Exechonella vieirai, and five, 17 and 18 introns were found in the partial mitogenomes of Parantropora penelope, Discoporella cookae and Cupuladria biporosa, respectively. Intron-encoded protein domains reverse transcriptase and intron maturase (RVT-IM) were identified in all species. Introns in E. vieirai and P. penelope had conserved Group II intron ribozyme domains V and VI. Conserved domains were lacking from introns in D. cookae and C. biporosa, preventing their further categorization. Putative origins of metazoan introns were explored in a phylogenetic context, using an up-to-date alignment of mitochondrial RVT-IM domains. Results confirmed previous findings of multiple origins of annelid, placozoan and sponge RVT-IM domains and provided evidence for common intron donor sources across metazoan phyla. Our results corroborate growing evidence that some metazoans with regenerative abilities  are susceptible to intron integration, most likely via horizontal gene transfer.Animal mitogenomes are typically devoid of introns. Here, we report the largest number of mitochondrial introns ever recorded from bilaterian animals. Mitochondrial introns were identified for the first time from the phylum Bryozoa. They were found in four species from three families (Order Cheilostomatida). A total of eight introns were found in the complete mitogenome of  Since this origin, the various evolutionary trajectories of eukaryotes have produced mitochondrial (mt) genomes (mitogenomes) that vary substantially in their sizes, gene content, genetic code, organization and physical shape2. Plant, fungal and other non-metazoan eukaryote mitogenomes range in size from 12 to 236 Kb, 66–11.3 Mb and 34–113 Kb, respectively and harbour a large proportion of non-coding DNA, including introns. In contrast, bilaterian mitogenomes are typically circular, streamlined molecules of approximately 16 kb (range: 11–50 Kb), which are typically devoid of introns3. The lack of introns in bilaterian mitogenomes has been attributed to the relatively high mutation rate of animal mtDNA, which can be ~ 9 to 25 times faster than that of corresponding nuclear DNA4. Introns, like all non-coding DNA, provide a substrate for potentially harmful mutations in the presence of high mutation rates, and are therefore hypothesised to have been purged from most animal mitogenomes5.A crucial step in early eukaryote evolution was the origin of mitochondria, which arose by incorporating α Proteobacteria endosymbionts as cellular organelles9. Group II introns occur in bacterial, archaeal and organellar genomes , but are not found in nuclear genomes11. Both types are self-splicing, mobile ribozymes, but they are distinguished by their splicing mechanisms. Group I introns use external guanosine nucleotides as cofactors, whereas Group II introns use a mechanism more akin to that found in spliceosomal introns8 and are believed to be precursors of eukaryote spliceosomal introns, retroelements and spliceosome components12. Both types frequently harbour intron-encoded proteins (IEPs) that facilitate intron mobility; Group I introns typically encode homing endonucleases, whereas Group II IEPs include reverse transcriptase (RVT) and intron maturase (IM) domains12. In terms of their secondary structures, Group II introns form six domains (DI—DVI) that radiate out from a central loop. Based on the intricacies of their secondary structures, five Group II introns have been identified 14. Conversely, Group I introns typically form secondary structures of P1–P10 stems15.In addition to spliceosomal introns, which occur in the nuclear genomes of eukaryotes and require ribonucleoprotein complexes called spliceosomes to facilitate their excision from precursor mRNA, there are two other types of introns. Group I introns occur in bacterial, archaeal, viral and organellar genomes as well as some eukaryote nuclear genomes22 and Placozoa26, whereas Cnidaria are only known to harbour Group I introns29. Conversely, in bilaterians, mitogenome introns are apparently very rare, but Group II introns have been recognised in the cox1 gene of annelids Nephtys (Phyllodocida)30, Glycera fallax, G. unicornis (Phyllodocida)31, Decemunciger sp. (Terebellida)32 and the myzostomid Endomyzostoma sp.33. Furthermore, high-throughput DNA sequencing has revealed additional introns of unknown type(s) in other protein-coding genes (PCGs) of Decemunciger sp. (nad1 and nad432) and the mollusc Cucullaea labiata (Arcoida) (cox135).Although typically not found in metazoans, both intron types have been recorded in the mitogenomes of non-bilaterian phyla Porifera36, are modular invertebrates, found worldwide in aquatic habitats, including freshwater bodies, shallow coastal waters, and the deep sea38. Almost exclusively colonial, they form encrustations on a variety of substrates, construct erect, three-dimensional structures or produce free living disks40. There are three classes of bryozoans. The least diverse of these is the exclusively freshwater class Phylactolaemata with ~ 86 Recent species41. The Phylactolaemata forms the sister group to all remaining bryozoans42. The class Stenolaemata, whose only surviving order is the exclusively marine Cyclostomatida, constitutes ~ 543 Recent species41 and they form the sister group to the class Gymnolaemata42. Although predominantly marine, the Gymnolaemata also includes a few brackish and freshwater species. It is the most speciose class with their ~ 5240 Recent species, of which ~ 319 belong to the order Ctenostomatida and ~ 4921 belong to the order Cheilostomatida41; the Cheilostomatida nest within a paraphyletic Ctenostomatida42. Apart from phylactolaemates, which can disperse via asexually produced propagules called statoblasts, and some gymnolaemates which can form resting structures called hibernacula43, bryozoan colonies are typically established by sexually produced larvae, which settle and metamorphose into the founding module (zooid) of the colony, the ancestrula37. The ancestrula then buds the adjacent zooids, which in turn continue the colony growth via asexual budding of further zooids44. Reparative growth of damaged colony parts frequently occurs45, and some species propagate largely via colony fragmentation and subsequent regeneration46.Here, we reveal the widespread occurrence of introns in the mitogenomes of taxa of the lophotrochozoan phylum Bryozoa. Bryozoans, whose fossil record goes back as far as the Cambrianhttps://www.ncbi.nlm.nih.gov/), none of which contain introns . As part of a wider study sequencing hundreds of mitogenomes from species of the bryozoan order Cheilostomatida, genome-skimming has revealed the occurrence of introns in multiple PCGs in single mitogenomes, multiple introns within single genes, as well as in intergenic regions. Here, we document introns discovered in the mitogenomes of four cheilostome bryozoans: two encrusting species – Parantropora penelope  and Exechonella vieirai  and two free-living species from the family Cupuladriidae , Cupuladria biporosa (AW817) and Discoporella cookae (AW3739)  and Contig B . Missing genes were: PCGs atp6 and nad4 and tRNAs trnA, trnC, trnE, trnH, trnR, trnT, trnV and trnW; genes nad2 and nad6 were incomplete at their 5’ ends. For diagrammatic representation of gene order of mt fragments, see Fig. Numbers of raw Illumina reads per species, contig sizes and coverage, and GenBank accession numbers are given in Table A large number of introns were found within several PCGs, sometimes multiple introns per gene, across multiple species of cheilostome bryozoans Fig. . We elimE. vieirai harboured single introns in three genes , cytb (PP-cytb) and nad5 (PP-nad5) and two introns in cox1 (PP-cox1-i-ii)  Fig. . FurtherC. biporosa and D. cookae Although there are substantial similarities in the intron distribution across these two species , D. cookae harboured three introns in nad5, whereas C. biporosa harboured only two introns in this gene. A further two introns were found in nad4 in C. biporosa, but due the partial nature of the D. cookae mitogenome, nad4 was missing from its contigs, thus, no inferences can be made about shared introns for this gene. Furthermore, both species have one intergenic region that harbours an open reading frame containing RVT and IM domains  IEP with RVT-IM domains, b) conserved Group II ribozyme domains V and VI, c) Group II intron-typical start and stop motifs (except the non-standard start motif in PP-cox1-ii). All introns in these two species finish on YAY, which is a typical Group IIA end motif, versus RAY, which is typical for Group IIB introns13. However, the secondary structures of the two IEP-containing introns 50. Although the secondary structure drawing of EV-cox1 , AU-rich with a base bias of U > A > G > C, have degenerate Group II intron-like boundaries , and lack any consistent secondary structure54, although they have been shown to have a structure resembling Group II intron domain VI55. Regarding the cupuladriid introns, their lengths, although shorter than introns in the non-cupuladriids, exceed the typical Group III intron size. Intron sizes range from 221–519 bp (average 272 bp) and 240–397 bp (average 273 bp) in C. biporosa and D. cookae, respectively . This was found toward the 5’ end of intron CB-cox3-ii (AACUAAG). Similarly, only two of the 15 introns in D. cookae for which the 5’ ends were available, had the degenerate Group III intron motif towards their 5’ ends: DC-nad5-i (UGACUUUUG) and DC-nad4L (GUAUG); nucleotides not emboldened are currently considered parts of the introns as they are the nucleotides immediately following the preceding exons. The degeneracy of the 3’ end motif means that it is present too frequently to make any meaningful inferences. Lastly, the secondary structure drawings frequently show a stem towards the 3’ end  of bryozoan mitochondrial introns in an evolutionary context, a phylogeny of RVT-IM domains with representatives from across the tree of life was constructed. The following description of phylogenetic interrelationships of RVT-IM domains Fig.  focussesCupuladria biporosa and Discoporella cookae is strongly supported (91% bootstrap support [bs]). They formed strongly supported Clade I together with RVT-IM domains from polychaete taxa Glycera unicornis , Glycera fallax  and Nephtys sp. (94% bs); this close association of annelid terminals and the non-monophyly of the two tandemly repeated Glycera fallax FS14 intron copies I1 and I2 (see Clade IV description below for copy I1 position) was also found by Richter et al.31. This clade was sister to a paraphyletic assemblage of fungal sequences that also included the diatom Halamphora calidilacuna, and a strongly supported clade (99% bs) composed of RVT-IM domains of rhodophyte genera Pyropia, Bangia and Neoporphyra and the sponge Axinella verrucosa (copy 96620), which formed the moderately supported sister (63% bs) to a clade of five placozoan sequences . As in Signorovitch et al.24 and Huchon et al.20, an independent origin of Placozoa sp. strains BZ49  and BZ2423 + Trichoplax adhaerens  was found in our analysis. Furthermore, this close association of the abovementioned rhodophyte genera Bangia and Pyropia, placozoans and sponges is in agreement with findings by Huchon et al.20.The common origin of RVT-IM domains in the two cupuladriids 26, Placozoa sp. strain BZ242324 and Trichoplax adhaerens23 nested in a moderately supported clade (67% bs) with charophyte Coleochaete, chlorophyte Ulva and fungi Auricularia and Termitomyces. In the wider context of a well-supported node (80% bs) that included members of Rhizaria, Fungi, Chlorophyta, Charophyta, Marchantiophyta and Bryophyta as well as placozoan Clade III, our results, although using a more elaborate taxon sampling, broadly agrees with results obtained by Huchon et al.20. All genera recovered as close relatives of Clade III in Huchon et al.20, i.e. Marchantia, Treubia, Klebsormidium, Chlorokybus and Schizosaccharomyces were also found to be part of this clade. A close association  of Trichoplax adhaerens with Marchantia, Chlorokybus and Schizosaccharomyces was also found by Vallès et al.30. A novel finding of the present study, as a result of a broader taxon sampling, is the strongly supported (89%) sister-group relationship of placozoan Clade III with Ulva compressa.A second clade of placozoan sequences composed of Placozoa sp. haplotypes H2, H17Exechonella vieirai was placed with strong nodal support (82% bs) in a clade with polychaete taxa Glycera fallax  and Decemunciger sp. The sister to Clade IV was formed by the tracheophyte Selaginella, but support for this was weak (26% bs). Similarly, neither Richter et al.31 nor Bernardino et al.32 were able to determine a supported position for Glycera fallax , although it formed a weakly supported clade with RVT-IM domains from Marchantia and land plants  in both of their analyses. Considering that both used the alignment by Zimmerly et al.56 as a starting point , this is to be expected. In contrast to the present analysis, in Bernardino et al.32 the RVT-IM copies of Decemunciger sp. formed a strongly supported clade with Nephtys sp. (91% bs), nesting in a weakly supported clade with Glycera unicornis (FS15 isolate31) and Glycera fallax . This difference in topology might be explained by the high sequence divergence in Decemunciger sp. copies, as evidenced by the long branch leading to the clade of the three Decemunciger sp. copies in Bernardino et al.32 and in Fig. Exechonella vieirai copy, might have led to this difference in topology.Clades I-IV, there were two terminals that did not group with any other metazoan RVT-IM copies. The first, Axinella verrucosa (copy 114120), formed a weakly supported clade (40% bs) with rhodophyte genera Hildenbrandia, Ahnfeltia, Pyropia, Bangia, Neoporphyra , all of which also formed the sister group in Huchon et al.20. In addition, in the present analysis, this rhodophyte clade also included Paralemanea, Grateloupia. This A. verrucosa + Rhodophyta clade formed the sister group with strong support (95% bs) to a strongly supported clade (99% bs) composed of eustigmatophycean Nannochloropsis, a gamma-proteobacterium, and diatom genera Psammoneis, Pseudo-nitschia, Thalassiosira and Cylindrotheca; in Huchon et al.20 this corresponding sister group was formed of Thalassiosira and Chattonella. Thus, there is broad agreement with our analysis and that by Huchon et al.20, except that our broader taxon sampling added additional genera. We also observed, judging by the associated taxa in our analysis and that by Huchon et al.20, the two intron copies of Axinella verrucosa  came out in switched positions in ours and their topologies. We suspect that this could either be due to a mislabelling of Fig. 7 in Huchon et al.20 or a mislabelling of their GenBank accession records. The position of the second terminal, Parantropora penelope, could not be unambiguously resolved as it nested on a very long branch in a poorly supported clade (16% bs) together with representatives of Fungi, Haptophyta, Bacillariophyceae, Chlorophyta, Charophyta and Marchantiophyta.In addition to Exechonella vieirai and Parantropora penelope. Furthermore, we infer a separate but possibly shared origin in the two cupuladriid taxa Cupuladria biporosa and Discoporella cookae . Moreover, the monophyly of bryozoan and annelid RVT-IM domains  and vertical transmission58. However, much evidence points to intron insertion via HGT from microbial or algal donors. In sponges, putative intron donors include fungi22, rhodophytes, diatoms and raphidophytes20. Additionally, placozoans have been proposed as possible intron donors in sponges21. Our analysis confirmed a close association of RVT-IM domains in sponges and placozoans (Clade II), as well as a close relationship of both with red algae  which is consistent with their particular reproductive life history strategy. The cupuladriid family are all free-living bryozoans that rely heavily on clonal propagation by fragmentation and regeneration of their disc-shaped colonies (e.g.46). This mode of reproduction often results in zooids being split open60 which could provide intron donors easy access to undifferentiated somatic cells. Discoporella cookae has undergone rates of clonal propagation exceeding 95% for at least 8 million years61 and clonal propagation in free-living bryozoans extends into the Cretaceous62. More generally, bryozoans are a rich source of bioactive compounds, many of which are likely produced by microbial symbionts64. Although the associated microbial species are mostly unknown, detailed studies on some bryozoan species have shown their colonies to harbour symbiotic bacteria on the surfaces of rhizoids65, intercellularly in the pallial sinus of their larvae and across larval surfaces66, as well as in tissue strands (funicular cords) that connect colony modules to one another67. Furthermore, bryozoans are often associated with microbial films68 and have been found to be infected by fungal species69. These close relationships between bryozoan hosts and microbes may have facilitated the acquisition of introns in this group.A commonality of the metazoan taxa harbouring Group II introns is their ability to bud and regenerate. The idea that budding and regeneration ability may favour intron transmission via somatic cells/tissues was already formulated by Szitenberg et alE. vieirai and P. penelope showed that the five copies of the latter formed a monophyly with strong nodal support . Thus, there is only evidence for a species-specific common ancestry of IEP-less Group II introns in P. penelope. This suggests that these introns may have either been inserted multiple times from the same type of source organism or that the introns self-propagated within the mitogenomes post initial insertion. Conversely, phylogenetic analyses of the IEP-less introns of the two cupuladriid taxa was completely unresolved and showed no grouping by species  and five, 18 and 17 in the incomplete mitogenomes of P. penelope, C. biporosa and D. cookae, respectively. This is the largest number of introns ever recorded in bilaterians . Furthermore, RVT-IM domains have only ever been found in cox1 in metazoans . Although this was also the case in our E. vieirai and P. penelope mitogenomes, open-reading frames with RVT-IM domains were found in intergenic regions in C. biporosa and D. cookae. Thus, this is the first time that these domains have been found residing outside of cox1 introns in metazoans.There are several features in the bryozoan mitogenomes investigated here that distinguish them from other bilaterian and metazoan intron-harbouring mitogenomes. In the context of bilaterians, our observed intron frequency in the four species of Bryozoa is unprecedented: eight in 31, increased genome sequencing has revealed more Group II introns within the Bilateria, with more likely to be uncovered in the future. Nevertheless, for now at least, the frequency of mitochondrial introns in bryozoans is exceptional when compared to other bilaterians. This provides an unparalleled opportunity for bryozoans to perhaps become not only a model for studying introns but also bilaterian mitogenome architecture overall, challenging our understanding of their function and evolution. Initial observations of intron absence versus presence in a broad phylogenetic context point towards a random acquisition process, rather than one guided by shared common ancestry (unpublished data). However, future work that dissects the process of intron gain and loss amongst closely related species ought to be explored. Furthermore, seeing that HGT from microbial donors is a likely mechanism for intron integration, studying the intraspecific distribution and variability of introns could provide interesting insights into their heritability and persistence.As foreseen by Richter et al.Parantropora penelope  for E. vieirai, P. penelope and D. cookae. Sequencing was performed by Novogene (HK) Company Limited on the Illumina HiSeq 4000 platform  using 2 × 150 bp paired-end sequencing.Total genomic DNA (gDNA) was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following manufacturer’s instructions. Double stranded (ds) DNA concentration was quantified using a Qubit™ fluorometer using either the Qubit™ dsDNA BR (Broad Range) or dsDNA HS (High Sensitivity) assay kits. Dual-indexed libraries were prepared using the TruSeq DNA Nano Library Prep Kit  for 70 and assembled de novo using SPAdes 3.13.071 with k-mer sizes of 33, 55, 77, 99 and 127. Contigs of putative mitogenomes were identified by conducting blast searches72 against a local custom database of reference bryozoan mitogenomes in Geneious 11.1.4 (https://www.geneious.com). Candidate mt contigs were further verified by blastn searches against the NCBI database (https://blast.ncbi.nlm.nih.gov).Paired-end reads were trimmed using Trimmomatic Version 0.39http://mitos.bioinf.uni-leipzig.de/)73. PCG boundaries (start/stop codons) were refined manually in Geneious following alignment with curated sets of reference bryozoan sequences using TranslatorX (http://translatorx.co.uk/)74. Mitogenomes were circularised where possible using the Repeat Finder plug-in in Geneious. Assembly quality and read coverage were assessed by reference mapping of trimmed reads to mitogenome contigs under strict settings in Geneious. The settings used were as follows: allow gaps = maximum 1% per read, maximum gap size = 3, minimum overlap = 50, minimum overlap identity = 95%, word length = 18, index word length = 13, ignore words repeated more than 12 times, maximum mismatches per read 2%, maximum ambiguity = 4.Mitogenome contigs were annotated using MITOS (https://www.ebi.ac.uk/Tools/st/emboss_transeq) and searched using blastp (https://blast.ncbi.nlm.nih.gov) to identify additional exons. In cases where blastp failed to identify matches, the amino acid translation was scanned for conserved motifs as gleaned from reference alignments.Introns were identified through the observation of fragmented exons. Exons were knitted together in light of alignments with non-intron containing reference data. In cases where exons had not been identified by MITOS, nucleotide data in between exons were translated into amino acids using EMBOSS Transeq (http://pfam.xfam.org/75) and blastp. No RVT-IM domains were initially found for Cupuladria and Discoporella. However, blastx searches of the complete mt fragments against the ucalgary.ca database  identified intergenic regions that were subsequently confirmed as RVT-IM domains by blastp and Pfam searches. Significant as well as insignificant Pfam results were considered. Conserved Group II ribozyme domains were identified using Rfam (https://rfam.xfam.org76). Intron boundaries were refined by searching for conserved 5’ (GUGYG) and 3’ terminals ([Y]AY)13.In order to identify intron-encoded RVT-IM domains, intron sequences were translated into amino acids using EMBOSS Transeq from all six reading frames. Translated sequences were subjected to searches using Pfam 33.1 , with a final extension step at 72 °C for 10 min (see Supplementary Table ann). Two PCRs (EV-cox1 and PP-cox1-i) failed and were repeated with Takara Long-range PCR kit (Takara Bio Inc.). Total reaction volume was 50 μl, using 0.5 μl enzyme, 5 μl buffer, 8 μl dNTPS, 2 μl of 10 μM of each primer and 4 μl gDNA. PCR cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles at 94 °C for 20 s, 51 °C for 30 s, 68 °C for 3 min, with a final extension at 68 °C for 10 min. Sequencing using the PCR primers was performed using an Applied Biosystems 3730 DNA Analyser, using BigDye version 3.1. Sanger reads were edited in Geneious. Sequence identity was verified by mapping to the reference contigs in Geneious.The E. vieirai and P. penelope were subjected to secondary structure folding at 20 °C using mfold (www.unafold.org)79. The consensus Group II secondary structures on the Zimmerly website 50 were taken as guides for subsequent manual secondary structure manipulation using forna (http://rna.tbi.univie.ac.at/forna/)80 in conjunction with iterative folding of parts of the sequences in mfold. Regarding intron sequences of C. biporosa and D. cookae, secondary structure folding was conducted at 20 °C using mfold, but no manual manipulations were performed.Following the excision of IEP ORFs, intron sequences of Trichoplax adhaerens (DQ112541), Placozoa sp. , Hoilungia sp. , Axinella verrucosa , Nephtys sp. (EU293739), Glycera unicornis , Glycera fallax  and Decemunciger sp. ; this is the first time that RVT-IM domains of placozoan haplotypes H2, H9, H11, H17, H19 and of the previously unsampled genus Hoilungia (H24)26, have been put into a ‘tree-of-life’ phylogenetic context. These ORFs were trimmed following Pfam analyses. No Pfam match for RVT-IM could be obtained for Endomyzostoma sp. In order to maximise our chances of identifying metazoan Group II intron origins, we also included blast matches of the abovementioned accessions and the bryozoans RVT-IM domains in our analyses. Duplicate accessions were removed in Mesquite v.3.5181. Amino acid sequence duplicates were removed using the web server for FASTA tools unique sequences (https://www.ncbi.nlm.nih.gov/CBBresearch/Spouge/html_ncbi/html/fasta/uniqueseq.cgi).Phylogenetic analyses of Group II intron RVT-IM domains were carried out as follows: Genbank searches of the protein database were conducted using terms ‘reverse transcriptase AND mitochondrion’  and ‘maturase AND mitochondrion’ . Furthermore, RVT-IM domains of the following metazoans were added: 82 using the --auto setting. Alignments were examined by eye in Geneious and obvious outliers were removed. Initial neighbour-joining trees, produced in PAUP* v.4.0a83, identified a large clade composed of plant RVT-IM sequences. As none of the focus sequences were nesting in this clade, it was removed from further analyses. The final alignment was carried out using the MAFFT settings --amino --bl 30 --genafpair --maxiterate 100. Ambiguously aligned positions were excluded using the stand-alone version of Gblocks v. 0.91b85 using the following settings: minimum number of sequences for a conserved position = lowest; minimum number of sequences or a flank position = lowest; maximum number of contiguous non-conserved positions = 10; minimum length of a block = 5; allowed gap positions = with half. The final alignment consisted of 1059 terminals and 3947 positions, of which 367 remained included. ModelTest-NG v.0.1.686 was used to determine the best model of amino acid substitution. Maximum likelihood (ML) phylogenetic analysis with 1000 fast bootstrap replicates was carried out using RAxML v.8.2.1287 under the LG + G4 + F model. Uncorrected p-distances were calculated using PAUP* v.4.0a83.Initial alignments were constructed in MAFFT v.7.453Exechonella and Parantropora, and (b) IEP-less introns of Cupuladria and Discoporella were carried out. The outgroup for analysis (a) was the well-annotated Group II intron Nephtys sp. (EU29373930) from which the IEP ORF had been excised according to their secondary structure drawing ; no suitable outgroup was available for analysis (b). Alignments were constructed in MAFFT with settings --genafpair --maxiterate 1000. Ambiguously aligned positions were excluded using the stand-alone version of Gblocks as outlined above. ModelTest-NG was used to determine the best model of nucleotide substitution. ML with 1000 fast bootstrap replicates and BI analyses were conducted in RAxML and MrBayes v.3.2.688 under the HKY + G model. Two parallel MrBayes runs were performed for 5 million generations. The burn-in was defined as the point at which the average standard deviation of split frequencies was < 0.01.To assess common ancestry of introns within species, phylogenetic analyses of (a) Group II IEP-less introns of Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Figure S8.Supplementary Figure S9.Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.


Noble nanomaterials also present special physical and chemical properties and, therefore, they provide model building blocks in modifying nanoscale structures for various applications, ranging from nanomedicine to catalysis and optics. The introduction of noble metal nanoparticles (NPs)  into chemically derived graphene is important in opening new avenues for both materials in different fields where they can provide hybrid materials with exceptional performance due to the synergistical result of the specific properties of each of the materials. This review presents the different synthetic procedures for preparing Pt, Ag, Pd and Au NP/graphene oxide (GO) and reduced graphene oxide (rGO) composites.In recent years, research has focused on developing materials exhibiting outstanding mechanical, electrical, thermal, catalytic, magnetic and optical properties such as graphene/polymer, graphene/metal nanoparticles and graphene/ceramic nanocomposites. Two-dimensional sp Considering its superior electronic characteristics, graphene sheets offer potential solutions for the manufacturing of nanocomposites . Th. Th13]. Abdulhusain et al.  prepared3O4-decorated graphene oxide and Au-Mn3O4 nanocomposite  is reacted for 24 h at 180 °C inside a high-pressure reactor. Afterward, a formulated (BNrGO)5-(CNT)5 support material is inserted into the ethylene–glycol mixture containing K2PtCl4, and additional processing at 120 °C is performed for a duration of 12 h in order to obtain the final product. Another ration of BNrGO-CNT was also prepared. The results obtained through ICP-MS determined the Pt loadings for the Pt/(BNrGO)3-(CNT)7, Pt/(BNrGO)5-(CNT)5, Pt/(BNrGO)7-(CNT)3, Pt/(BNrGO)9-(CNT)1, Pt/rGO, Pt/CNT and Pt/C catalysts at 18.20, 18.80, 18.70, 19.40, 18.40, 19.00 and 19.60 wt%, respectively. Pt particles show interplanar spacings of 0.223 nm and 0.195 nm, accurately corresponding to the (111) and (200) planes of face-centered cubic Pt crystals. Through TEM it can be observed that the Pt/BNrGO-CNT hybrid is composed mainly of the four types of elements , with these four components being uniformly distributed throughout the whole sheets. XRD reveals that the aforementioned diffraction peak appears at approximately 2θ = 25.0° for Pt/rGO and Pt/BNrGO-CNT. The three characteristic diffraction peaks of metallic Pt correspond to the (111), (200) and (220) crystal planes within the cubic Pt structure. The intensity ration between D and G (ID/IG) of the GO, rGO and Pt/rGO samples was determined as 0.87, 1.04 and 0.96, respectively, proving the presence of several topological flaws in the carbon structures. Conversely, the ID/IG of Pt/BNrGO-CNT proved a low 0.76, hinting towards the fact that the incorporation of low-defect CNTs into the rGO skeletons can be used as a means of reducing the density of defects of the hybrid system. The specific surface areas are 226 m2 g−1 and 240 m2 g−1 for Pt/BNrGO-CNT and BNrGO-CNT hybrids, respectively.Xue et al. used the thermally assisted method to anchor Pt nanocrystals onto three-dimensional (3D) porous boron and nitrogen double-doped reduced graphene oxide–carbon nanotube frameworks (Pt/BNrGO-CNT) . They us2 + Ar) mixture [2 solution in diluted HCl by wet impregnation, resulting in 10 wt.% palladium on rGO. The target Pd-Au/rGO with metal concentrations of 7.5 wt.% Pd and 2.5 wt.% Au was obtained similarly through wet impregnation, employing a mixture comprising PdCl2 and HAuCl4 aqueous solutions. In both cases, the impregnated samples were dried at ambient temperature followed by thermal reduction in Ar. Afterward, it was thermal treated in a (H2 + Ar) mixture (10 vol.% H2) for 30 min at 250 °C. The resulting surface areas determined using the BET method are 210 m2/g for Pd/rGO and 206 m2/g for Pd-Au/rGO. The XRD profiles for both of the catalysts display the (002) graphene reflexion situated at 23° for Pd/rGO and at 23.7° for the Pd-Au bimetallic catalyst, as well as the metal reflexions: Pd (111) at 39.9°, Pd (200) at 46.4° for Pd/rGO and one metal reflexion situated at 38.9° for Pd-Au/rGO. Size of the metal crystallite calculated by XRD is 5 nm for Pd/rGO and 3.5 nm for Pd-Au/rGO. The bimetallic catalyst displays a distance between the carbon layers of 0.378 nm, whereas the Pd/rGO catalyst displays a distance of 0.386 nm, with the medium number of graphene layers for Pd/rGO being 4 and for Pd-Au/rGO being 5.5. The burning temperature for the palladium containing catalyst is 517 °C, while the Pd-Au bimetallic has a much lower value at 447 °C. The ID/IG intensity ratio of the D and G bands is 0.90 for Pd/rGO and 0.85 for Pd-Au/rGO; values were obtained from Raman spectra [Grad et al. prepared Pd/rGO and Pd-Au/rGO catalysts through wet impregnation of GO using an aqueous mixture of metal salts. The resulting mixture of GO and metal ions was then processed through thermal treatment in  presented by the Raman spectra of the investigated samples proved to be equal to 0.60 for GO, 0.63 for rGO and 0.81 for Au/rGO, respectively. The obtained ID/IG values are directly proportional with the number of structural defects in the sample.Wojnicki et al. synthesized Au/rGO. They first dissolved metallic gold in aqua regia to obtain the Au(III) chloride complex . A MagnuGold nanoparticles decorated on rGO were prepared using the microwave-assisted process (MW). The procedure has demonstrated remarkable advantages as eco-friendly method for Au/rGOs obtaining with simultaneous reduction in graphene oxide and formation of gold nanoparticles by an innovative one-step process. The characterization of prepared samples demonstrated good chemical stability and controllable morphology. The samples were used for membrane electrode assembly development and tested in operation of proton exchange membrane fuel cells. The electrochemical stability of the innovative Au/rGO-based cathode was analyzed using several accelerated stress tests (ASTs) by considering the cycling potential protocol. The electrochemical analysis considering the I-V study, cyclic and linear voltammetry has provided improved performances in comparison with the standard commercial cathode. The aggressive AST indicated an excellent stability; thus, the authors reported an improved electrocatalyst for oxygen reduction reaction with higher stability and durability for fuel cells. Moreover, the paper indicates the possibility of extending the protocol using the microwave-assisted process for obtaining other noble metal nanoparticles supported on rGO ,37,38,39A single-step route to obtain platinum/platinum-cobalt uniformly distributed nanoparticles supported on reduced graphene oxide was developed recently. This route provides significant advantages such as its low cost, low time-consuming nature and high yield in comparison to state-of-the-art chemical methods used to prepare efficient Pt/rGO catalyst. The morphology of prepared samples has been evaluated by specific techniques, while the electro catalytic durability has been evaluated using the electrochemical performances in fuel cells ,41. SignThe ultrasonic method  leads to−1~3200 cm−1 [Li et al. synthesized the AuPs/rGO through a mixture of 4 mg/mL GO suspension and 0.48 mg/mL AuPs solution with a volume ratio of 1:1; it was then sonicated for 1 h. After the AuPs/rGO was reduced by hydroiodic acid, it was washed with deionized water and then dried in air. The images of AuPs/rGO composite by SEM showed the uniform distribution of the AuPs on the surface of rGO. XRD measurements and the corresponding calculated results are consistent with the SEM images. The AuPs composites are inserted into the layered graphene sheets. The diffraction peak is 2θ = 8.84° for rGO to 2θ = 7.86° for AuPs-rGO. XRD measurements are consistent with the SEM images. The Raman spectra of both the AuPs-rGO and pure rGO film show a wide band at 2400 cm200 cm−1 .3 was added. Centrifugation at 12,000 rpm was used to separate the final Ag/GO nanocomposites, which were then washed with double-distilled water [−1 for hydroxyl -OH, carboxyl -COOH, epoxy C-O-C and alkoxyl C-O. After anchoring with AgNPs, the epoxy stretching mode at 1226 cm−1 is no longer present, while peaks that are indicative of different oxygen functional groups remain well-preserved. Nanocomposites of GO and Ag/GO reveal a diffraction peak at 2θ of 10.1° in the XRD patterns relating to the (002) crystal plane of GO nanosheets. In addition to the distinctive diffraction peak of GO, the Ag/GO nanocomposites also exhibit a number of separate peaks at 2θ of 38.1, 44.2, 64.5 and 77.5 degrees, which can be attributed to the (111), (200), (220) and (311) facets of typical fcc metallic Ag (JCPDS No. 04-0783), respectively. The patterns revealed by XRD demonstrate the effective adhesion of AgNPs to GO nanowires. The ID/IG ratio of the GO increased from 0.87 to 0.92 when the GO was anchored with AgNPs. The C/O atomic ratio found in the Ag/GO nanocomposites is greater than that of the GO, with the values obtained by XPS analysis being 2.6 for Ag/GO and 2.0 for GO. FTIR showed the characteristic peaks for Ag/GO centered at 284.8, 287.0 and 288.5 eV, respectively, for C=C, C-O and C=O. XPS analyses determined an atomic percentage of Ag of 2.46.Tran et al. prepared the Ag/GO nanocomposites through the ultrasonication method. Ag/GO nanocomposites were synthesized with GO, double-distilled water and sonicated for a duration of 10 min, after which AgNOed water . The FTI+-decorated PCN-222  using ZrCl4, meso-tetra(4-carboxyphenyl) porphine, benzoic acid and AgNO3. The solution was ultrasonically dispersed and dissolved in a mixture of N, N-dimethylformamide and acetic acid. Preparing the Ag+-decorated PCN-222@EDTA-GO-CS (CS-chitosan) foam required AgNO3, ZrCl4, H2TCPP and benzoic acid that were ultrasonically solvable in the N, N-dimethylformamide [+-decorated PCN-222 showed clear crystal lattices and the TEM-EDS mappings revealed the even dispersion of Ag, C, N, O and Zr elements. The characteristic peaks of PCN-222 at 1709, 1640, 1603, 1549, 1402, 967, 801 and 719 cm−1 were obtained through the use of FT-IR spectra. The peaks in binding energies at 367.5 eV and 373.5 eV arose from the presence of Ag+ ions.Bi et al. prepared PCN-222 and Agormamide . The Ag+2PtCl6 was added to the solution. In the XRD patterns, the characteristic peaks of (002) obtained at 11.0° (d002 = 0.85 nm), 28.9° and 24.0° were for GO, CQDs and Pt/CQDs/rGO, respectively. After the reduction process, the diffraction peaks are observed at, respectively, 46.9°, 55.7° and 81.6°, corresponding to (111), (200) and (220) planes of the face-centered cubic (fcc) structure of Pt. The crystallite sizes of GO, CQDs and Pt/CQDs@rGO are 54 nm, 17 nm and 38 nm, respectively. The large crystallite size of Pt/CQDs@rGO shows the aggregation of Pt nanoparticles. D and G bands have been detected using Raman spectroscopy at 1350 cm−1 and 1580 cm−1 for graphene oxide, 1350 cm−1 and 1580 cm−1 in the case of GQDs and at 1357 cm−1 and 1590 cm−1 for Pt/N-CQDs@rGO nanocomposite. The values of the ratios between the intensities of the D and G bands (ID/IG) are 0.88 and 0.94 and have been calculated for GO and Pt/CQDs@rGO, respectively. The EDX investigation exhibits all essential elements such as C, O, Na, Cl and Pt individually from Pt/CQDs@rGO nanocomposite.Mehmandoust et al. prepared Pt/CQDs@rGO nanocomposite (CQDs—carbon quantum dots) by ultrasonication method . The Pt/4 as reducing agents through the ultrasonication method [3 and glucose are added. The same experimental procedure is repeated for vitamin C and NaBH4. The Ag/rGO samples have peaks at 2θ values of 38.13°, 44.34°, 66.44° and 77.44°, which agree with the cubic crystal structure of the Ag NPs. The grain sizes of the Ag NPs are 28 nm, 25 nm and 22 nm for GAg_G, GAg_V and GAg_S, respectively. Through Raman spectroscopy, a graphitic band (G band) at 1590 cm−1 and a disorder band (D band) at 1365 cm−1 can be observed. The absorption bands at 257 nm for GAg_G, 268 nm for GAg_S and 270 nm for GAg_V. ID/IG ratios have been measured at 1.26 for GAg_S, 1.19 for GAg_V and 0.94 GAg_G.Mariappan et al. presented the study of Ag/rGO prepared with glucose, vitamin C and NaBHn method . The Ag/Aljafari et al. prepared Pd/GO using the sonication technique. This process used GO, palladium acetate and glucose. The pH was adjusted using NaOH . The dif3 and cetyl trimethylammonium bromide. The final product is graphene oxide sheets that are decorated by silver nanoparticles. The peaks at 38.31°, 44.41°, 63.51° and 77.71° can be assigned to the (111), (200), (220) and (311) crystalline planes of silver, respectively, which shows that the silver nanoparticles are composed of pure crystalline silver. The particle diameter of silver nanoparticles is about 10 nm. The band of silver nanoparticles is at about 414 nm.Mao et al.  synthesiFor the preparation of graphene materials doped with metal nanomaterials, the synthesis methods are very important to ensure the best dispersion of the metal particles and the narrowest distribution of their size, because both significantly affect the electrocatalytic activity.In In recent years, different methods have been proposed for the synthesis of nanoparticles deposited on a graphene support. The choice of the most suitable method has the greatest importance in terms of the structure and catalytic efficiency of the catalysts. In conclusion, the most valuable method among the preparation methods of graphene-deposited nanomaterial catalysts is microwave field irradiation, especially due to the short synthesis time, the fast and uniform heating and the significant challenge in controlling uniformity of the metal nanoparticle’s decoration on the graphene surface. By applying irradiation in the microwave field, under the influence of temperature, homogeneous reaction centers are formed in the reaction medium at the interface between the irradiation-sensitive graphene support and the metal precursor. Additionally, the presence of a reducing agent in the reaction medium means that the precursor can be converted to its metallic form by microwave irradiation.The qualities of noble metals have demonstrated a special efficiency in the electrocatalytic activity and the electrochemical stability of compounds based on carbon and graphene oxide. In order to improve the oxygen reduction reaction (ORR) and the quality of hydrogen adsorption and desorption, a higher electrochemical active surface area (ECSA) of the catalyst based on noble metals is necessary. The intrinsic increase in the active surface is proportional to the metal content in the chemical compound and to the dispersion of metal nanoparticles on the rGO sheets. The uniform distribution and surface morphology of noble metal nanoparticles on rGO have an effect on the ORR. An excessive reaction energy can cause an agglomeration of the noble metal nanoparticles, leading to particle sizes over 10 nm and the suppression of catalytic activity by reducing the active surface. This review presents the most used and up-to-date methods for the synthesis of graphene functionalized with noble metals  as well as the relevant methods for the characterization of catalysts. The potential of capitalizing on the improved catalytic properties of graphene functionalized with noble metals was also discussed. The studies presented in this review were carried out in order to understand how the metal–support interaction drives chemical catalysis. The preparation technique, the type and amount of metal, the nature of the support, the type of dopant and the technique of applying the catalyst, all these are dependent on the metal–support relationship. Following this review, it was found that the noble metals demonstrated a special efficiency in the electrocatalytic activity and the electrochemical stability of the compounds based on carbon and graphene oxide. It was also observed that to improve the oxygen reduction reaction (ORR) and the quality of hydrogen adsorption and desorption, a higher electrochemical active surface area (ECSA) of the noble metal catalyst is required. The uniform distribution and surface morphology of noble metal nanoparticles on rGO were found to have an effect on the ORR. Most applications of noble metal functionalized graphene are in fuel cells, renewable energy  and supercapacitor applications.


Graphene is a two-dimensional nanomaterial with excellent mechanical, electrical and thermal properties. The application of graphene in cement-based materials has good prospects. However, the mechanical properties of cement-based materials are difficult to be significantly enhanced by ordinary graphene nanoplates. In this paper, nitrogen-doped graphene is first reported as an additive with dosages of 0.01, 0.02, 0.03, 0.04 and 0.05 wt.%, respectively, to prepare iron ore tailings–based cementitious composites. The iron ore tailings–based cementitious composite with 0.02 wt.% graphene shows an extremely high flexural strength of 15.05 MPa at 3 days, which is 134.4% higher than that of the iron ore tailings–based cementitious composite without graphene. The effects of graphene content and curing age on the flexural strength and microstructure of iron ore tailings–based cementitious composites were studied. In particular, the scanning electron microscope was adopted to observe the micromorphology of the composites. It is helpful to understand the graphene reinforcement mechanism for the high early flexural strength of iron ore tailings–based cementitious composites. By altering the morphology of iron ore tailings–based cementitious composites, graphene plays two roles in the composites. One role is to connect C-(A)-S-H gels, ettringite and other hydrated crystals to construct a three-dimensional structure. The other is to attract iron ore tailings distributed on its platform to enhance its flexural strength properties. These findings provide favorable guidance for the performance enhancement and mechanism replenishment of graphene-reinforced cementitious composites. It endows graphene with good conductivity [Ordinary cement-based mortar and concrete materials are generally hard and brittle with low bending strength. It is difficult to meet the growing engineering needs ,2. To souctivity ,10. The uctivity .Studies have shown that graphene and its derivatives have a certain attraction to cement hydration products, and they could be used to effectively strengthen cement-based composites ,13,14. GHowever, some recent studies have reported that graphene can well connect C-S-H gels, ettringite and other hydration products together to form a three-dimensional structure and can improve the mechanical properties of composites by filling pores and bridging cracks in the cementitious system ,42. SilvIn addition, the production of cement and concrete industries faces problems such as resource depletion and high energy consumption. In response to this problem, many researchers are committed to studying how to effectively use solid wastes to prepare cement or concrete ,48,49,50In this paper, nitrogen-doped graphene was used as an additive with dosages of 0.01, 0.02, 0.03, 0.04 and 0.05 wt.%, respectively, to reinforce iron ore tailings–based cementitious composites, which were prepared by a cement and iron ore tailings aggregate. Their mechanical properties were evaluated by conducting flexural and compressive strengths tests. It was found that compared with the standard sand-cement cementitious system, the early flexural strength of iron ore tailings–based cementitious composites was improved more significantly with the addition of graphene. The microstructure of graphene and its reinforced iron ore tailings–based cementitious composites were observed by a scanning electron microscope (SEM). A new reinforcement mechanism is proposed to support the high early flexural strength of graphene-iron ore tailings–based cementitious composites.The raw materials for producing graphene-iron ore tailings–based cementitious composites (GICC) are nitrogen-doped graphene, iron ore tailings, Portland cement and Belite cement. In addition, polycarboxylate superplasticizer was adopted as a strengthening agent. It also played an important role in dispersing the graphene .−3, and the particle size is 6~60 μm. The chemical composition is mainly C and O.Graphene was provided by Guangdong Minglu Tianyuan New Material Technology Co., Ltd. . It consists of 1~5 layers. Its packing density is 0.08~0.12 g·cm2, Fe2O3, Al2O3 and CaO. Iron ore tailings were obtained from Ili Kazak Autonomous Prefecture of Xinjiang, China. 2/kg and 3.15 g/cm3, respectively. Belite cement was supplied by Tangshan Polar Bear Building Materials Co., Ltd. . Its Blaine specific surface area and specific gravity are 450 m2/kg and 2.97 g/cm3, respectively. Portland cement was supplied by Tangshan Jidong Cement Co., Ltd. . Its Blaine specific surface area and specific gravity are 390 mPolycarboxylate superplasticizer was supplied by Qinfen Building Materials Co., Ltd. . It is in a liquid form with a water reduction rate of 28%, and its gas content is 5%.The experimental materials were weighed at the proportions shown in The GICC mixture was stirred by the mortar mixer slowly at 75 rpm for 30 s, and then stirred at a fast speed of 300 rpm for 90 s. Subsequently, the mixture was poured into a standard mold of 40 mm × 40 mm × 160 mm. The mixture in the mold was vibrated for 120 s with a shaking table to expel the entrapped air from the mortar. Finally, plastic wrap was used to prevent moisture from evaporating from the mortar. The samples were demolded after 24 h and cured at 20 ± 2 °C with a relative humidity of 95%. To obtain representative mechanical strength measurements, five replicates were performed for each batch.The mineralogical components of iron ore tailings were analyzed by X-ray powder diffraction (XRD)  using Cu Kα Radiation (λ = 1.54056 Å) at 40 kV and 40 mA. The scanning range was 5°~70°, and the scanning speed was 6°/min.The mechanical properties test was carried out according to GB/T 17671-1999 . The fleAfter the mechanical properties test, the typical samples were analyzed by a SU 8020 field-emission-scanning electron microscope . Before the SEM characterization, the selected samples were soaked with ethanol for 72 h to stop the hydration process, and then the samples were placed in an oven at 55 °C, drying for 12 h to eliminate the ethanol. Before SEM images were taken, the samples were treated with gold sputtering to improve the SEM image quality.In order to study the reinforcing effect of graphene on iron ore tailings–based cementitious composites, the mechanical properties of composites before and after adding graphene were analyzed. The effect of graphene content on the flexural and compressive strengths of GICC is presented in As shown in As shown in Given the complex composition of iron ore tailings, an SEM-EDS test was carried out for iron ore tailings. The micromorphology and element composition of iron ore tailings are presented in The micromorphology of an iron ore tailings–based cementitious composite (the XB0 sample) is shown in As shown in 2 to form C-(A)-S-H gels [However, the graphene-iron ore tailings–based cementitious composite is different from the graphene-standard sand-cement composite. It can be seen in S-H gels ,69. SomeS-H gels ,62,70. T2 are also found in the 28-day hydrated sample in 2 is also linked to graphene through the bridging effect of ettringite. A tight connection system established by the ettringite meshes connects various hydrated crystals with graphene sheets to enhance the strength of cementitious composites. It can also be seen that the C-S-H gels increase with the prolongation of hydration time. In the interface transition zone between the graphene and the ettringite, some deposited crystals and ettringite are covered by C-(A)-S-H gels, further consolidating the composite structure [As shown in tructure . With thIt can be seen from the XB2 sample that the three-dimensional structure theory found in the previous GSCC is also applicable to GICC . Both of2 with graphene to enhance the flexural strength of GICC. The main hydration product, C-(A)-S-H gels, not only can make the connection between the graphene, iron ore tailings and the ettringite tighter but also can play a fixed role that makes the connection between the graphene and the substrate stronger. In addition, adding an appropriate amount of graphene can effectively deposit iron ore tailings particles onto its surface, because they show a tendency to attract each other [The flexural strength enhancement mechanism of GICC is presented in ch other . The finFinally, graphene nanoplates have large specific surface areas and flat planes. Their dense electron clouds lead to the absorption of cations, thus providing a substrate for crystal formation and growth . A moder(1)The early mechanical properties of iron ore tailings–based cementitious composites were enhanced by using graphene. Graphene showed a great effect on the flexural strength development of iron ore tailings–based cementitious composites while keeping the compressive strength basically unchanged.(2)Graphene can significantly improve the flexural strength of iron ore tailings–based cementitious composites in a short hydration time. When adding a 0.02 wt.% dosage of graphene, the 3-day flexural strength of GICC attained 15.05 MPa. It was 134.4% higher than that of the iron ore tailings–based cementitious composite without graphene.(3)There was a three-dimensional structure of graphene-ettringite-C-(A)-S-H gel in the GICC system. This structure can effectively improve the flexural strength of iron ore tailings–based cementitious composites.(4)A proper amount of iron ore tailings deposited onto the graphene can solidify the three-dimensional structure. However, when the graphene content exceeded 0.02 wt.%, large amounts of iron ore tailings were likely to deposit onto the graphene surface. They separated the graphene from the cement matrix, thus reducing the flexural strength of GICC.In this paper, the flexural strength development and microstructure of iron ore tailings–based cementitious composites (GICC) were deeply investigated. Additionally, the flexural strength reinforcement mechanism of GICC was discussed and clarified. The following conclusions were drawn:Our results showed that nitrogen-doped graphene had a greater effect on improving the early flexural strength of iron ore tailings–based cementitious composites. At present, the research and application of graphene nanoplates in solid wastes-based cementitious composites are still few. The development of long-term mechanical properties and the quantitative analysis of the cementitious components in the nitrogen-doped graphene-reinforced cementitious composites should be further investigated in our future work.


Pseudomonas aeruginosa are global antimicrobial resistance threats. Cefepime-taniborbactam (FTB) is an investigational β-lactam/ β-lactamase inhibitor combination that is active against Enterobacterales and Pseudomonas aeruginosa expressing serine and metallo-β-lactamases. CERTAIN-1 (Cefepime Rescue with Taniborbactam in cUTI) evaluated FTB efficacy and safety in the treatment of cUTI.Carbapenem-resistant Enterobacterales and multidrug resistant CERTAIN-1 was a randomized, double blind, double dummy, Phase 3 study comparing FTB to meropenem (MEM) in adults hospitalized with cUTI or AP. The primary endpoint was the composite  response at the test of cure (TOC) visit in the microITT population. Patients were randomized 2:1 to FTB 2.5g IV q8h or MEM 1g IV q8h for 7 days or up to 14 days in patients with bacteremia. The non-inferiority margin was -15.0% and a pre-specified test for superiority for the primary endpoint was performed following confirmation of non-inferiority.> 65 years represented 38.0% of the microITT population and bacteremia was present in 13.1% of patients. Composite success was achieved in 70.0% and 58.0% of FTB and MEM patients, respectively . FTB was statistically superior to MEM for the primary endpoint at the TOC  and statistical superiority was sustained at the late follow up visit (LFU). Analyses of secondary endpoints and subgroups were consistent with the primary efficacy analysis. Treatment-emergent adverse events (TEAEs) were observed in 35.5% of FTB patients and 29.0% of MEM patients. Serious adverse events occurred in 2.0% and 1.8% of FTB and MEM patients, respectively. The most common TEAEs were headache  and diarrhea .A total of 661 patients were randomized and 436 patients (66.0%) were included in the microITT population, including 42.2% with AP and 57.8% with cUTI. Patients Following 7 days of therapy, FTB was statistically superior to MEM for the primary endpoint at the TOC. Composite success for FTB remained statistically superior to MEM at the LFU visit. FTB was safe and well-tolerated with a safety profile similar to MEM.Paul C. McGovern, MD, Paratek Pharmaceuticals: Stocks/Bonds|Venatorx Pharmaceuticals: employee|Venatorx Pharmaceuticals: Stocks/Bonds Florian Wagenlehner, MD, Glaxo Smith Kline: Advisor/Consultant|Spero Pharmaceuticals: Advisor/Consultant|Venatorx Pharmaceuticals: Advisor/Consultant Leanne Gasink, MD, MSCE, Amplyx Pharmaceuticals: Advisor/Consultant|CSL Behring: Advisor/Consultant|Pfizer: Advisor/Consultant|Spero Therapeutics: Advisor/Consultant|Venatorx Pharmaceuticals: Advisor/Consultant|Vera Therapeutics: Advisor/Consultant Greg Moeck, PhD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Patrick L. McLeroth, MD, Labcorp: Full time employee|Labcorp: Stocks/Bonds Mary Beth Dorr, PhD, Merck: Past employee|Merck: Stocks/Bonds|Pfizer: Stocks/Bonds|Venatorx: Employee Aaron Dane, MSc, Amplyx: Advisor/Consultant|AN2 therapeutics: Advisor/Consultant|Artizan: Advisor/Consultant|Cidara: Advisor/Consultant|ContraFect: Advisor/Consultant|Correvio: Advisor/Consultant|Davolterra: Advisor/Consultant|Destiny Pharma: Advisor/Consultant|Entasis: Advisor/Consultant|F2G Limited: Advisor/Consultant|GSK: Advisor/Consultant|Humanigen: Advisor/Consultant|Kymab: Advisor/Consultant|Modis: Advisor/Consultant|Orca: Advisor/Consultant|Pfizer: Advisor/Consultant|Phico: Advisor/Consultant|Pled Pharma: Advisor/Consultant|Rare Thyroid: Advisor/Consultant|Roche: Advisor/Consultant|Scynexis: Advisor/Consultant|Sinovent: Advisor/Consultant|Spero Therapeutics: Advisor/Consultant|Transcrip: Advisor/Consultant|Venatorx: Advisor/Consultant Tim Henkel, MD, PhD, Venatorx Pharmaceuticals: Stocks/Bonds.


Guidelines recommend regular physical activity (PA) and decreased sedentary time (SED) for patients after myocardial infarction (MI). Therefore, valid self-assessment of PA is vital in clinical practice. The purpose of this study was to assess the convergent validity of commonly used PA and SED questions recommended by the National Board of Health and welfare (NBHW) and national SWEDEHEART-registry using accelerometers as the reference method in patients after MI.Data were obtained 2017–2021 among Swedish men and women (180 assessments). Participants answered five commonly used PA and SED-questions (by NBHW and SWEDEHEART) and wore an accelerometer (Actigraph GT3X) for seven days. Convergent validity was assessed gradually by; Kruskall Wallis-, Sperman rho, Weighted Kappa- and ROC-analyses. Misclassification was explored by Chi-square analyses with Benjamini–Hochberg adjustment.The strongest correlation (r = 0.37) was found for the SED-GIH question (NBHW). For PA, no specific question stood out, with correlations of r = 0.31 (NBWH), and r = 0.24–0.30 (SWEDEHEART). For all questions (NBHW and SWEDEHEART), there was a high degree of misclassification (congruency 12–30%) affecting the agreement (0.09–0.32) between self-report and accelerometer assessed time. The SED-GIH, PA-index and SWEDEHEART-VPA had the strongest sensitivity for identifying individuals with high SED (0.72) or low PA (0.77 and 0.75).The studied PA and SED questions may provide an indication of PA and SED level among patients with MI in clinical practice and could be used to form a basis for further dialogue and assessment. Further development is needed, since practical assessment tools of PA and SED are desirable.The online version contains supplementary material available at 10.1186/s13102-022-00509-y. Cardiovascular disease (CVD) is the major cause of death in Europe . There iInternational guidelines strongly recommend individualised PA and SED recommendations for patients with CVD , 7. Ther“The Swedish National Board of Health and Welfare” (NBHW) recommends two PA questions (NBHW-PA questions) to assess levels of everyday PA, exercise, and a combination of these . They alThus, the current questions seem to have an acceptable predictive validity for identifying individuals with a higher risk of rehospitalization and all-cause mortality , 3. HoweThis was a single center study performed at the Department of Cardiology at Danderyd’s hospital, Stockholm, Sweden. Individuals were informed of the study and invited to participate by a nurse at the first routine follow-up visit, six to eight weeks after MI.Data were collected via questionnaires, accelerometers, diaries, and the national quality registry SWEDEHEART between October 2017 and May 2021. The participants answered a questionnaire concerning PA and SED both at six to eight weeks and at 10–12 months post MI. Immediately after the visits, an accelerometer with a diary were posted to the participant, with instructions on how to wear the device and to start the accelerometer assessment directly. After the measuring period, participants returned the accelerometer and the diary by prepaid postage to the Swedish School of Sport and Health Science. Covariates were collected from a sub-register of SWEDEHEART, the SEPHIA-registry, focusing on outpatient cardiac rehabilitation up to one year after MI from the responsible cardiac department . The folIndividuals aged 18–80 years with newly diagnosed MI (ICD I21) registered in the SWEDEHEART registry were included in the study. Individuals who were physically disabled (wheelchair dependent) or with reduced ability to answer the questions were excluded by not being asked for participation.Six commonly used PA and SED questions were included in the study .The NBHW-PA and SED questions use different time frames. The NBHW-PA questions focus on PA in an ordinary week while the SED question focuses on an ordinary day.Everyday PA: “During a regular week, how much time (in minutes) are you physically active in ways that are not exercise, for example walks, bicycling, or gardening? Add together all activities lasting at least 10 min”. Seven fixed answers were available; “no time”, ” < 30”, “30–60”, “60–90”, “90–150”, “150–300” and “ > 300”.Physical exercise: During a regular week, how much time do you spend exercising on a level that makes you out of breath, for example running, fitness class, or ball games? The questions had six fixed answers; “no time”, “ < 30”, “30–60”, “60–90”, “90–120” and “ > 120”.The questions of everyday PA and physical exercise formed a validated PA-index (3–19 points) of total PA level . This waSedentary time: “How much time (in hours) do you sit during a normal day, excluding sleep?” . There wSWEDEHEART-MVPA: “Number of physical activity sessions of at least 30 min (two 15-min sessions can be combined into one 30-min session) in the last 7 days, with a minimal intensity of fast walking”.The PA questions in the SWEDEHART registry focus on PA during the previous week and are listed below. The individuals can choose between 0–7 days/week SWEDEHEAR: How many days in the last week did you do at least 30 min, total time (at least 10 min at a time) of physical activity that made you slightly out of breath and gave slightly elevated heart rate?SWEDEHEART-MPASWEDEHEART-VPA: How many days during the last week did you do some form of continuous vigorous physical activity /exercise, (at least 20 min), that made you out of breath and gave you elevated heart rate?The following two questions separate moderate and vigorous intensity levels of PA.An accelerometer provides information on body movements, which can be translated to data on PA, using validation algorithms. Such translation gives reasonably valid data of duration, frequency, and absolute intensity of PA and SED , 17. AccThe accelerometers and data files were processed using the software Actilife, version 6.13.4 . Data were collected tri-axially using a sampling rate of 30 Hz and, after extraction, data were down-sampled and saved as 60 s epochs, with activity intensity based on vector magnitude. Normal frequency filter was applied. Additionally, the Choi algorithm was used for validate non-wear time , definedCut-points were used to describe the daily PA behaviour, using the following components: time spent in sedentary , light physical activity , moderate PA (MPA 2690–6166) and vigorous physical activity  . The meaTo be included in the analyses, individuals had to answer the questionnaire and have valid accelerometer data for at least seven days defined as ≥ 600 min of data per day after non-wear time had been excluded.The descriptive data are presented as median and 25th and 75th percentiles (Q1–Q3), or number and proportions. Before Spearman and weighted Kappa analysis, the continuous accelerometer data were divided into the same categories as the different answer options for PA  and SED questions. For the PA-index, the average number of minutes in MVPA was kept as a continuous variable.Convergent validity was explored gradually:Multiple linear regressions were performed to control for repeated measurements among individuals with two assessments. The study-id was used as a covariate.Correlations between the categorised accelerometer data and the PA and SED-GIH questions were calculated using Spearman’s rho (r). The associations were interpreted as weak (r < 0.10), modest (r 0.1–0.3), moderate (r 0.3–0.5), strong (r 0.5–0.8) or very strong (r 0.8–1.0) . To explWeighted kappa analyses were conducted to assess the agreement between PA and SED-GIH questions with the categorized accelerometer data (n.a. for the PA-index). The agreement (Kappa) was interpreted as poor (< 0.20), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80) or almost perfect (0.81–1.00) .Receiver operating characteristics (ROC) curves were calculated. The answer options were kept as ordinal variables and the accelerometer data were used as state variable (golden standard) and dichotomised at a cut-off value. The cut-off for achieving the PA recommendations was ≥ 150 min a week of MPA for the everyday PA question, ≥ 75 min a week of VPA for the exercise question and ≥ 150 min of MVPA for the PA-index (NBHW-PA questions). For the SWEDEHEART questions the accelerometer cut-off was set at ≥ 20 min of total VPA for at least 3 days a week and ≥ 30 min of total MPA or MVPA for at least 5 days a week. Individuals with ≥ 9.5 h of SED per day were categorized as high SED, while individuals with less time spent sedentary were categorized as low SED. This cut-off was chosen based on international PA , 7 and SKruskal–Wallis analyses with Bonferroni correction were performed to investigate the differences in median accelerometer derived time between the different categories of the PA and SED questions.Lastly, calculations in Excel were performed to assess how well the PA and SED-GIH questions corresponded to the accelerometer derived data, either congruent, or over- and underestimating. Then, Chi square analyses with Benjamini–Hochberg adjustments were used to assess differences in congruence, under-, and overestimation between gender, age, LVEF, systolic blood pressure, diabetes, and BMI. All statistical analyses were performed using SPSS 27.0 software .n = 2), (n = 3) everyday-PA and exercise (n = 2) questions.A total of 123 individuals answered the questionnaire and provided complete accelerometer data were included  in accelerometer collected time for different categories of the NBHW-PA and SED questions Fig. a. For evIn the multiple linear regression , but the agreement was weak (0.09). However, the ability to classify individuals sitting ≥ 9.5 h was good, with an acceptable level of both sensitivity 72%) and specificity (62%)  and no specific question had a significantly stronger correlation (p > 0.47). In general, the weighted kappa analyses showed a poor to fair agreement between the answer categories and categorised accelerometer data (agreement 0.10–0.32) with the strongest agreement (fair) for the SWEDEHART-MVPA and the NBHW everyday PA questions. Results from the ROC analyses are presented in Table 4–0.31 anCongruence between individuals’ self-reported PA and SED levels compared to the accelerometer measured data varied between 12 and 30% .There were few other factors affecting the reporting pattern. Older individuals over-reported everyday PA to a higher degree than younger 63% vs 44%). Men under-reported the number of PA sessions in MVPA (SWEDEHEART) compared to women (39% vs 18%)  frequently used in clinical practice, there was a high degree of misclassification (over- and underreporting), with modest to moderate correlation and poor to fair agreement. Nonetheless, they may provide some broadly indication about PA and SED level, which could form the basis for further discussion and assessment on an individual level in clinical practice.The updated European guidelines of CVD prevention highlight the importance of decreasing SED; therefore it is important that the convergent validity of the SED-GIH question has an acceptable level. The correlation to accelerometer assessed SED (r = 0.37) was somewhat lower compared to a study in a general population that showed a correlation between the SED-GIH question and total stationary time (r = 0.48) . ConvergThe agreement between the SED-GIH question and SED time assessed by accelerometer was weak. This might be due to eight out of ten participants underestimating their sedentary time causing misclassification. The difficulty to estimate SED has been shown in other studies, with misclassifications of minus 85 min per day  and undeFor PA, the correlation for the NBHW and SWEDEHEART questions with accelerometer derived data was modest to moderate. There were no significant differences in correlation for the NBHW-PA questions compared to the SWEDEHEART ones. The correlation of NBHW PA-index with accelerometers as a criterion method showed similar results to a study in a general population, r = 0.27 (vs 0.31) for MVPA min/day . To our For the PA questions, agreement with time assessed by accelerometer was poor to fair (0.10–0.32) with the strongest agreement for the SWEDEHEART-MVPA and the NBHW everyday PA question. As with a previous study , there wROC-curve analysis of the PA-index was in line with a study in a general population (0.66) . TheretoThe most important strength of this study is that it includes a large cohort of CVD patients, focusing on individuals with MI. It also includes a higher proportion of older individuals compared to previous studies , 11. GenA limitation is the lack of consensus for cut-off values of accelerometer data for individuals with CVD. This is troublesome and the time spent at different levels of PA intensity should therefore be interpreted with caution, especially in patients with reduced cardiorespiratory fitness, which leads to differences in absolute and relative intensity . In thisAccelerometers were used as the criterion method. However, accelerometer measurements do have disadvantages. For example, information about relative intensity and activities such as bicycling, strength training, and swimming, all common types of exercise within cardiac rehabilitation , are omiAnother limitation is that participants may have become more conscious about their PA behaviour during the study, affecting how they answered the questions and their PA behaviour during the measurement period. The participants answered the questionnaire prior to wearing the accelerometer, this may be a limitation when the SWEDEHEART-PA questions focus at PA the last week, however several studies indicate that accelerometer data collected over seven days use to be consistent between weeks (high reliability) –31. Non-Exploring an individual’s total PA behaviour is complex and these questions have a limited convergent validity and low, precision. Therefore, a combination of both accelerometer derived data and questionnaires could be recommended , 9.The present study is clinically important as it focuses on commonly used SED and PA questions to patients with CVD, where regular PA and low SED both have a central role in cardiac rehabilitation. The convergent validity of the SED and PA questions is poor to moderate compared to accelerometer assessed data. The SED-GIH question had the strongest correlation. However, for PA we could not identify any preferable question. In spite of the risk of misclassification when using questionnaires, the questions seem to be a practical and acceptable method that may provide some indication about PA-level, which could form the basis for further discussion and assessment on an individual level.Additional file 1. Correlations between self-rated PA level, SED and study-id with accelerometer assessed PA and SED time.Additional file 2. Graphical results of the ROC analyses for the specificity and sensitivity of the PA and SED questions compared to accelerometer collected data.Additional file 3. Convergent validity for self-reported estimates of physical activity and sedentary using accelerometer assessed time as reference for time point one (n=123) and two (n=56).Additional file 4. Congruence between self-reported PA and SED compared to the accelerometer assessed time for different strata.


Bone defects and fractures heal slowly compared with injuries to other tissues, creating a heavy burden for patients, their families, and society. Alongside conventional treatment methods for fractures and bone defects, adjuvant therapies play an important but underappreciated role. In a previous study, we found that systemic administration of flufenamic acid promoted osteogenesis in vivo, but its side effects limited the application of our findings. In the present study, we assess the effects of external butyl flufenamate ointment on the healing of cranial defects in mice. We found that application of butyl flufenamate ointment on the surface of the skin accelerated the healing of cranial defects in mice by promoting BMP2 secretion from mouse-skin mesenchymal stem-cells. These findings indicate that butyl flufenamate ointment has potential therapeutic value for treating superficial fractures or bone defects while avoiding the toxicity and side effects of systemic medication, representing a safe and convenient adjuvant therapy to promote healing of superficial bone defects and fractures. Bone defects caused by fractures, tumors, congenital malformation, trauma, and infection are common in the clinic and can cause serious harm to patients’ physical and mental health . Since tLocal administration has several benefits compared with systemic administration, including decreased side effects and toxicity, greater convenience, and improved patient compliance ,5. Thus,As a member of the non-steroidal anti-inflammatory drug family, flufenamic acid (FFA) is a representative of fenamic acid. It is commonly used in the clinic to treat inflammatory diseases, such as rheumatic arthritis ,9. Oral In the present study, we aimed to explore whether the topical application of butyl flufenamate ointment on the skin can promote healing of cranial defects in mice. Our results showed that topical application of butyl flufenamate ointment accelerated the healing of cranial defects in mice. Mechanistically, butyl flufenamate ointment accelerated healing by promoting BMP2 secretion from mouse-skin mesenchymal stem-cells (mSMSCs). This study provides valuable data on the potential application of butyl flufenamate ointment to treat superficial bone defects, representing a safe and convenient adjuvant therapy to promote healing.All animals used in this study were obtained from Vital River Corporation . The mice were kept in a pathogen-free facility on a 12:12-h light: dark cycle with water and food provided ad libitum. The Institutional Animal Care and Use Committee of the Peking University Health Science Center (LA2019019) approved the in vivo experiments in the present study, and all experiments were performed under relevant guidelines.Collage I, dispase V, fetal bovine serum (FBS), modified Eagle’s medium (MEM), and 100× penicillin/streptomycin solution were purchased from Gibco . Other reagents and materials were obtained from Sigma-Aldrich  unless stated otherwise.2 in area was carefully removed. The skin was then disinfected with 75% ethanol and washed with PBS. Then, the skin was cut as finely as possible and digested using a mixture of 2 mL collagenase I (4 mg/mL) and 2 mL dispase V (8 mg/mL) at 37 °C for approximately 1.5 h. After adding 4 mL MEM, the mixture was centrifuged at 1500 rpm for 5 min. The sediment was then resuspended in medium, filtered through a 70 μm sieve, and seeded in 100 mm dishes. The cells were then cultured in a 5% CO2 atmosphere at 37 °C in proliferation medium (PM) consisting of MEM, penicillin/streptomycin, and 20% (v/v) FBS. After 24 h, the cells were washed twice with PBS, and new PM was added. Cells from passages 2–4 were used in subsequent experiments.Eight-week-old female C57BL6 mice were used to isolate mSMSCs. After the mice were sacrificed, the skin on their backs was prepared and disinfected. An area of skin on the back approximately 1.5 × 1.5 cmv/v) FBS, penicillin/streptomycin, 10 mM β-glycerophosphate, 10 nM dexamethasone, and 0.2 mM L-ascorbic acid. The adipogenic medium (AM) was prepared by mixing MEM, 10% (v/v) FBS, penicillin/streptomycin, 100 nM dexamethasone, 200 μM indomecin, 10 μM insulin, and 500 μM 3-isobutyl-1-methylxanthine. The osteogenic medium (OM) was prepared by mixing MEM, 10% (v/v) FBS, penicillin/streptomycin, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.2 mM L-ascorbic acid. The chondrogenic medium (CM) was prepared by mixing MEM, 10% (v/v) FBS, penicillin/streptomycin, 1 μg/mL ascorbic acid, 100 nM dexamethasone, 1% sodium pyruvate, 10% insulin–transferrin–selenium (ITS-Premix), and 10 ng/mL mouse transforming growth factor β1 . T. T20]. Togenesis e. The rebmp2 but not bmp4 -β protein superfamily . BMP2 haOur study still has several limitations. We used a mouse cranial defect model to assess the effects of external use of butyl flufenamate ointment; different bone defect/fracture models should be used to verify these effects. While we showed that butyl flufenamate ointment promoted BMP2 secretion in mSMSCs, further studies are required to comprehensively determine the mechanism of its effects.Overall, our results demonstrated that the external use of butyl flufenamate ointment promoted the healing of cranial defects in mice, suggesting that it might have therapeutic value as an adjuvant for external use to promote healing of superficial bone defects and fractures.The present study showed that topical application of butyl flufenamate ointment accelerates the healing of cranial defects in mice by promoting BMP2 secretion in mSMSCs. These findings suggest that butyl flufenamate ointment may have therapeutic value for treating superficial fractures or bone defects while avoiding the side effects of systemic medication, representing a safe and convenient adjuvant therapy.


LC and SN integrity was assessed using neuromelanin-sensitive MRI. Response inhibition was measured using fMRI with a stop-signal task. In healthy controls, LC (but not SN) integrity correlated with the stopping-related activity of the right inferior frontal gyrus (IFG) and right subthalamic nucleus (STN), which further correlated with stop-signal reaction time (SSRT). PD patients showed reduced LC integrity, longer SSRT, and lower stopping-related activity over the right IFG, pre-supplementary motor area, and right caudate nucleus than healthy controls. In PD patients, the relationship between SSRT and the fronto-subthalamic pathway was preserved. However, LC integrity no longer correlated with the stopping-related right IFG or right STN activity. No contribution of SN integrity was found during stopping. In conclusion, LC (but not SN) might modulate inhibitory functions of the right IFG-STN pathway. Damage to the LC might impact the right IFG-STN pathway during stopping, leading to response disinhibition in PD.A long-running debate concerns whether dopamine or noradrenaline deficiency drives response disinhibition in Parkinson’s disease (PD). This study aimed to investigate whether damage to the locus coeruleus (LC) or substantia nigra (SN) might impact inhibitory functions of the fronto-subthalamic hyperdirect or fronto-striatal indirect pathway. Patients with PD ( ResponsA long-held debate concerns whether dopamine or noradrenaline deficiency (or both) drives response disinhibition in PD. A series of human and animal studies using the selective noradrenaline reuptake inhibitor atomoxetine has shown the role of noradrenaline in response inhibition. In rats, atomoxetine can produce a dose-dependent speeding of SSRT . In healin vivo using neuromelanin-sensitive MRI. Neuromelanin is a by-product of catecholamine synthesis, existing in LC noradrenaline neurons and SN dopamine neurons , pramipexole (n = 16), piribedil (n = 8), rasagiline (n = 8), selegiline (n = 2), and amantadine (n = 5).We recruited 29 patients with idiopathic PD at the Zhongshan Hospital Department of Neurology between 2020 and 2021. Inclusion criteria were (a) PD diagnosed by senior neurologists according to the Movement Disorder Society clinical diagnostic criteria for PD , (b) Hoen = 2), violation of the race model for computing SSRT (n = 1), excessive head motion (n = 6), or feeling of discomfort during scanning (n = 1).2.2We recruited 29 healthy controls (HCs). Inclusion criteria were (a) age 50–75 years, (d) education > 6 years, and (c) right-handed. Exclusion criteria were (a) a history of significant neurological or psychiatric diseases, (b) alcohol or drug abuse, (c) possible current depression, (d) possible mild cognitive impairment (MoCA-B < 26/30), or (e) contraindications to MRI. Three additional HCs were excluded due to a violation of the race model for computing SSRT.2.3The stop-signal task was designed, analyzed, and reported following a recent consensus . The tas2.4We controlled behavioral data quality by monitoring whether the task performance met the independence assumption of the race model: (a) p is between 0.25 and 0.75, and (b) the reaction time of failed Stop is numerically shorter than the reaction time of Go . All inct tests .We estimated SSRT using a modified integration method that replaces Go omissions with the maximum reaction time, given the presence of Go omissions . We repo2.5Neuroimaging data were acquired on a Siemens Tim Trio 3T MRI scanner with a 32-channel head coil at the Brain Imaging Center of the Center for Excellence in Brain Science and Intelligence Technology.2, resolution 1 × 1 × 1 mm3).Structural T1-weighted images used a magnetisation-prepared rapid gradient-echo sequence .Neuromelanin-sensitive T1-weighted images used a fast spin-echo sequence with magnetization transfer pulse .Functional T2*-weighted images used a standard echo-planar imaging sequence . The first three images were discarded to allow magnetization equilibration. All other images were corrected for slice acquisition time difference, realigned to a mean functional image, registered to the high-resolution T1-weighted image, normalized to the Montreal Neurological Institute (MNI) coordinate system, smoothed with a Gaussian kernel of 4-mm full-width half-maximum, and filtered with a 128-sec high-pass filter.fMRI data were processed using SPM12 .We controlled fMRI data quality by monitoring the total displacement  and spatversus correct Go (SS > Go). A whole-brain two-sample t test was conducted at the group level .First, we computed the stopping-related regional activation. The general linear model convolved a design matrix with a canonical hemodynamic response function at the subject level. The design matrix included successful Stop, failed Stop, correct Go, Go commission errors, and Go omissions as separate regressors. Each trial was time-locked to its onset. The total displacement was included as a nuisance regressor. Classical parameter estimation was applied with a one-lag autoregressive model. The stopping effect was defined as successful Stop t tests .Second, we examined whether PD patients showed lower stopping-related activity than HCs in the fronto-subthalamic or fronto-striatal pathway. Regions of interest were derived from a meta-analysis of 99 stop-signal task fMRI studies (NeuroSynth) , includip < 0.05), and (b) whether the stopping-related activity of the fronto-subthalamic pathway correlated with LC or SN integrity or levodopa equivalent daily dose .Third, we explored relationships between LC integrity, fronto-subthalamic pathway, and SSRT. We examined: (a) whether SSRT correlated with the stopping-related activity of the fronto-subthalamic or fronto-striatal pathway . They manually identified the LC and SN in the native space to avoid potential distortion in normalization following previous studies .2 each) and pontine (20 mm2) were identified adjacent to the fourth ventricle in three contiguous slices. The LC contrast-to-noise ratio (CNR) was the difference between the mean LC signal intensity and mean pontine signal intensity divided by the SD of the pontine signal intensity . The SN area was summed up across sides.The SN was identified through a semi-automatical procedure to minimize subjective bias . The SN r = 0.98, p < 0.001; SN, r = 0.98, p < 0.001). Their mean measurements were used for statistical analysis. We examined whether PD patients showed reduced LC or SN integrity than HCs using one-tailed two-sample t tests .Measurements of the two raters were highly consistent  = −2.92, p = 0.002) and SN integrity than HCs (t(55) = −5.33, p < 0.001). The group difference in LC integrity was confirmed by an atlas-based analysis (see supplement)  .Fig. 2Lo3.2p = 0.42) or Geriatric Depression Scale score (p = 0.97).3.3t test). The IFG, preSMA, and caudate nucleus were more activated for successful Stop than correct Go. The STN did not show significant stopping-related regional activation at the group level, probably due to a larger inter-individual variability. Given the importance of the fronto-subthalamic pathway, we applied region-of-interest analysis to the STN.t tests, t(56) = −1.81, p = 0.038), preSMA (t(56) = −1.99, p = 0.025), and right caudate nucleus (t(56) = −2.25, p = 0.015), and marginally in the right STN (t(56) = −1.50, p = 0.07). However, these tests were only significant using one-tailed t tests and not corrected for multiple comparisons.PD patients showed lower stopping-related activity than HCs in the fronto-subthalamic and fronto-striatal pathways B, includ3.4F = 5.04, p = 0.004, R2 = 0.23) included the stopping-related right IFG , preSMA  and right STN activity  but excluded the stopping-related caudate activity (p = 0.78). Four outliers were analyzed separately (see supplement). Participants with higher stopping-related preSMA and right STN activity and lower stopping-related right IFG activity showed shorter SSRT.First, SSRT correlated with the stopping-related activity of the right IFG, preSMA, and right STN across groups C. A stepF = 4.76, p = 0.038, R2 = 0.15) included the LC CNR  but excluded the SN area (p = 0.757). A stepwise regression model for the stopping-related right STN activity  = 5.81, p = 0.023, R2 = 0.18) also included the LC CNR  but not the SN area (p = 0.466). HCs with better LC integrity showed higher stopping-related right IFG and right STN activity. However, no such correlation was found for PD patients (ps > 0.27).Second, LC (but not SN) integrity correlated with the stopping-related activity of the right IFG and right STN in HCs A. A stepF = 4.33, p = 0.024, R2 = 0.26) included the daily levodopa dose  and levodopa equivalent daily dose of D2/3 receptor agonists  but excluded the LC CNR and SN area (ps > 0.38). PD patients with daily exposure to more levodopa and fewer receptor agonists showed higher stopping-related preSMA activity.Third, daily doses of dopaminergic drugs correlated with the stopping-related preSMA activity in PD patients B. A step4Cognitive decline in PD correlates with the progression of α-synuclein pathology . Causal Damage to the LC may impact the right IFG-STN hyperdirect pathway during stopping. In PD patients B, the re4.1The STN is a strong candidate for inhibiting an initiated response within hundreds of milliseconds as the conduction velocity of ipsilateral cortico-subthalamic axons is ∼ 7 m/s, much faster than that of the cortico-striatal axons (∼1.5 m/s) . The STNBeyond the STN, we observed a complex regulation of response inhibition in frontal regions projecting along the hyperdirect pathway. In healthy adults and PD patients, higher stopping-related preSMA activity and lower stopping-related right IFG activity correlated with faster SSRT. This observation is in line with diffusion MRI findings that both right IFG-STN and preSMA-STN white matter tracts are engaged in response inhibition . HoweverIt remains unclear which fronto-subthalamic pathway is dominant or how the two fronto-subthalamic pathways interact during stopping. Recent human studies with invasive EEG and single-unit recordings have emphasized the importance of the right IFG-STN pathway . Chen etIn contrast, human imaging studies suggested that the right IFG modulates the excitatory projection from the preSMA to STN . Rae et 4.2Our novel finding is that greater LC integrity correlated with greater stopping-related right IFG and right STN activity in HCs. This finding thus fills an important gap in the literature. Human imaging studies have shown that the elevation of extracellular noradrenaline levels  can enhance stopping-related right IFG activity . Populat4.3SN integrity did not contribute to the preSMA-STN pathway . However, levodopa and D2/3 receptor agonists may impact the stopping-related preSMA activity in PD patients. PD patients with daily exposure to more levodopa and fewer D2/3 receptor agonists showed higher stopping-related preSMA activity, potentially leading to faster SSRT. This observation is consistent with previous findings that both D1 and D2 receptors can modulate SSRT in humans and animals , and tha5n = 58). Causal evidence is needed to confirm the model or to investigate hidden factors driving the correlations.First, LC and STN are small structures, but the spatial resolution of 3T MRI was limited. Further research with ultra-high field MRI or brain stimulation is needed to confirm our findings and determine the contribution of different LC and STN subregions . Second,6This study demonstrated that damage to the LC (but not SN) might impact the fronto-subthalamic hyperdirect pathway during stopping. In HCs, LC integrity correlated with the stopping-related right IFG and right STN activity, which further correlated with SSRT. PD patients showed reduced LC integrity, longer SSRT, and lower stopping-related activity over the right IFG and preSMA than HCs. In PD patients, the relationship between SSRT and the fronto-subthalamic pathway was preserved. However, LC integrity no longer correlated with the stopping-related right IFG or right STN activity. No contribution of SN integrity was found during stopping. LC might modulate inhibitory functions of the right IFG-STN pathway. Damage to the LC might impact the right IFG-STN pathway during stopping, leading to response disinhibition in PD.Biman Xu: Methodology, Investigation, Formal analysis, Writing – original draft. Tingting He: Investigation, Writing – review & editing. Yuan Lu: Investigation, Formal analysis, Writing – review & editing. Jia Jia: Investigation, Writing – review & editing. Barbara J. Sahakian: Writing – review & editing. Trevor W. Robbins: Writing – review & editing. Lirong Jin: Conceptualization, Resources, Writing – review & editing, Funding acquisition. Zheng Ye: Conceptualization, Methodology, Formal analysis, Writing – original draft, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.