Viburnum japonicum (Adoxaceae) is an evergreen shrub distributed in Japan, Korea, and Taiwan. We sequenced its complete chloroplast (cp) genome to examine its phylogenetic relationship within Dipsacales. This genome is 158,614 bp long and features a large single-copy region  and a small single-copy region , separated by two inverted-repeat regions . It contains 128 genes, including 84 coding genes, eight rRNAs, and 36 tRNAs. The overall GC content is 38.1%. Our phylogenetic tree showed that V. japonicum is closely related to V. utile and is clustered together with four species in the family Adoxaceae.The wax-leafed  Viburnum genus has traditionally been regarded as a group within Caprifoliaceae along with Sambucus and Sinadoxa  Spreng. is an evergreen shrub distributed in the subtropical regions of Korea, Japan, and Taiwan . The voucher specimen was stored at the herbarium in the Department of Biology Education, Chonnam National University (BEC: Lee. 16101504). Total genomic DNA was extracted with a DNeasy Plant Mini Kit , and pair-end sequencing (300 × 300 bp) was performed using the Illumina Miseq platform . This presented 13,920,280 raw reads. After trimming the sequences, clean reads were mapped with the reference cp genome for V. utile (NC_032296), using Geneious 10.2.3  regions of 26,516 bp that separate a large single-copy region  and a small single-copy region . The genome contains 128 genes: 84 protein-coding genes, eight rRNA genes, and 36 tRNA genes. Seventeen genes are duplicated in the IR region. The ML phylogenetic tree, with 1000 bootstrap replications, is monophyletic with two independent clades in Dipsacales. Viburnum japonicum is closely related to V. utile and clusters together with four species in the family Adoxaceae (V. japonicum provides information for further phylogenetic studies and genome resources within Dipsacales.The cp genome of doxaceae . The chl


Apis cerana is one of the main honeybee species in artificial farming, which is widely distributed in Asian countries. The genome of A. cerana has been sequenced by several different research groups using second generation sequencing technologies. However, it is still necessary to obtain more complete and accurate genome sequences. Here we present a chromosome-scale assembly of the A. cerana genome using single-molecule real-time (SMRT) Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated assembly is 215.67 Mb in size with a contig N50 of 4.49 Mb, representing an 212-fold improvement over the previous Illumina-based version. Hi-C scaffolding resulted in 16 pseudochromosomes occupying 97.85% of the assembled genome sequences. A total of 10,741 protein-coding genes were predicted and 9,627 genes were annotated. Besides, 314 new genes were identified compared to the previous version. The improved high-quality A. cerana reference genome will provide precise sequence information for biological research of A. cerana. Apis cerana is one of the main honeybee species that can be systematically raised by human beings and has been widely cultivated in the eastern countries such as China, Japan, and India for a long time, bringing considerable economic benefits to beekeepers. Compared with western honeybees, A. cerana is more sensitive to smell, better at using sporadic honey sources, and more suitable for collecting a variety of honey plants, while western honeybees usually prefer to collect large single honey source. Moreover, the A. cerana has stronger disease and stress resistance, stronger cold tolerance, lower feed consumption and longer collection period compared to western honeybees .Genome sequences are of great significance to the basic biological research of a species. The first genome assembly of  in 2015 . Then, i28.79 Mb . Also, tThe third generation sequencing (TGS) technologies do not need PCR amplification, which can effectively avoid systematic errors caused by bias of PCR amplification. At the same time, the lengths of DNA sequence fragments sequenced from a single run are so long that the average length of the reads can reach 10,000 bp, which is very helpful for assembling repetitive sequences. Compared with the second generation sequencing technologies, the TGS technologies also maintain the advantages of high throughput and low cost. Typical TGS technologies are single-molecule real-time (SMRT) sequencing technology developed by Pacific Biosciences company and Nanopore sequencing technology launched by Oxford Nanopore company, which are now widely used in genome sequencing of animals and plants .de novo assembly of the eastern honeybee, A. cerana. The PacBio long reads from haploid drones were assembled into contigs, then, they were further assembled into scaffolds using Hi-C proximity ligation data. The completeness and contiguity of this new assembly was greatly improved compared to previous genome assemblies.Here we used PacBio and Hi-C technologies to generate a highly contiguous A. cerana apiary raised by a local bee keeper in Fulong Township, Baisha Li Nationality Autonomous County, Hainan province , China, in October 2018. The intestines of the pupae were removed to avoid contamination of gut microbes before construction of SMRTbell and Hi-C libraries. Two SMRTbell libraries were constructed and sequenced. For each library, DNA was extracted from a single intestine removed drone pupa by AxyPrepTM Multisource Genomic DNA Miniprep Kit . Genomic DNA concentration was measured using the Qubitfluorimetry system with the High Sensitivity kit for detection of double-stranded DNA . Fragment size distribution of the genomic DNA was assessed using the Agilent 2100 Bioanalyzer with the 12000 DNA kit . Then, 5 μg of high molecular weight genomic DNA was sheared using g-Tube  to 10 kb, and the sheared DNA was used as input into the SMRTbell library preparation. SMRTbell library was prepared using PacBio 10 kb library preparation protocol. Once library was completed, it was size selected from 10 kb using the Blue Pippin instrument  to enrich for the longest insert size. The library was sequenced on a PacBio Sequel system using Sequencing Kit 3.0, 1200 min movies with 120 min pre-extension and software v6.0 (PacBio).The experimental bees used in this study were 6-day old drone pupae sampled from a single colony in a A. cerana. Briefly, the drone pupa was fixed with formaldehyde for 10 mins and then was lysed in lysing solution . Then, the cross-linked genomic DNA was digested with HindIII overnight. Sticky ends of the genomic DNA were biotinylated and proximity-ligated to form chimeric junctions that were enriched for and then physically sheared to a size of 300–700 bp. Chimeric fragments representing the original cross-linked long-distance physical interactions were then processed into paired-end sequencing library, and sequenced on the Illumina HiSeq X Ten platform.One Hi-C library was created from a single intestine removed drone pupa of After PacBio sequencing and removal of low quality reads, the remaining PacBio clean reads were assembled into contigs using HGAP4 of SMRT Link (version 6.0) . To asseRRID: SCR 015027) (RRID: SCR 012954) , with parameters setting at E-value 1e-5. Gene ontology analysis was performed using Blast2GO   and Repe 012954)  were use 012954)  with an BLAT v35 , then thBlast2GO  through Blast2GO  with a pBlast2GO  with E-vBlast2GO  by BBH mRRID: SCR_015008)   with the_015008)  to map t_015008)  with defA. cerana in our previous research 1 was performed using Mummer  size of 1,519 bp. The average exon and intron sizes were 247 bp and 688 bp, respectively. 9,627 genes occupying 89.63% of the whole set of predicted genes were annotated through KEGG, KOG, Pfam and uniprot databases .The quality of the assembled genome and annotated gene sets were assessed using BUSCO. Among the 4,415 BUSCO groups searched, 4,116 BUSCO groups (including complete and fragmented BUSCOs) were identified, occupying 93.23% of the total BUSCO groups. Among them, 3,801 groups were complete BUSCO groups, occupying 86.09% of the total BUSCO groups supporting the high quality of the genome assembly .A. cerana genome.The completeness of the predicted genes was further assessed by mapping Illumina RNA-seq data from our previous study  to the pA. cerana genome reported by us  domain containing genes showed great difference between A. cerana and A. mellifera, 63 genes vs. 137 genes, which is consistent with the results reported in the previous two genome assemblies of A. cerana (A. cerana, and 314 Pfam family domains are unique to A. mellifera.We compared the domains of all the predicted genes between ellifera . The tot. cerana , suggestA. cerana and A. mellifera. Finally, 111 genes unique to A. mellifera and 71 genes unique to A. cerana were identified (A. mellifera unique genes, 55 genes have KOG annotation, and “Signal transduction mechanisms” “General function Translation” are the major KOG categories. For A. cerana unique genes, most of them are annotated as “hypothetical protein,” just 30 genes have KOG annotation and half of them belong to “transcription” category, suggesting that these genes might promote the formation of unique characters of A. cerana by regulating gene transcription.In addition, we analyzed unique genes in entified . Of the A. cerana using an integrated strategy of PacBio and Hi-C technologies. This new genome assembly has an about 212-fold improvement in contiguity over the previous version based on second generation sequencing technologies, and many new genes were identified. This genome assembly will be an important genome reference for studies of functional genes in A. cerana. Also, it will provide important data resource for evolutionary and comparative genomic studies.We reported the newly assembled genome of A. cerana data has been submitted to the NCBI under Bio-Project number PRJNA579740. The Hi-C and PacBio sequencing data are available from NCBI via accession numbers SRR10377217, SRR10377218, and SRR10377219.The genome assembly 3.0 of Z-JZ and H-JZ conceived and designed the research. Z-LW and W-YY prepared the experimental materials. Y-QZ conducted the experiments. Y-QZ, H-JZ, QY, and Z-LW analyzed the data. Z-LW and Y-QZ wrote the manuscript. All authors read and approved the publication of the manuscript.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.


Haloferax volcanii, a halophilic species belonging to the phylum Euryarchaeota, has provided many useful tools to study Archaea, including easy culturing methods, genetic manipulation and phenotypic screening. This review will focus on DNA replication and DNA repair pathways in H. volcanii, how this work has advanced our knowledge of archaeal cellular biology, and how it may deepen our understanding of bacterial and eukaryotic processes.The tree of life shows the relationship between all organisms based on their common ancestry. Until 1977, it comprised two major branches: prokaryotes and eukaryotes. Work by Carl Woese and other microbiologists led to the recategorization of prokaryotes and the proposal of three primary domains: Eukarya, Bacteria and Archaea. Microbiological, genetic and biochemical techniques were then needed to study the third domain of life.  Th. ThH. vong light , implicafor UvrD . Furthergent MMC . Similarp. NRC-1  where deH. volcanii have revealed a link between the bacterial NER protein UvrA and NreA (HVO_0734), a member of the archaea-specific Nre family of proteins [H. volcanii encodes only NreA, which is not essential but cells lacking NreA are sensitive to MMC treatment. Double deletion mutants of nreA and uvrA are no more sensitive than single mutants, suggesting NreA and UvrABC act in the same pathway in H. volcanii [Genetic experiments in proteins . Most arproteins . H. volcvolcanii . NreA havolcanii .S. islandicus and T. kodakarensis mutants lacking XPB and XPD helicases are only slightly sensitive to UV radiation, MMC and MMS treatments, indicating that these enzymes do not play an essential role in NER [Methanosarcina mazei [H. volcanii encodes homologues of the bacterial Uvr system. However, it also encodes homologues of endonucleases XPF  and XPG  [H. volcanii has been shown to play no role in NER, instead being implicated in the restart of stalled replication forks [The majority of archaeal species, including hyperthermophiles, carry homologues of eukaryotic-like NER proteins, including endonucleases XPF/XPG and helicases XPB/XPD ,309–313.e in NER ,314. Somna mazei  and varina mazei . As prevVO_2873) . Unlike on forks .S. solfataricus does not favour repair of transcribed strands suggesting that the system for TCR in this species (if present) is no faster than that of GG-NER [T. kodakarensis has been shown to pause at a variety of DNA lesions suggesting damage recognition by the RNAP itself, akin to mechanisms of TCR in other domains of life [H. volcanii, TCR has been shown to occur in the absence of UvrA, unlike Halobacterium sp. NRC-1, which is unable to efficiently repair UV damage without UvrA [H. volcanii is UvrA independent; this could be performed by the RNA polymerase itself (as seen in eukaryotes and T. kodakarensis) or by an as yet unidentified coupling factor [TCR, while observed in some archaeal species, does not seem to be universally conserved throughout the domain. The crenarchaeon f GG-NER ,316. How of life . To date of life . While Tout UvrA . In the g factor ,319.2.6.3.mutSL genes are thought to have been acquired from bacteria [mutS and mutL genes in Halobacterium sp. NRC-1 does not increase the mutation rate [The MMR machinery is conserved across bacteria and eukaryotes, but most archaea lack key components of the canonical pathway . In bactbacteria –325. Arcbacteria ,326, indion rate , suggestin vitro in P. abyssi, where it acts on branched DNA structures containing flapped and splayed DNA [S. solfataricus XPF and P. abyssi and T. kodakarensis EndoMS/NucS are able to interact with PCNA, and this interaction is required for their specificity and endonuclease activity [EndoMS  is an endonuclease first characterized ayed DNA –331. Endayed DNA . Both S.activity ,332,333.et al. [T. kodakarensis EndoMS has the ability to specifically cleave dsDNA containing a base pair mismatch in vitro, indicating a role for EndoMS in a novel MMR pathway. Other structural studies have supported the hypothesis of EndoMS specifically recognizing and binding mismatched bases by a unique dual base flipping mechanism [T. kodakarensis, the endoMS gene is in an operon with the radA recombinase gene [Thermococcus. Nevertheless, a functional link between EndoMS and HR has been observed in Sulfolobus acidocaldarius, where EndoMS has been shown to act in HR-mediated stalled fork repair to remove helix-distorting DNA lesions, overlapping with the role of XPF and suggesting a potential role for EndoMS in NER [T. gammatolerans, where NucS is capable of cleaving uracil- and hypoxanthine-containing dsDNA, indicating an alternative to BER for the repair of deaminated bases in this species [Ishino et al.  have demechanism ,335. In ase gene , but thiS in NER . A new f species .H. volcanii; it encodes four MutS , two MutL  and one NucS protein (HVO_0486). NucS is not essential and no phenotype is observed in cells lacking nucS [mutS genes (namely mutS1a and mutS1b) are located in operons with a mutL partner and are predicted to function in MMR [mutSL genes leads to an increase in the spontaneous mutation rate of H. volcanii . The other two MutS proteins belong to a subfamily that does not seem to be involved in DNA repair in other organisms [H. volcanii remains to be elucidated.Interestingly, some haloarchaeal species encode both NucS and MutSL homologues, including ing nucS . Two of rganisms ,338. The2.6.4.T. kodakarensis, this pathway has been identified using computational methodology [H. volcanii remains to be demonstrated.Ribonucleotides (rNMPs) that are misincorporated into genomic DNA are recognized and removed by the RER pathway. Archaeal D family DNA polymerases have been shown to incorporate 1 rNTP every approximately 1000 bases, and archaeal B family DNA polymerases every approximately 2500 bases ,340. RNa2.7.−1–10−3 bases [Family Y DNA polymerases have a specialized function, whereby they are able to bypass various forms of DNA damage that block DNA synthesis by canonical replicative polymerases; this process is known as translesion synthesis (TLS) . Family −3 bases ,345,346.−3 bases . Thus, tMost eukaryotes encode four-family Y DNA polymerases, each with specificity for different types of the lesion ,349. Meain vitro under non-physiological temperatures. The TLS polymerases found in archaea belong to the DinB subfamily of family Y DNAPs. This subfamily is the most diverse and is found throughout all domains of life. DinB polymerases are prone to making single-nucleotide deletions in vivo and in vitro, caused by template slippage where repetitive sequences are present [The properties, roles and functions of archaeal family Y DNAPs are not well understood, since most have been tested  present ,351,352.S. solfataricus encodes either Dpo4 (polymerase IV) or Dbh (DinB homologue) [cis-syn cyclobutene thymine dimer and 8-oxo guanines with relative efficiency, compared with other family Y DNA polymerases, since it can accommodate two adjacent template bases into its active site [Hyperthermophilic archaea encode only one family Y DNA polymerase. Depending on strain, mologue) ,353–355.mologue) ,356. Dpoive site ; similarive site  and humaive site . This feive site –363. Simive site ,365.S. islandicus has implicated a family B enzyme, Dpo2, in TLS repair of helix-distorting lesions [S. solfataricus, a complex has been described where both family Y and family B polymerases are bound to PCNA and DNA [Until now, only family Y polymerases were believed to act in TLS, but recent work in  lesions . The pot lesions . In S. s and DNA . This is and DNA  and euka and DNA . TetheriH. volcanii encodes one family Y polymerase, PolY (HVO_1302), which shares homology with bacterial DinB. As in eukaryotic family Y polymerases, H. volcanii PolY encodes a C-terminal PIP motif, but it has diverged somewhat from the canonical sequence [polY is possible in H. volcanii , suggesting that other pathways are available for the repair or bypass of bulky lesions that block DNA synthesis by canonical polymerases.sequence . Degenersequence . Deletio2.8.DSBs are considered to be one of the most critical forms of DNA damage that cells can suffer. A break in both strands of the DNA double helix can lead to inhibition of key processes including DNA replication, transcription and cell division, alongside major genome rearrangements. Therefore, the major pathways of double-strand break repair (DSBR) are crucial for maintaining genome stability. The most accurate form of DSBR is homologous recombination (HR), but this is a complex and energetically costly process, and therefore less-demanding pathways of DSBR operate alongside HR .Figure 2.8.1.Classical non-homologous end joining (NHEJ) repairs DSBs by ligating DNA ends using little or no complementary base pairing. The first step is recognition of the DSB by Ku protein, comprising a Ku homodimer in bacteria and a Ku70/Ku80 heterodimer in eukaryotes. Ku acts as a scaffold protein to recruit other proteins involved in DSB repair, including nucleases to resect the damaged DNA, family B and family X DNAPs to fill the gap, and DNA ligase to ligate the DNA strands . NHEJ isMethanocella paludicola [H. volcanii [Ku proteins are conserved in bacteria, yeast and higher eukaryotes, but to date only a few archaeal species have been shown to encode Ku proteins, which have most likely been acquired from bacteria by LGT ,371. To volcanii ,373; insvolcanii ,375.2.8.2.a and While NHEJ does not require any homology, microhomology-mediated end joining (MMEJ) involves the annealing of short homologous sequences at broken DNA ends. The products of MMEJ are always associated with gene deletions and contribute to chromosome translocations and genome rearrangements. The basic mechanism for MMEJ involves resection of DNA ends, annealing of the micro-homologous region, the removal of the DNA flaps by the structure-specific nuclease Fen1, filling of the gap by DNA polymerases and ligation by DNA ligase –378  by the Rad50-Mre11 complex, which restrains HR and instead promotes MMEJ as the immediate pathway of DSB repair [MMEJ has been observed directly in a small number of archaeal species, including landicus ,375,379.B repair . HR is l2.8.3.b and The mechanism of HR is conserved throughout the three domains of life. HR is an essential process in archaea that provides a high-fidelity DSB repair mechanism, compared with other error-prone end-joining processes. This high fidelity is due to the use of homologous DNA duplex as the template for repair. Regulation of HR is essential to maintain genome integrity and avoid DNA rearrangements ,381. Whefigure 4H. volcanii genome is highly polyploid with a genome copy number of approximately 20 copies per cell [HR is the best-studied DSB pathway in archaea . Alongsiper cell ; having 2.8.4.2.8.4.1.The Rad50-Mre11 complex is present in all domains of life ,386. In S. islandicus and T. kodakarensis), Rad50 and Mre11 are essential for cell viability [Halobacterium sp. NRC-1, Mre11 is essential while Rad50 is dispensable, indicating a Rad50-independent function of Mre11 [H. volcanii, deletions of rad50 (HVO_0854) and mre11 (HVO_0853) are viable and mutants show increased resistance to various types of DNA damage, including UV, ionizing radiation and MMS. However, these mutants recover slowly and exhibit higher rates of HR at DSBs than the wild type [H. volcanii, the action of Rad50-Mre11 in temporarily restraining HR may help prevent DNA ends from engaging with multiple homologous partners; after the number of available DNA ends has been reduced by MMEJ, DSBs are ultimately repaired by HR [Rad50-Mre11 processes DSB ends by resecting the 5′ strand, generating a short single-stranded 3′ overhang. This overhang is then coated by a recombinase protein (RadA in archaea) to facilitate strand invasion ,393. In iability . By contof Mre11 . In H. vild type . In a poed by HR .H. volcanii Rad50-Mre11 complex has been shown to be involved in nucleoid compaction following genomic stress. Such nucleoid compaction is another mechanism to ensure DSB ends remain within close proximity [In addition, the roximity , which mroximity ,396,397.roximity , unlike roximity ,399.2.8.4.2.herA and nurA are encoded in the same operon as rad50 and mre11 [in vitro and in vivo [Sulfolobus viability [H. volcanii. Bacterial homologues of HerA and NurA have been identified and were initially thought to be essential for cell viability [Deinococcus radiodurans and Thermus thermophilus [nurA and herA has no effect on cell growth. Unexpectedly, T. thermophilus cells lacking nurA and herA show increased resistance to UV irradiation and MMC treatment [In thermophilic archaea, nd mre11 . HerA an in vivo . The Her in vivo ,402–404.iability . HerA aniability . Geneticmophilus ; in the reatment .2.8.5.2.8.5.1.The 3′ ssDNA tail generated during pre-synapsis acts to invade a homologous duplex; this is facilitated by coating of the ssDNA with a recombinase protein. The strand exchange protein, or recombinase, promotes homology search and catalyses strand invasion, giving rise to a D-loop (displacement loop) ,407. ArcH. volcanii cells show growth defects, a lack of homologous recombination, and increased sensitivity to DNA damage agents [Archaeal RadA is more similar to eukaryotic Rad51 than to bacterial RecA . Like bae agents –412, inde agents , suggest2.8.5.2.Paralogues of RecA-family proteins are present in many organisms where they generally function as recombination mediators –418. EukH. volcanii, RadB (HVO_2383) acts as a recombination mediator that interacts with RadA and promotes its polymerization on ssDNA [radB has been deleted show a phenotype similar to cells lacking radA; defects include growth retardation, low levels of recombination and DNA damage sensitivity [ΔradB phenotype have been identified in H. volcanii RadA, S101P and A196 V, which suggest that RadB induces a conformational change in RadA, promoting its polymerization on ssDNA [In archaea, RadB is a paralogue of RadA that is present only in Euryarchaea . In H. von ssDNA . Strainssitivity ,412. Two2.8.6.2.8.6.1.During HR, four-way DNA intermediates  are formed by strand exchange between homologous DNA molecules. Resolvases, a group of highly specialized structure-specific metal-dependent endonucleases catalyse the cleavage of HJs into two DNA duplexes . ResolvaE. coli, HJs are resolved by the RuvABC complex. The initial RuvAB complex, comprising a RuvA tetramer and two RuvB hexamers, specifically binds the HJ and promotes branch migration, following which RuvC cleaves the junction [In junction . Eukaryojunction ,428.Sulfolobus species contain an additional resolvase Hje, which shows higher DNA cleavage activity than Hjc [H. volcanii Hjc (HVO_0170) is non-essential, has been shown to act in the same pathway as RadA and is necessary for efficient growth in the presence of cross-linking agent MMC [In archaea, the Hjc resolvase has been implicated in the cleavage of HJs. Hjc is present in all archaeal species and has been shown to interact with various proteins involved in DNA replication and repair, including the helicase Hel308 , clamp pthan Hjc ,432. H. gent MMC .S. islandicus, Hjc is regulated by phosphorylation, which acts to inhibit its catalytic activity [S. islandicus exhibit increased resistance to DNA damaging agents, suggesting that phosphorylation acts to redirect repair to avoid HR that is dependent on Hjc [S. islandicus Hjc are not conserved in other archaeal species, modification of Hjc and other HR proteins by phosphorylation in S. acidocaldarius is well documented [. This regulatory mechanism could have evolved early during evolution, as the activity of several HJ resolvases is also regulated by phosphorylation in eukaryotes [H. volcanii.In activity . Interest on Hjc . While tcumented . This rekaryotes –437. How2.8.6.2.Hef (helicase-associated endonuclease fork-structure DNA) is a member of the XPF family of structure-specific endonucleases known to act on branched, flapped and forked DNA structures . Hef feaH. volcanii, Hef (HVO_3010) is not involved in NER and instead a bacterial Uvr system is used [H. volcanii and T. kodakarensis mutants deleted for hef show sensitivity to MMC [H. volcanii, Hef is non-essential but cannot be deleted from cells lacking Hjc, suggesting these two proteins participate in alternative mechanisms for the resolution of recombination intermediates [S. islandicus are redundant [H. volcanii, Hef is required for cell viability in the absence of HR and is recruited to sites of DNA replication fork arrest [T. kodakarensis [P. abysii [T. acidophilum [H. volcanii, Hef in this species features a PIP box, indicating that Hef : PCNA interactions are common to most euryarchaea.In  is used . However is used . Both H.y to MMC ,307. In mediates . Redundaedundant . In H. vk arrest ,442. Thikarensis , P. abys. abysii  and T. adophilum . This inT. kodakarensis, although Hef cannot bind both PCNA and HAN simultaneously [han in H. volcanii  results in increased sensitivity to DNA damage agent MMS, but not to other genotoxic agents such as H2O2, 4NQO or MMC [Alongside Hef, archaea from the phylum Euryarchaea encode a conserved protein HAN (Hef-associated nuclease), a RecJ-like protein displaying 3′–5′ exonuclease activity . Hef andaneously . DeletioO or MMC . It has O or MMC ,442.2.8.6.3.in vitro, showing the preference for forked DNA and D-loops, and can remove bound proteins during translocation; these activities suggest a role in HR and the restart of stalled replication forks [S. tokodaii [M. thermautotrophicus [Hel308 is a Ski2 family 3′–5′ helicase found in archaea and metazoans (where it is named HelQ/PolQ) –450; howon forks . Hel308 on forks , Hjc restokodaii  and RPA rophicus . Hel308 S. tokodaii and S. islandicus [H. volcanii (HVO_0014) are viable but slow-growing and show sensitivity to DNA cross-linking agents such as MMC ; a similar phenotype has been seen in human ΔhelQ mutants [H. volcanii Hel308 in the processing and repair of inter-strand cross-linked DNA lesions.Hel308 is not essential in the majority of archaeal species that encode it, with the exception of landicus ,453. Mut mutants . This im2.9.E. coli showed that in the absence of origins, DNA replication can be primed from D-loops or R-loops (RNA displacement loops); these structures undergo remodelling to form a canonical replication fork [Replication origins were originally thought to be indispensable for cellular life, and deletion of origins was found to lead to impaired growth or cell death . Work inion fork . For utiion fork ,455. Botion fork .H. volcanii without affecting viability; in fact, strains deleted for origins grow 7.5% faster than wild type [E. coli, cells deleted for replication origins become dependent upon the recombinase protein (here RadA), indicating that originless strains use RDR and hence HR becomes essential [H. volcanii shows a flat replication profile, indicating a lack of specific initiation and termination sites [T. kodakarensis has been shown to be capable of DNA replication in the absence of origins, again using a mechanism dependent on HR [H. volcanii, MFA analysis of T. kodakarensis shows a flat replication profile in originless strains. But unlike H. volcanii, a flat replication profile is also seen in wild-type T. kodakarensis, suggesting that the origin, while present, is not used under laboratory conditions [Surprisingly, it is possible to delete all chromosomal origins of replication in ild type . As in Essential . Marker on sites . Similarnt on HR . As in Hnditions .H. volcanii and T. kodakarensis are highly polyploid species. Having a large number of genome copies increases the chance of finding a homologous DNA duplex to invade and carry out HR; this may explain why RDR can occur in these organisms with relative ease. Similar findings have been made with polyploid species of cyanobacteria, which are able to carry out efficient DNA replication in the absence of the initiator protein DnaA [E. coli and higher eukaryotes, are reliant upon origins to replicate their DNA; the lack of homologous DNA sequences would restrict the potential for RDR. Within archaea, members of the crenarchaea have been shown to contain only 1–2 genome copies and, unlike polyploid euryarchaea, have a defined cell cycle [S. islandicus, where one of its three replication origins must be maintained for cell viability [Both ein DnaA . Such a ll cycle ,460. Wheiability .H. volcanii: Haloarcula hispanica and Haloferax mediterranei. The two replication origins of H. hispanica cannot be deleted at the same time, indicating that at least one origin is essential for viability [H. mediterranei, deletion of all three chromosomal origins is possible but this leads to the activation of a dormant origin [H. volcanii. The variety of responses to origin deletion in archaea hints at complex differences between species in their capacity for RDR, and further work is needed to elucidate the specific mechanisms involved.However, not all polyploid Euryarchaea are capable of originless replication, including two close relatives of iability . In H. mt origin ; the dor3.H. volcanii have accelerated. These tools have, in turn, increased our understanding of the mechanisms of DNA replication and repair, both in this organism, in halophiles and in archaea generally. The increasing number of model archaeal species has provided additional insights into diversity across the domain, where species within a phylum may use radically different mechanisms for basic processes such as DNA replication.Over the past three decades, the development and application of genetic and biochemical tools for The identification and study of archaeal DNA repair and replication enzymes has provided invaluable information on their eukaryotic counterparts. On the other hand, the development of tools specific for archaea has shed light on adaptations and proteins specific to this domain. Such a two-pronged approach—to determine what is common with eukaryotes and what is unique to archaea—is needed to uncover the evolutionary history of DNA replication and repair.H. volcanii. The pathways used are counterparts of either entire eukaryotic or bacterial systems, with limited substitution within a pathway. Such preservation of pathway integrity is explained by the complexity hypothesis [H. volcanii, LGT has led to wholesale displacements , alternative activities that are used interchangeably  or acquisitions that are not used . These three scenarios illustrate how H. volcanii is not just a good model organism for the study of DNA replication and repair mechanisms, but is also invaluable for the study of LGT and evolution.The ease of genetics and availability of DNA damaging agents have allowed the identification of key DNA repair enzymes in pothesis , which sH. volcanii has undoubtedly increased, numerous questions remain. The ever-increased repertoire of genetic and biochemical tools for H. volcanii cements its place as a bona fide archaeal model organism. These tools will prove invaluable to answer open questions and thereby increase our understanding of DNA replication and repair in archaea.While our understanding of DNA replication and repair in


E. coli and S. enterica than in monocultures. While we observed similar mechanisms of resistance arising under rifampicin selection, under ampicillin selection different resistance mechanisms arose in co-cultures and monocultures. In particular, mutations in an essential cell division protein, ftsI, arose in S. enterica only in co-culture. A simple mathematical model demonstrated that reliance on a partner is sufficient to slow the rate of adaptation, and can change the distribution of adaptive mutations that are acquired. Our results demonstrate that cooperative metabolic interactions can be an important modulator of resistance evolution in microbial communities.With antibiotic resistance rates on the rise, it is critical to understand how microbial species interactions influence the evolution of resistance. In obligate mutualisms, the survival of any one species (regardless of its intrinsic resistance) is contingent on the resistance of its cross-feeding partners. This sets the community antibiotic sensitivity at that of the ‘weakest link’ species. In this study, we tested the hypothesis that weakest link dynamics in an obligate cross-feeding relationship would limit the extent and mechanisms of antibiotic resistance evolution. We experimentally evolved an obligate co-culture and monoculture controls along gradients of two different antibiotics. We measured the rate at which each treatment increased antibiotic resistance, and sequenced terminal populations to question whether mutations differed between mono- and co-cultures. In both rifampicin and ampicillin treatments, we observed that resistance evolved more slowly in obligate co-cultures of  Little is known about how ecological interactions between bacteria influence the evolution of antibiotic resistance. We tested the impact of metabolic interactions on resistance evolution in an engineered two-species bacterial community. Through experimental and modeling work, we found that obligate metabolic interdependency slows the rate of resistance acquisition, and can change the type and magnitude of resistance mutations that evolve. This work suggests that resistance evolution may be slowed by targeting both a pathogen and its metabolic partners with antibiotics. Additionally, we showed that community context can generate novel trajectories through which antibiotic resistance evolves. Any random numbers less than 0.5 meant those individuals gained a mutation. In all cases, mutations could be either a growth rate mutation or an MIC mutation with a 50:50 chance. Growth rate mutations were simulated by adding a random number from a normal distribution with mean 0 and standard deviation 0.1 to the current growth rate . MIC mutations altered the MIC of the individual, as described in the results. We tracked the genealogy of all individuals.Once the new population was generated, some of the new individuals may be mutated. Each simulation used a pre-determined mutation rate except that there must be individuals of all species with an MIC greater than the antibiotic concentration for any species to grow.To simulate interdependence, we simulated >1 species per well. Each species had N individuals, and the dynamics of each species were independent of the other species, The second time scale—the transfer scale—was simulated to approximate the wet-lab experiments. Individuals were transferred from one well to two wells: the same antibiotic concentration, and the next higher antibiotic concentration. Therefore, wells received individuals from two wells. If there were surviving species in both source wells, N/2 individuals were randomly chosen from each well to populate the new well. If there were surviving species in only one source well, all N individuals were transferred from that well to the new well. If no source wells had surviving species, the destination well was sterile. Simulations were conducted in R.Fig 5C), we first found the mean and standard error in the tolerance in each set of simulations. Then, the mean of the two-species community was divided by the mean of the one-species community. The error was propagated using (mean_relative_tolerance)*sqrt((SEM[two species]/mean_tolerance[two_species])^2 + (SEM[one species]/mean_tolerance[one_species])^2). The average MIC mutation size  was the average MIC in a surviving population divided by the average number of mutations in that population. In all cases, since species were functionally equivalent, we arbitrarily chose one species to assess. Statistics were performed in R. Example code to run simulations is available as supplementary files .Statistical analyses of the simulation data: We analyzed the rate of evolution of antibiotic tolerance as was done in the wet-lab experiments. A linear mixed- effects model with a random slope for each replicate within a treatment group was used to test the relationship between the response variable MIC and explanatory variables transfer, culture type (monoculture or co-culture), and a transfer-culture type interaction. A one-way analysis of variance tested the amount of mutations observed in the most-tolerant population, with an independent variable of the # of interdependent species. To calculate the relative tolerance of two-species simulations versus one-species simulations (S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigOD equivalent is calculated from the fluorescent protein in each strain, allowing calculations of yield in the co-culture. Error bars represent standard deviations.(PDF)Click here for additional data file.S2 FigA. MICs of monoculture- and co-culture- evolved E. coli isolates containing wild-type or mutant prc. B. Monoculture and coculture growth rates of monoculture-evolved E. coli isolates containing wild-type or mutant prc. C. Monoculture and coculture growth rates of co-culture-evolved E. coli isolates containing wild-type or mutant prc. All p-values based on Mann-Whitney U tests. Each data point represents the average MIC for three isolates obtained from a single population. For each species- culture type combination, there are six populations total, and the statistical comparisons represent MIC comparisons between populations with wild type vs. mutant alleles.(PDF)Click here for additional data file.S3 FigA. MIC of mdoG wild-type vs. mutant E. coli isolates. B. Monoculture and co-culture growth rates of mdoG wild-type vs. mutant E. coli isolates. C. MIC of mdoH wild-type vs. mutant E. coli isolates. D. Monoculture and co-culture growth rates of mdoH wild-type vs. mutant E. coli isolates. E. MIC of mdoH wild-type vs. mutant S. enterica isolates. F. Monoculture and co-culture growth rates of mdoH wild-type vs. mutant S. enterica isolates.Each data point represents the average MIC for three isolates obtained from a single population. For each species- culture type combination, there are six populations total, and the statistical comparisons represent MIC comparisons between populations with wild type vs. mutant alleles. (PDF)Click here for additional data file.S4 FigA. MICs of wild-type vs. mutant E. coli with mutations in rne  and proQ . B. MICs of wild-type vs. mutant S. enterica with mutations in acrB/ompR, ramR, , and metL (evolved in co-culture only). C. MICs of wild-type vs. mutant S. enterica with mutations in ompF evolved in monoculture (gold) or co-culture (green). D. MICs of wild-type vs. mutant S. enterica with mutations in yoaE evolved in monoculture (gold) or co-culture (green).Each data point represents the average MIC for three isolates obtained from a single population. For each species- culture type combination, there are six populations total, and the statistical comparisons represent MIC comparisons between populations with wild type vs. mutant alleles. (PDF)Click here for additional data file.S5 FigP = 0.0614 for monocultures, p = 0.3545 for co-cultures, Mann-Whitney U test. Each data point represents the average MIC for three isolates obtained from a single population. For each culture type combination, there are six populations total, and the statistical comparisons represent MIC comparisons between populations with wild type vs. mutant alleles.(PDF)Click here for additional data file.S6 FigError bars represent standard deviation.(PDF)Click here for additional data file.S7 FigA) and ampicillin (B) population. Numbers next to the data points represent population numbers. High-frequency (>50%) genotypes can be found in S1 Table. P-values represent Mann-Whitney U test results.Average MIC of isolates from each evolved rifampicin ((PDF)Click here for additional data file.S1 Code(ZIP)Click here for additional data file.


The application of robotic surgery for rectal cancer is increasing steadily. The purpose of this meta-analysis is to compare pathologic outcomes among patients with rectal cancer who underwent open rectal surgery (ORS) versus robotic rectal surgery (RRS).We systematically searched the literature of EMBASE, PubMed, the Cochrane Library of randomized controlled trials (RCTs) and nonrandomized controlled trials (nRCTs) comparing ORS with RRS.P = 0.13), number of harvested lymph nodes , complete total mesorectal excision (TME) rates  and the length of distal resection margins (DRM)  did not differ significantly between the RRS and ORS groups.Fourteen nRCTs, including 2711 patients met the predetermined inclusion criteria and were included in the meta-analysis. Circumferential resection margin (CRM) positivity (OR: 0.58, 95% CI, 0.29 to 1.16, Based on the current evidence, robotic resection for rectal cancer provided equivalent pathological outcomes to ORS in terms of CRM positivity, number of harvested lymph nodes and complete TME rates and DRM. Colorectal cancer accounts for approximately 10% of diagnosed cancers and cancer-related deaths worldwide each year . SurgicaWe systematically searched EMBASE, PubMed and the Cochrane Library for relevant articles . We used a search strategy with a combination of the following MeSH terms: Da Vinci/robotic/robot-assisted/open, rectal/colorectal/total mesorectal excision. Search algorithms for each database are summarized in We evaluated prospective randomized controlled trials (RCTs) and non-randomized controlled trials (nRCTs) that compared RRS with ORS for rectal cancer; eligible studies with at least one of the following outcomes of interest: CRM positivity, harvested lymph nodes, complete TME and the length of distal resection margin (DRM), were included. Repeat publications of data from the same hospital, absent outcome parameters of interest, review articles, and case reports were excluded.Study selection, evaluation, and data extraction were checked independently by two investigators. Any disagreement was resolved through consultation with a third author. The outcomes of interest were complete TME, CRM positivity, number of harvested lymph nodes, and DRM. For each study, characteristics were extracted if available. If certain outcomes of the included studies were not reported, the corresponding authors were contacted via e-mail. The Jadad score, total score from 0 (poor) to 5 (excellent), was used to assess the quality of RCTs , otherwiP < 0.10, and heterogeneity was quantified using the I2 statistic. A fixed-effect model was used to pool data when statistical heterogeneity was not present. Otherwise, the random-effects model was selected. Subgroup analysis was performed to identify potential heterogeneity [P < 0.05 was considered significant.Weighted mean differences (WMD) with a 95% confidence interval (CI) and odds ratios (OR) with 95% CI were used for the statistical analysis of continuous and dichotomous variables, respectively. Statistical heterogeneity was assessed using the Chi-square test with significance set at ogeneity , 17. A sogeneity . All anaOur initial search identified 324 citations of potentially eligible studies; 67 of these were excluded due to duplication and 226 were removed after screening the titles and abstracts. Overall, 31 potentially relevant articles were retrieved for full-text review  and of tP = 0.13) (P < 0.0001). An assessment based on country/geographic region revealed that the RRS group had a significantly lower CRM positivity rate when compared with the ORS group in Asian countries   . The sub 0.0002) .P = 0.74)  . A subgr = 0.74) .P = 0.83)  . A subgr = 0.83) .P = 0.96) (P = 0.04). However, the RRS group had a significantly shorter DRM than the ORS group when studies were completed after 2014   . A subgr 0.0001) .P = 0.471; Begg’s test, P = 0.373)  .Robotics is an emerging technology with wide acceptance in colorectal surgery . This meCRM positivity is minimized if the principle of TME is followed. Positive CRM positivity can be used as an alternative predictor of local recurrence, while intraoperative accidental perforation can be used as a predictor of adverse oncologic outcomes , 35. OneA recent meta-analysis has identified significantly longer distal margins with the robotic approach compared with the open approach . The preThe length of the DRM is also thought to reflect the surgical quality and affect long-term oncological outcomes. We evaluated the quality of mesorectal excision with DRM parameters and found no significant difference between the two procedures. These results are considered oncologically acceptable , 39. SurThe remote control, along with the placement of wristed instruments in line with pelvic walls, allows the surgeon to perform the rectal resection much more ergonomically , 42. HowThe following limitations of our study need to be considered. First, four clinical trials of this meta-analysis had a sample size of less than 100, which were typically small studies. Second, the substantial heterogeneity of the included studies may reduce the power of the results. Third, two groups were comparable in age, sex, body mass index, and tumor stage. According to our quality assessment, part of observational studies included were prone to risk of selection bias, which have a potential impact on outcomes. However, subgroup analyses did not show significantly different results, supporting the findings of this meta-analysis. This study was conducted at an appropriate time, because enough data was available to use meta-analytical methods which enabled us to provide the most up-to-date information on this topic.In conclusion, our study showed that the robotic resection for rectal cancer provided equivalent pathological outcomes to the open approach in terms of CRM positivity, number of harvested lymph nodes, complete TME rate and DRM. Future, large well-designed RCTs, are warranted to confirm the findings of this study.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


Ottelia acuminate var. crispa is an endangered aquatic herb with extremely narrow distribution. In this study, we assembled the chloroplast genome of this species based on the second-generation high-throughput sequencing. The genome is 157,783 bp in length with a typical quadripartite structure including a large single-copy region (LSC) of 88,294 bp, a small single-copy region (SSC) of 49,379 bp, and a pair of inverted repeats (IRs) of 10,055 bp each. A total of 128 genes were annotated, including 83 protein-coding genes (PCG), 37 tRNA genes, and 8 rRNA genes. The phylogenetic tree shows that O. acuminate var. crispa has a close relationship with the genus Elodea. The chloroplast genome presented here provides a valuable resource to conserve this endangered species. Ottelia acuminate var. crispa (Hand.-Mazz.) H. Li  is an endangered aquatic herb that currently only exists in Lugu Lake in southwest China. As a submerged dioecious plant, water and light environment, climatic conditions, and nutrients are often a factor to it and now have been an endangered species. It was classified in the IUCN Red list as critically endangered species in Ottelia acuminate var. crispa flowering throughout the year could be an important ornamental aquatic plant attract tourists, while its leaves are also of certain edible value. In addition, O. acuminate var. crispa could be used in monitoring water pollution and environmental protection due to its high requirements for water quality environment. However, little is known about the complete chloroplast genome (cp-genome) information of such an endangered species. In this study, we reported the cp-genome of O. acuminate var. crispa. The annotated genome has been submitted to GenBank under the accession number of MT993696 (https://www.ncbi.nlm.nih.gov/nuccore/MT993696).O. acuminate var. crispa individual from Lugu Lake in Yunnan Province, China . Voucher specimen (MNU-PHO-0154) of the species was stored in the Ecological Security and Protection Key Laboratory of Sichuan Province, China. We used the DNA-secure Plant Kit (TIANGEN) to extract the total DNA from samples. The whole-genome sequencing was carried out with the HiSeq X Ten Platform . Finally, we generated about 10 G high-quality base pairs of raw data. The software fast QC  chloroplast genome. The mapped reads were extracted and then used to assemble the genome with the software NOVOPlasty v4.1 , 35 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes.The cp-genome of O. acuminate var. crispa, we reconstructed a phylogenetic tree with other 6 complete chloroplast genomes downloaded from NCBI (O. acuminate var. crispa. The sequences were aligned using the software MAFFT (Katoh and Standley O. acuminate var. crispa is closely related to genus Elodea with strong support (To infer the phylogenetic position of rom NCBI  includin support .O. acuminate var. crispa will not only provide valuable informative resource to facilitate the identification, conservation, and utilization of this extremely endangered species but also provide insightful information to further research on phylogenetic study of Hydrocharitaceae.In summary, the complete chloroplast genomic of


Background: In this era, worldwide interest has been directed towards using natural antioxidants to guard against drug side effects. Saussurea lappa is a famous medicinal plant with many biologically active compounds. Triamcinolone acetonide (TA) is an extensively used glucocorticoid. Hence, this study explored, for the first time, the possible beneficial effects of S. lappa ethanolic extract on TA-induced oxidative damage in the lung and spleen of rats. Methods: Five experimental groups were used: control group, S. lappa-treated group , TA-treated group (40 mg/kg/twice/week I/P), S. lappa + TA co-treated group, and S. lappa/TA prophylactic group. Results: TA exposure significantly induced leukocytosis and neutrophilia. In addition, TA significantly reduced the levels of C-reactive protein, interleukin-12, tumor necrosis factor α, and immunoglobulins. Lung Caspase-3 overexpression and splenic CD8+ downregulation were also noted in the TA group. TA treatment significantly increased malondialdehyde concentration but reduced superoxide dismutase and glutathione peroxidase activities. S. lappa counteracted the TA oxidative and apoptotic effects. The best results were recorded in the prophylactic group. Conclusions:S. lappa has a remarkable protective effect via its anti-inflammatory, anti-apoptotic, and antioxidant capacity. Thus, it could be a candidate as a natural antioxidant to face glucocorticoid’s harmful side effects. Saussurea lappa (S. lappa), family Asteraceae, which is commonly known as Costus. Sesquiterpene lactones such as costunolide, dehydrocostus lactone, and cynaropicrin are the main constituents of S. lappa [S. lappa can be utilized as an elective antioxidant operator in both the medical and food industries [S. lappa roots have been extensively suggested for treatment of inflammation-associated ailments such as asthma, chronic gastritis, rheumatoid arthritis, and bronchitis [S. lappa may be related to the maintenance of lysosomal membranes with an antiproliferative consequence [+ and CD8+ via conjugation with sulfhydryl groups of target proteins [Recently, various studies have highlighted the implication of oxidative stress in adverse side effects associated with drug use ,2. HenceS. lappa . S. lappdustries  via scavdustries . S. lapponchitis . The antsequence . Cynaropproteins .Various health disorders have been associated with drugs and xenobiotics that exist in the human environment ,14. GlucYersinia ruckeri [+ T cells showed less sensitivity to GC-mediated inhibition of mitogen-induced proliferation than CD4+ T cells [The anti-inflammatory activity of TA could be through the inhibition of the number of basal cells in the blood . Additio ruckeri . Human C T cells . Chung e T cells  reported T cells . Repeate T cells .Caspases are protease enzymes, which belong to the cysteine-aspartic acid endo-peptidases family . CaspaseS. lappa, its potency to counteract the TA-induced immunosuppression and oxidative damage of the lungs has not yet been studied. Little is known about the modulatory role of S. lappa on TA anti-inflammatory activity. In addition, apoptotic activities of the active components of S. lappa have been responsible for anticancer activity in cancer cell lines, but little is known about the apoptotic effect of S. lappa on normal cells. Hence, this study was designed to assess the effect of S. lappa ethanolic extract on hematological indices, immunoglobulin levels, and pro-inflammatory cytokines in TA-treated rats. In addition, oxidative stress and lipid peroxidation indices were evaluated. Additionally, the probable underlying mechanisms were investigated by assessing Caspase-3 and CD8+ immune expression in lung and spleen tissues, respectively. Furthermore, the study compared the prophylactic and therapeutic potency of S. lappa ethanolic extract.Despite the earlier reported biological activities of S. lappa roots were purchased from a herbal store in Sharkia governorate, Egypt. The plant was distinguished and verified within the Botany department at Zagazig University . Whole plants were air-dried, processed into a moderately coarse powder, and kept in tightly closed bottles for later use in the extraction procedure. About 100 g of the obtained powder was soaked in 1000 mL of ethanol (70%) at room temperature and soaked for three days. The filtration of this solution occured through Whatman grade-1 filter paper in a funnel under vacuum. After that, the filtrate was subjected to the rotary evaporator to dry and evaporate the liquid under reduced pressure. Crude ethanolic extract of S. lappa roots was collected, which was further lyophilized to a dry powder [y powder . FinallyS. lappa ethanol extract sample. The temperature of the column oven was first held at 55 °C and then raised by 5 °C/min to 250 °C, held for 2 min, then increased to 300 °C at 25 °C/min. The temperature of the injector was kept at 270 °C. A carrier gas of helium was used at a constant flow rate of 1 mL/min. The solvent interval was 4 min, and 1 µL diluted samples were automatically injected by AS3000 Autosampler coupled with GC in the split mode. Electron ionization mass spectra were composed at 70 eV ionization voltages over the range of m/z 50–650 in full scan mode. The transfer line and ion source were set at 280 °C and 200 °C, respectively. The components were recognized by matching their mass spectra and retention times with those of the NIST14 and WILEY 09 mass spectral databases.A GC-ISQ mass spectrometer  with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness) was used for characterization of the components of the TA was obtained in a commercial form  that had 40 mg of TA/mL. It is distributed by Egyptian International Pharmaceutical Industries Co. . Commercial enzyme-linked immunosorbent assays (ELISA) kits  were adopted to estimate IgM, IgG, and C-reactive protein (CRP) . All other chemicals/reagents used were of analytical grade and purchased from .Forty adult male albino rats weighing 150–160 g were acquired from the Laboratory Animal Unit of Veterinary Medicine, Zagazig University, Egypt. Rats were housed in metal cages and kept at room temperature (25±5 °C) with a normal 12 h light/12 h dark cycle. They were fed a typical pellet diet with water ad libitum and adapted to the laboratory conditions for seven days before the experiment started. The Institutional Animal Care and Use Committee of Zagazig University approved the present protocol with the reference number ZU-IACUC/2/F/37/2020.Gp. 1 (control): Each rat was orally administered 1 ml distilled water for three successive weeks.S. lappa): Each rat orally received 600 mg S. lappa ethanolic extract/kg daily via a gastric tube [Gp. 2 (ric tube  for threGp. 3 (TA): Each rat was orally administered distilled water (1 ml/rat) for one week, then intraperitoneally injected with 40 mg/kg of TA twice a week for two weeks .S. lappa extract for two weeks by the same previous doses and routes.Gp. 4 (co-treated): Each rat was orally administered distilled water (1 mL/rat) for one week, then concurrently treated with TA and S. lappa extract for one week, and then was concurrently treated with TA and S. lappa extract for two weeks by the same previous doses and routes. Rats were given their S. Lappa dose via gastric gavage needle in 1 mL distilled water.Gp. 5 (prophylactic group): Each rat received As described in The body weights of all rats in each group were recorded at the start and end of the experiment. Calculation of the spleen and lung indexes were made after sacrificing the animals according to this formula: organ index (mg/g) = organ weight (mg) / body weight (g) .+.After the end of the experiment, blood samples were obtained from the retro-orbital venous plexus of anesthetized rats and were drained into sterile ethylenediaminetetraacetic acid (EDTA)-tubes for hematological analysis. Another blood sample was collected into a sterile centrifuge tube devoid of anticoagulant for serum isolation for the biochemical assay. After sacrificing the animals, spleen and lung specimens were quickly collected and partitioned into two parcels. The 1st parcel was kept at −20 °C until used for determining the oxidant and antioxidant biomarkers. The 2nd portion was preserved in 10% neutral formalin for H&E histopathological and immunohistochemical investigation of lung Caspase-3 and spleen CD8The complete blood count (erythrogam and leukogram picture) was determined by an automated blood cell analyzer  .Serum IL-12 and TNF-α were estimated by a commercial quantitative ELISA measurement  based on Grassi et al.  and BeutMalondialdehyde (MDA) concentration and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were measured in the splenic and lung tissues by ELISA kits of Cusabio Biotech. Co., Ltd. , using the methods depicted by Kakkar et al. , Paglia Specimens of spleen and lung were collected from different groups. The collected specimens were fixed in 10% buffered neutral formalin solution, dehydrated in gradual alcohol (70%–100%), cleared in xylene, and implanted in paraffin. Then, five-micron-thick paraffin sections were prepared and stained with hematoxylin and eosin (H&E) dyes and then inspected microscopically .+ and lung Caspase 3  , respectively, overnight at 4 °C. Sections were incubated with a biotin-conjugated secondary antibody ; Cat. No. sc-2040) for 20 min at 32 °C after three washes with PBS, then incubated with horseradish peroxidase (HRP)-labelled streptavidin. The antibody binding was determined using a substrate/chromogen (diaminobenzidine) reagent and Mayer’s hematoxylin. The cellular reaction appeared as brown granules inside the nucleus of epithelial cells, the staining intensity of the samples was evaluated as mild, moderate, or strong and scored by calculating the positive cells/image percentage by a 5-point scale as follows: + = < 10% (negative to weak expression), ++ = 10%–25% (mild expression), +++ = 26%–50% (moderate expression), ++++ = 51%–75% (strong expression).Spleen and lung specimens were prepared for immunohistochemistry using the avidin-biotin- peroxidase (ABC) method . To deacp < 0.05), n = 5/group.All data were expressed as the mean ± standard error (SE) and were statistically analyzed using one-way ANOVA followed by Duncan’s posthoc test for multiple comparisons, with the help of the SPSS software program . The significant differences were detected at  in the weights of the S. lappa-treated group compared to the control group  was detected in the prophylactic group.Estimation of the bodyweight at the start and after the end of the experiment showed a significant increase  elevation in the RBCs count, Hb concentration, and PCV level were recorded following TA injection compared to control rats. However, S. lappa treatment either in combination with TA or as a prophylactic dose minimized the elevation of erythrogram elements evoked via a TA challenge in a significant manner (p < 0.05), which was more prominent in the protective group.Initially, no significant difference in the erythorgram elements including red blood cells (RBCs), hemoglobulin (Hb), and packed cell volume (PCV) in the S. lappa-treated group relative to the control group. However, no significant changes in the counts of other leukocyte types were recorded. On the other hand, significant (p < 0.05) leukocytosis and neutrophilia were obvious in the TA-exposed group compared to the control group. However, a significant reduction in the count of lymphocytes, monocytes, eosinophils, and basophils was evident in the TA-exposed group compared to control rats. Meanwhile, the S. lappa use with TA in both prophylactic and treated groups resulted in a significant enhancement in these indicators in comparison with the TA-only-exposed group.Concerning leukogram findings, a significant increase in the total leukocytic count (TLC) concomitant with lymphocytosis was apparent in the S. lappa ethanolic extract oral administration for three weeks significantly depressed the release of pro-inflammatory cytokines  and CRP relative to the control group. Moreover, a significant boost of the immunoglobulin levels (IgG and IgM) was evident in the S. lappa orally administered rats relative to the control group.As shown in S. lappa ethanolic extract application in both prophylactic and therapeutic groups significantly restored the levels of the depleted immunoglobulin. Still, it did not significantly alter IL-12 and TNF-α levels compared to the TA-only-exposed group. However, the S. lappa administration in the prophylactic group significantly reduced the CRP level relative to the TA-only-exposed group.On the other hand, TA administered rats showed a significant reduction in IL-12, TNF-α, CRP, IgG, and IgM levels relative to the control group. In contrast, the S. lappa ethanolic extracts for three weeks significantly (p < 0.05) increased the activities of antioxidant enzymes (SOD and GPx) in lung and splenic tissues compared to the control group. However, significant depression of the lipid peroxidation marker MDA was evident in S. lappa-treated rats relative to control rats.As shown in p < 0.05) reduction in the SOD and GPx content in lung and splenic tissues with an elevation of MDA concentration relative to the control group. Moreover, S. lappa showed a remarkable protective role against TA-induced oxidative stress through partial restoration of these enzyme levels near to the control values.However, intraperitoneal injection of rats with 40 mg TA/kg two times weekly for 14 days induced a significant  and cytotoxic (CD8+) populations were decreased in the blood of mice that were I/P injected with dexamethasone (25 mg/kg) for three days.In contrast, a significant drop in the serum levels of CRP, IL-12, TNF-α, IgG, and IgM indicated the capacity of TA to suppress the cells innate and adaptive immunity by inhibiting the synthesis of pro-inflammatory cytokines. CRP is an acute-phase protein generated in the liver, described as an inflammatory effector molecule. It may be one of the links between nonspecific innate immunity and specific clonal immunity because of its capacity to trigger the classical cascade that mediates phagocytosis and improves antigen presentation . Moreoveg et al.  indicateS. lappa. This immunostimulant activity of S. lappa may be ascribed to the several bioactive compounds present in the extract based on the GC-MS characterization, comprising dehydrocostus lactone, dihydrodehydrocostus lactone, caryophyllene oxide, saussurea lactone, costunolide, and beta-costol. Active sesquiterpenes lactones, including dehydrocostus lactone, dihydrodehydrocostus lactone, and costunolide, were formerly isolated from S. lappa methanolic extracts. These bioactives have been known to have beneficial immunomodulatory activities. For instance, dehydrocostuslactone and costunolide showed their immunomodulatory actions through inhibiting the killing activity of cytotoxic T lymphocytes by inhibiting the rise in tyrosine phosphorylation in response to the cross-linking of T cell receptors [S. lappa hydroethanolic extract in mice. Regarding the outcome of S. lappa administration on TA anti-inflammatory activity, in the S. lappa prophylactic group, there was a nonsignificant trend toward decreased release of the pro-inflammatory cytokines TNF-alpha and IL-12. However, a significant reduction in the CRP level was apparent in the prophylactic group compared to the TA-only-treated group. These results could suggest that S. lappa administration could potentiate the TA anti-inflammatory activity to a moderate extent in the prophylactic application. Similarly, another plant compound named Ginsenoside Rh1 potentiated dexamethasone’s anti-inflammatory effects in a collagen-induced arthritis mouse model [In contrast, significant restoration of the immunoglobulin titer was recorded in TA-exposed rats that received eceptors . These feceptors  that repse model .S. lappa ethanolic extracts for three weeks significantly augmented the activities of antioxidant enzymes (SOD and GPx) in lung and splenic tissues but depressed MDA accumulation, a lipid peroxidation biomarker. These results are in harmony with the results of Abdel-Rahman et al. [Costus pictus, which belongs to the same family as S. lappa (Costaceae), that efficiently prevented ROS generation, regulated GPx activities, and inhibited MDA formation. In addition, Benedetto et al. [S. lappa to donate electrons to reactive radicals, converting them into more stable and unreactive species.In the current study, the oral administration of n et al. . In thisn et al.  confirmeo et al.  demonstrConversely, a significant decrease in the concentrations of SOD and GPx activities but an increment in the MDA level was evident in both lung and splenic tissues of animals exposed to TA. Such changes could be a result of enhanced radicals generation during TA metabolism that overwhelms the natural antioxidant system in the body leading to oxidative damage and cell injury. These findings are compatible with Yi et al. , who revS. lappa pre- or concurrent administration in TA-exposed rats significantly reversed the restored antioxidant enzyme activity and suppressed the lipid peroxidation. In addition, activities of CAT, SOD, and GST enzymes were enhanced in deltamethrin-intoxicated animals fortified with aqueous extracts of S. lappa (300 mg) [S. lappa extract offered significant safeguards against toxicant-induced depletion of marker enzymes and oxidative stress to an extent similar to that of the standard natural antioxidant α-tocopherol. This effect may possibly be related to the decrease of membrane damage and membrane fluidity because of its high content of flavonoids, phenolic, steroids, and chlorogenic acid [S. lappa’s major bioactives, such as dehydrocostuslactone and costunolide, have been reported to play some crucial roles as antioxidant agents via conjugation with target proteins mercapto (SH)-groups to intervene in several main biological activities in cells [S. lappa-treated group may be due to their protective effect from the harmful effects of ROS-mediated lipid peroxidation of tissue macromolecules [Interestingly, the (300 mg) . Saleem (300 mg)  found thnic acid . In addiin cells ,98. The olecules .S. lappa and its major constituent dehydrocostus lactone have been studied in various types of cancers such as neuroblastoma [S. lappa has a different mode of action toward normal cells. For instance, in the study of Zhao et al. [S. lappa’s main bioactives significantly reduced the Caspase-dependent apoptotic pathway in neuronal cells, probably linked to the free radical scavenging activity. Besides, other natural products showed selective apoptotic activity [S. lappa-treated rats, suggesting its anti-apoptotic activity. Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key nuclear proteins [S. lappa apoptotic activity in normal cells could provide a basis on which further research in cancer treatment through S. lappa can be executed.The antitumor activity of blastoma , hepatocblastoma , leukemiblastoma , lung cablastoma , and problastoma . The plao et al. , S. lappactivity ,106. Foractivity . In the proteins . Additioproteins ,110. ThuS. lappa pre- or concurrent administration in TA-exposed rats significantly suppressed Caspase- 3 immunoexpression, which could be linked to their antioxidant activity.On the other hand, overexpression of Caspase- 3 as an apoptotic marker in the lung tissue of the TA-administered group was confirmed microscopically in the current study. This is in agreement with Harada et al.  who showS. lappa extracts either before or in combination with TA administration significantly counteracted TA-evoked weight reduction. Such improvement could be the consequence of the improvement of the immune functions and antioxidant activities in rats.Collectively, the oxidative stress and immunosuppression induced by TA were accompanied by a remarkable decline in body weight gain, which is clear from the significant reduction in the spleen and lung indexes recorded in this group. These findings are in harmony with the previous studies of Kumari et al.  and Ling50 of S. lappa in Swiss Albino mice to establish its safety margin and verified that no death or signs of toxicity were observed in animals treated with the extract up to doses of 2000 mg/kg b.wt., which established its safety for use in further pharmacological screening. Although the published evidence to date and traditional use in various countries support the safety of S. lappa, more investigations are warranted on bioavailability, pharmacokinetics, physiological pathways, and the importance to human health [Importantly, the safety profile of any herbal medicine is an important factor that should be considered to determine its therapeutic utility . In thisn health .S. lappa oral administration could efficiently rescue TA-induced immunosuppression. In addition, S. lappa could moderately augment TA-anti-inflammatory activity. Additionally, S. lappa could efficiently combat the TA-induced oxidative stress and apoptotic consequences in lung tissues. In addtion, the reclamation from the baleful effects of TA is highly dependent on the method of S. lappa supplementation. S. lappa application as a prophylactic regime achieved better results than as a therapeutic one.Taken together, the combined biochemical, histopathological, and immunohistochemical results suggested that the


Progress in genome mining has recently shown that Streptomyces species encode for many biosynthetic gene clusters which are mostly unexplored. Here, we selected three Actinomycetes species for whole genome sequencing that are known to produce potent thiopeptide antibiotics. Streptomyces actuosus biosynthesizes nosiheptide, Streptomyces sioyaensis produces siomycin, and Actinospica acidiphila is a member of the Actinomycete subfamily. Bioinformatic analyses demonstrated diverse secondary metabolomes with multiple antibiotic-encoding gene clusters. Detailed mass spectrometry analysis of metabolite extracts verified the active expression of nosiheptide and siomycin from S. actuosus and S. sioyaensis while fractionation of the bacterial extracts and subsequent challenge against Staphylococcus aureus demonstrated potent antibiotic activity of fractions containing these compounds. Whole genome sequencing of these species facilitates future bioengineering efforts for thiopeptides and characterization of relevant secondary metabolites. In 2015More than 75 % of antibiotics are derived from natural products ,7. NaturIn the past ten years, a renaissance has occurred in natural product discovery, driven by knowledge of biosynthesis, bioinformatics, and whole genome sequencing ,11. SincStreptomyces species and both Illumina and PacBio sequencing have been used for WGS of these bacteria. Whereas Illumina is economical and gives short reads, PacBio is relatively expensive but provides long reads. Long reads enable the assembly of high-quality genomes with few contigs and bypasses the inaccuracies generated by the high G + C content of Streptomyces’ genomes 2+  with a m/z of 1222.1113 [M+H]+ . MSe spectra analysis showed fragments with m/z values of 771.0480 and 1206.1369 Da, corresponding to the internal intersection of the macrocycles with the amino tail and a single fragment from the tail of nosiheptide, respectively . After fractionation, nosiheptide was also found in fractions 8–10 which eluted with a mixture of 50 % EtOAc: 50 % methanol and 100 % methanol from the column, matching its polarity .The 3.6Staphylococcus aureus ATCC 29213 (S. aureus) in a growth curve assay. When S. aureus was challenged with the fractions of S. actuosus, fraction 6 resulted in a significant growth delay while fractions 8–10 resulted in complete bactericidal activity . This result demonstrates the importance of the new approach to natural product discovery to identify antibiotics encoded in the genomes but not produced by these species under laboratory conditions. Together with the LCMS and bioassay analyses, each species was predicted and shown to produce antibiotics with potent activity against the common opportunistic pathogen S. aureus.The fractionated culture extracts were used to challenge the opportunistic pathogen activity h. This daction 8 i. By LCMaction 8 g, confiractivity j, despit4Streptomyces species as shown by sequencing technology analysis of deposited genomes at NCBI .Access to high quality whole genome sequences is a requirement for the new natural product discovery pipeline. Here, we sequenced three Actinomycetes that are known producers of thiopeptides, or in the family thereof, which not only revealed the BGCs for these thiopeptides but also a large amount of novel and uncharacterized BGCs  and S1. S. actuosus is known to produce nosiheptide and an avermipeptin analogue [S. aureus. In line with this, we observe that polar fractions of S. actuosus inhibit growth of S. aureus  of S. sioyaensis DSM 40032 shows 8 uncharacterized BGCs spread over 289 contigs [ contigs . In our  contigs . There i contigs . The com contigs . These t contigs ; however contigs ,75. ChemS. azureus and S. cyaneus CGMCC 4.1671 are known producers of thiostrepton [S. cyaneus B-2296T revealed that the strain we received was misannotated as S. cyaneus and should be reclassified as A. acidiphila B-2296 . A. acidiphila strains do not encode for any thiopeptides, but antiSMASH analysis shows the presence of 17 secondary metabolite BGCs, 9 of which encode metabolites with known antibiotic activity (Table S2). The whole genome relatedness of all reported Actinospica species against A. acidiphila B-2296 was analyzed by ANI . Interestingly, A. acidiphila B-24431 demonstrated the highest percentage of similarity to the A. acidiphila B-2296 species reported here with ∼95 % similarity; however, a relatively high percent similarity is observed across all Actinospica species. In parallel, a low percent similarity from Streptomyces azureus B-2655 and Streptomyces cyaneus CGMCC 4.1671 was observed. Coupled to the ANI analysis is A. acidiphila B-2296, this data suggests the presence of numerous Streptomyces species with a high percentage of similarity to the Actinospica sub-family.A third member of the unique thiopeptides harboring both RiPP and NRPS character in their BGCs is thiostrepton. strepton ,76,77; hStreptomyces species with identical 16S sequences show very different secondary metabolomes [S. sioyaensis, S. actuosus, and A. acidiphila as reservoirs for multiple classes of natural products, some with potent antibiotic activity. With high quality genomes, our data facilitates a genomic approach to natural product discovery. Future studies will focus on development of the known thiopeptide antibiotics into clinically relevant compounds as well as expand the reservoir of potential new antibiotics not previously characterized from these organisms.The advent of affordable whole genome sequencing combined with a better understanding of biosynthesis, allows for computational mining of bacteria for natural product production . The Actabolomes . Togethe5Streptomyces sioyaensis, Streptomyces actuosus, and Actinospica acidiphila revealed numerous secondary metabolite biosynthetic gene clusters. Liquid chromatography mass spectrometry coupled to antibiotic activity assays verified the production of potent thiopeptides siomycin and nosiheptide, produced by S. sioyaensis and S. actuosus, respectively. These results expand the field of natural product discovery and provide a genomic platform in which to bioengineer relevant new antibiotics to combat the antimicrobial resistance crisis.Whole genome sequencing and bioinformatic analyses of Haley M. Majer: Investigation, Data curation, Formal analysis, Writing - original draft, Visualization, Software, Validation. Rachel L. Ehrlich: Data curation. Azad Ahmed: Investigation. Joshua P. Earl: Data curation. Garth D. Ehrlich: Funding acquisition. Joris Beld: Supervision, Funding acquisition, Conceptualization, Methodology, Software, Data curation, Writing - review & editing.The authors report no declarations of interest.