzon spp., etc.) have been involved in related studies [H. medicinalis, several alternative names have been used: hirudin-like factors that have no anticoagulant activity [Haemadipsa spp. [Hirudinaria spp. [H. manillensis.Leeches have been used as a medical and pharmaceutical resource for many centuries, massive bioactive proteins have been reported, and many species , we broadly classified the antithrombotic proteins of leeches in this study into three categories: inhibitors of coagulation, inhibitors of platelet aggregation, and enhancers of fibrinolysis. Of the 21 antithrombotic protein families identified, 14 (67%) were involved in coagulation inhibition, including 2 thrombin inhibitors (hirudin and granulin), 3 Factor Xa inhibitors , and 9 serine protease inhibitors . Three gene families  were involved in the inhibition of platelet aggregation. Another three  were involved in enhancing fibrinolysis. The remaining family, hyaluronidase, does not belong to these three categories, but since it facilitates tissue penetration and diffusion for the other 20 protein families, its indirect antithrombotic function is not negligible.H. manillensis hirudins in this study showed that three  out of five hirudins had anticoagulant activity. We also collected the sequence and functional information of hirudins from previous reports and, then, tested the phylogenetic relationships of the reported hirudins and those from this study. The results showed that the proteins with and without anticoagulant activity clustered into separate clades. For example, the active hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5 had closer relationships with proteins that were confirmed to have anticoagulant activity. In contrast, the inactive hirudin_Hman3 and hirudin_Hman4 were more phylogenetically related to proteins confirmed to have no anticoagulant activity. These results repeatedly confirmed that at least three functionally active hirudins were simultaneously distributed in a single H. manillensis genome. These findings, together with the presence of two pseudogenetic genes (lumbrokinase_Hman3 and destabilase_Hman2), remind us that some other antithrombotic proteins may also suffer from loss of biological activity. The actual biological function of the other 67 proteins will need to be validated by laboratory experiments in the future.Hirudin is the most potent thrombin-specific inhibitor identified to date and is a representative pharmacologically active substance in leeches ,136. In H. manillensis. Combined with automatic and manual prediction, we identified 72 antithrombotic genes involving 21 gene families. The functions of the corresponding proteins include anticoagulation, anti-platelet aggregation, fibrinolysis, and drug diffusion. We also provided the complete CDS and protein sequences and their variation information for all 72 antithrombotic genes/proteins. This is the most comprehensive collection of genomes and leech antithrombotic biomacromolecules to date. Our results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes of thrombosis.In conclusion, in this study, we provided an almost complete, high-quality genome of


Ptecticus tenebrifer , which is endemic in East Asia, plays a critical role in natural processing of organic waste. In this study, we found that the complete mitochondrial genome of P. tenebrifer is 15681 bp in length obtained from the MGIseq system (MGI) with 150 bp paired-end reads. As with typical animal mitochondrial genomes, it contains 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic analysis was performed using the maximum likelihood method with a bootstrap value of 500 and the Tamura-Nei model and reveals the close relationship with P. aurifer. Hermetia illucens  (Diptera: Stratiomyidae) has attracted worldwide attention for its role in digesting various types of organic waste, i.e. cycling nutrients   , have beP. tenebrifer, collected at Seocheon-gun(county), Chungcheongnam-do, Republic of Korea  in August 2019. The specimen and gDNA were deposited at National Institute of Ecology (NIE hereafter) . Library preparation was performed using the MGIEasy DNA library prep kit (MGI). Sequencing was conducted on the MGIseq system (MGI) to generate 150-bp paired-end reads. To isolate the proper seed sequence for mitochondrial genome assembly using Novoplasty, trimmed reads were mapped to the Midori COI reference database . Mapping coverage and depth at each locus were calculated using the genomecov module of Bedtools v2.27.1. A candidate seed longer than 150 bp was manually selected for mitochondrial genome assembly. Using candidate seeds, Novoplasty v4.2 was repeatedly applied with various K-value parameters until a complete circularized mitochondrial genome was obtained. The annotation module from Mitoz v2.3(or Mitos) was used to annotate and visualize the constructed mitochondrial genome.Here, we report the first complete mitochondrial genome of Apis mellifera as an outgroup. They were aligned using MUSCLE followed by removing all gaps resulting in total of 10,596 bp on MEGA (ver.11) . Like other typical animal mitochondrial genomes, it contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes (P. tenebrifer mitochondrial genome is significant, similar to those of H. illucens and Nasimyia megacephala (Yang and Yang The complete genome of NA genes . The oveP. tenebrifer and H. illucens having evolved the ability to decomposing organic wastes (Jeong et al. D. melanogaster form the basal clade with A. mellifera (P. tenebrifer species. Except the organism of this study and H. illucens, life history of other Stratiomyomorpha species is largely unknown. Therefore, our study could be utilized for correct identification of the species as the future counter-measure of the current energy-consuming organic waste treatment, and for proving potential ecological roles of other closely related species.This phylogenetic analysis shows that Stratiomyomorpha and Tabanomorpha species are diverged from Schizophora counterpart at the basal node. Stratiomyomorpha and Tabanomorpha are monophyletic based on the full genome data . Most ofellifera . The phy


Malic acid (MA) is an important flavor acid in fruits and acts as a mediator in a series of metabolic pathways. It is important to understand the factors affecting MA metabolism for fruit flavor improvement and to understand MA-mediated biological processes. However, the metabolic accumulation of MA is controlled by complex heredity and environmental factors, making it difficult to predict and regulate the metabolism of MA. In this study, we carried out a genome-wide association study (GWAS) on MA using eight milestone models with two-environment repeats. A series of associated SNP variations were identified from the GWAS, and 15 high-confidence annotated genes were further predicted based on linkage disequilibrium and lead SNPs. The transcriptome data of candidate genes were explored within different tomato organs as well as various fruit tissues, and suggested specific expression patterns in fruit pericarp. Based on the genetic parameters of population differentiation and SNP distribution, tomato MA content has been more influenced by domestication sweeps and less affected by improvement sweeps in the long-term history of tomato breeding. In addition, genotype × environment interaction might contribute to the difference in domestication phenotypic data under different environments. This study provides new genetic insights into how tomato changed its MA content during breeding and makes available function-based markers for breeding by marker-assisted selection. In ripe fruits, malic, citric, and tartaric acids are the main organic acids, and act as important indicators of the flavor and nutritional qualities of fruits. Fruit acidity is due to the presence of organic acids, and different acidic substances are not equally acidic and differ significantly in acidity . DiffereMdVHA-Bs, MdVHA-E2, and MdtDT12 to control MA content in apple . Fruit spatiotemporal expressions of candidate genes were also provided in another profile with the transcript of cultivated variety S. lycopersicum cv. ‘M82’ [http://hemi.biocuckoo.org/down.php).We further cross-checked the expression patterns of candidate genes. Gene expression in different organs and fruits at different stages in tomatoes with three genomic backgrounds was studied, including cro-Tom’ , S. pimpv. ‘M82’ . HemI soπ) is often used as a measure of the degree of variation in a population [FST) is also applied to confirm highly differentiated regions of molecular diversity [π and FST were performed to identify the selective sweeps about MA content at two key stages (domestication and improvement) in tomato evolution [πPIM/πCER) as well as between CER and BIG (πCER/πBIG) , and population differentiation ratios FST (PIM versus CER) and FST (CER versus BIG)  were identified as selective sweep regions.Nucleotide diversity ; the National Natural Science Foundation of China ; the Wuhan Biological Breeding Major Project (2022021302024852); the Key Project of Hubei Hongshan Laboratory (2021hszd007); the Hubei Key Research & Development Plan ; the Fundamental Research Funds for the Central Universities (2662022YLPY001) and the International Cooperation Promotion Plan of Shihezi University (GJHZ202104)..W.G. and Y.Z. conceived and designed the experiments. W.G. and F.Y. analyzed the data and carried on the analyses. J.T. and X.Z. performed the data collection. S.H., Yaru W., Ying W., F.Y., L.Y., L.S., F.L., P.G., F.W. and H.D. conducted the literature review and edited the manuscript. Y.Z. was responsible for funding acquisition and article revision.The datasets supporting the findings in this study are available in the supplementary materials.The authors declare no competing interests.Horticulture Research online.Web_Material_uhad021Click here for additional data file.