Covering: up to 2014–2022.E,E,E)-geranylgeranyl diphosphate (GGPP) for photosynthetic pigment production, with diterpenoid biosynthesis arising very early in land plant evolution, enabling stockpiling of the extensive arsenal of (di)terpenoid natural products currently observed in this kingdom. This review will build upon that previously published in the Annual Review of Plant Biology, with a stronger focus on enzyme structure–function relationships, as well as additional insights into the evolution of (di)terpenoid metabolism since generated.Diterpenoid biosynthesis in plants builds on the necessary production of terpenoid natural products currently observed in this kingdom. However, GGPP also serves as general precursor to more specific diterpenoid biosynthesis where its constituent four isoprenyl units form the core of the derived natural product. The first appearance of such metabolism in plants appears to have been for phytohormone biosynthesis, but has since undergone dramatic expansion. Accordingly, the vast majority of the over 12 000 currently known diterpenoids are produced by this kingdom.1 As befitting their origins from photosynthetic pigments in planta diterpenoid biosynthesis is initiated in plastids, including proplastids and leucoplasts as well as chloroplasts.2,3 Hence, the relevant isoprenyl units are generally derived from the plastid-localized 2-C-methyl-d-erythritol 4-phosphate (MEP)-dependent isoprenoid pathway.4 Given its even more central role in metabolism that pathway will not be further discussed here. This review focuses on evolution of the enzymatic families that operate more selectively in (di)terpenoid metabolism, most notably those for plant terpene synthases (TPSs) as well as cytochromes P450 (CYPs) that insert oxygen into the TPS-derived hydrocarbon backbones. Although less is known about subsequently acting enzymes, which generally decorate the functional groups introduced by CYPs, these also are reviewed, as is the presence of diterpenoid biosynthetic gene clusters. In each case, this review emphasizes the additional insights gained since publication of the previous review.1GGPP can be considered a central metabolite in photosynthetic organisms, as it is required for production of light absorbing pigments (ent-kaurenoic acid in such metabolism.5 This further provides an example of the large labdane-related diterpenoid super-family (∼7000 currently known), whose production is characterized by initial bicyclization catalyzed by class II diterpene cyclases.6 In this case the relevant enzyme produces ent-copalyl diphosphate (ent-CPP), whose labdane backbone inspired this nomenclature. By contrast, the biosynthesis of other diterpenoids is initiated by class I TPSs that act directly on GGPP and necessarily function in more specialized metabolism. For example, in biosynthesis of the rice phytoalexin 10-oxodepressin, whose production further provides an example of biosynthetic gene cluster assembly, as also depicted here.7,8To provide context two examples of diterpenoid metabolism are presented here . The fir2trans GGPP it has been shown that diterpenoid biosynthesis also can proceed from the all-cis -nerylneryl diphosphate (NNPP). At the time of the previous review only a single example from the Solanum genus was known.9 More recently NNPP-derived diterpenoids have been reported from other plant species as well  family member. While CPTs more typically produce long-chain isoprenoids, those forming NNPP fall within a phylogenetically distinct clade of short-chain (≤4 isoprenyl units) producing CPTs regardless of species. This indicates that use of such all-cis isoprenyl diphosphates, presumably as alternative terpenoid precursors, arose before separation of the relevant genera, all of which fall within the Asterid clade of the Eudicots. However, examination of a wider range of species will be required to more precisely determine when the underlying CPT gene duplication and neofunctionalization to production of shorter-chain isoprenyl diphosphate precursors arose, as well as any subsequent gene loss, along with the possibility of independent evolution of such functionality in other plant lineages.While generally derived from the all- as well .10,11 Intrans terpenoid precursors. These seem to have given rise to terpene synthases, leading to their common designation as class I enzymes. Briefly, this was first suggested by homology between their divalent magnesium (Mg2+) binding regions (a pair of DDxxD motifs in IDSs), which was further consistent with use of these co-factors to ionize allylic diphosphate ester bonds to initiate carbocation-based reactions in both cases, and latter supported by the observation of structural homology between the catalytically relevant domain in each family.12 This helical-bundle fold has been termed the α domain.13 Although the IDS gene duplication and neofunctionalization event giving rise to terpene synthases clearly occurred well before the origin of land plants, examples of more recent diversion of IDSs to terpene synthase activity also have been reported.14By contrast, GGPP is produced by members of a distinct family of isoprenyl diphosphate synthases (IDSs), which gave rise to the more prototypical all-via condensation forming alternative branching, cyclopropyl or cyclobutyl linkages have been found in plants  a much more complex cyclization reaction  contain at least one additional domain. Nevertheless, microbial-like terpene synthases composed of just an α domain whose sequence phylogenetically groups with those from microbes rather than the plant TPSs are found in nonseed plants (representing early diverging lineages). Notably, these are involved in mono- and sesqui-, but not di-terpene biosynthesis.24 Accordingly, these enzymes will not be further discussed here. However, this functional split, with smaller (mono- and sesqui-) terpenes produced by a separate enzymatic family in nonseed plants, highlights the use of TPSs for at least diterpenoid biosynthesis throughout all land plants, suggesting the ancestral TPS served such a function. Indeed, given the use of ent-kaurene derived diterpenoids as phytohormones in all extant land plants it has been hypothesized the evolution of diterpenoid metabolism in plants was initiated by acquisition of a bifunctional cyclase that first converts GGPP to ent-CPP terpenoids, ent-CPP . Indeed,ent-CPP synthase (CPS) as well as subsequently acting ent-kaurene synthase (KS), with extant examples of such bifunctional CPSKSs found in some nonseed plant species. Consistent with retention of the allylic diphosphate ester bond in ent-CPP, the KS exhibits class I terpene synthase activity . In addition, as indicated by the enantiomeric (ent-) nomenclature relevant to phytohormone biosynthesis, the initial bicyclization of GGPP yields one of four distinct stereoisomers of labda-13-en-8-yl+-15-diphosphate. Altogether DTCs can form close to 100 different compounds, although those identified to-date only yield 20 distinct products . These prototypically produce a labda-13-en-8-ylproducts .6-barrel domains, which have been hypothesized to share a common origin – i.e., via gene duplication and fusion.12 Consistent with this hypothesis it has recently been shown that a single such domain is capable of catalyzing an analogous protonation-initiated cyclization reaction with a geranylgeranyl dihydroxybenzoate derivative.27 The more typical pair of domains have been termed β and γ, with a primary sequence order of γβ.13 Fusion of a di-domain DTC, as still found in bacteria, to create the ancestral CPSKS provides the origin of the additional domains found in the TPS family  activity.28 A particularly early example of such domain loss seems to have occurred prior to establishment of seed plants (spermatophytes), leading to the TSP-d1 group found in gymnosperms and, hence, TPS-a, b & g subfamilies found in angiosperms.29Notably, protonation-initiated carbocationic cyclization reactions also are observed in triterpenoid biosynthesis, as catalyzed by oxido-squalene cyclases (OSCs) and squalene-hopene cyclases (SHCs), which share structural homology with the DTCs, leading to their common grouping as class II terpene cyclases. In particular, catalysis is carried out in an active site situated between a pair of (αα)S family . The relvia protonation. In the case of the ancestral SHCs as well as DTCs this is the ‘middle’ residue of a highly conserved DxDD motif, reflecting their common need to carry out the more energetically difficult protonation of an olefin relative to the less strenuous epoxide protonation mediated by OSCs (2+-binding motifs in class I terpene synthases (matching the pair of DDxxD motifs from the IDS family), both a highly conserved DDxxD but also less well conserved (derived) NSE/DTE motifs,30 serve as signatures of such activity. While loss of the γ domain is clearly indicative of monofunctional class I TPSs, these motifs are useful in elucidating basic activity of the tri-domain TPSs found in all land plants – i.e., at least one such TPS is required for phytohormone biosynthesis. Before spermatophyte divergence the ancestral CPSKS underwent gene duplication and subfunctionalization to yield the separate CPSs and KSs found in all extant species. Both of these retain the ancestral γβα tri-domain architecture, albeit with some degradation of the no longer relevant domains, particularly including loss of the corresponding catalytic motifs. Moreover, the KS and/or CPS(KS) required for phytohormone biosynthesis have given rise, via repeated gene duplication and neofunctionalization to more specialized metabolism, to extended sub-families of TPSs – i.e., CPS(KS)s to TPS-c and KSs to TPS-e − in a lineage specific manner. For example, as has been discussed for the Poaceae31 and Lamiaceae plant families.32The homology between DTCs and triterpene cyclases (SHC and OSC) extends to use of a conserved aspartic acid residue, located in the β domain, as the catalytic acid initiating cyclization  by OSCs . Accordient-kaurene, with substitution of threonine leading to production of an isopimaradiene instead, representing premature deprotonation of the usual carbocation cascade reaction. However, this hypothesis had only been tested in KSs from spermatophytes,33,34 but has now been demonstrated to hold true for KSs across the full phylogenetic range of land plants,35 where it is conserved within a KS-specific PIx motif .37 This result not only highlights the requirement for an otherwise inert active site, but also suggests how enzymatic engineering might be accomplished. In addition, it has been recently recognized that the KSs involved in phytohormone biosynthesis contain a similarly KS-specific extension of the DDxxD motif to TTxxDDxxD s involved in phytohormone biosynthesis have been recently defined. In the case of the KSs it had been previously proposed that a particular isoleucine was important for production of Ix motif . MoreoveTxxDDxxD , althougArabidopsis thaliana (AtCPS),39,40 it has now been determined the CPSs specifically involved in phytohormone biosynthesis contain a histidine and asparagine dyad, located in the γ domain across the active site cleft from the DxDD motif, which help tightly coordinate a water molecule likely serving as the catalytic base.41 These two residues appear to be functionally conserved across the full phylogenetic range of land plants, with alanine substitution invariably leading to an alternative hydroxylated product derived from the addition of water to ent-labda-13-en-8-yl+-15-diphosphate  and ferns (polypodiophytes). The absence of this subfamily in these two disparate plant lineages is hypothesized to stem from retention of the bifunctional CPSKS at least until the divergence of ferns, consistent with the presence of such enzymes in extant nonseed species including ferns. Given the lack of channeling between the class II and I active sites in bifunctional enzymes,49 a monofunctional KS can react with the ent-CPP released by the remaining CPSKS, although it is unclear if this activity is sufficient to explain retention of both.Notably, the observed conservation patterns suggest these motifs should be predictive for KS and/or CPS(KS) activity. Indeed, this hypothesis has recently been explicitly tested ectively .29 In adi.e., the other subfamilies . Given their dedication to more specialized metabolism and apparently monophyletic origin, these are termed here clan S. In addition, this recent analysis reveals deep divisions within the TPS-d and TPS-h subfamilies. While those from TPS-h largely follow plant lineage, those from TPS-d (by definition limited to gymnosperms) are informative. In particular, while the previously defined TPS-d3 group largely consists of bifunctional diterpene cyclases (much like the TPS-h subfamily), it is now clear this gave rise to the TPS-d2 group, characterized by monofunctional class I mono- or sesqui-terpene synthase activity yet retaining the ancestral γβα tri-domain architecture, this then gave rise to the TPS-d1 group wherein the now defunct γ domain is lost, which in turn gave rise to all the remaining subfamilies found in angiosperms  product outcome in abietane synthases.50,51 This alanine along with the surrounding residues defines a VSIAL motif highly conserved in these enzymes and is just four residues upstream of the key isoleucine in KSs. While no structures have been reported for KSs or any other members of the TPS-e subfamily , structural analysis of the abietane synthase from the gymnosperm Abies grandis (AgAS) demonstrates that the key alanine sits at a widely conserved (G1/2) helix-break.52 The key isoleucine in KSs then sits lower in the active site, consistent with the distinct (enantiomeric) stereochemical configuration of the relevant substrates. Regardless, it is notable that the two TPS-h subfamily members from lycophytes recently shown to produce abietanes (HsLS and PdLS) contained all three of these abietane synthase specific motifs and are relatively closely related to TPS-d3, suggesting that such activity may have evolved prior to the divergence between lycophytes and gymnosperms  stemming from addition of water -ent-kolav-3-enyl diphosphate  that serves as the catalytic base was uncovered not only by structural analysis of AtCPS,of water .41 This e OlIAS; .29 Similhosphate 48 – whics OpKOS; .29e.g., in KSLs from rice (Oryza sativa) and wheat (Triticum aestivum),53,54 as well as the Lamiaceae plant family,55 and even more generally.25,56,57 Accordingly, it seems likely changes in DTC product outcome may be immediately accommodated by subsequently acting extant enzymes, providing a means for facile metabolic evolution of labdane-related diterpenoids.Given the ease with which DTCs are converted to alternative product outcomes, how readily these can be further elaborated upon is a key aspect of metabolic evolution. Notably, at least some substrate promiscuity has been shown with the subsequently acting class I TPSs – i.e., labdane-related diterpenoid biosynthesis) is not confined to just the TPS-e subfamily and TPS-d3 group, as such functionality has been shown for TPS-b subfamily members, at least in the Tripterygium genus.58–60 Conversely, beyond the noted derivation of TPS-e subfamily members to react directly with GGPP that was used to define the TPS-f subfamily, similar reactivity also is evident within TPS-d3 given the production of taxadiene by members of this group.61 The increased number of carbon–carbon double bonds in GGPP (and NNPP) relative to the precursors for smaller terpenoids , enables a dizzying array of routes for the catalyzed carbocation cascades, which is even further expanded upon by the ability to catalyze isomerization to the tertiary intermediate geranyllinalyl diphosphate (GLPP), enabling 1,6-cyclization  > 8, requiring the addition of at least two spatially separated oxy groups to acquire reasonable solubility (log P ≤ 5), matching the usual composition of the simplest diterpenoids with known biological activity – e.g., rice oryzalexins  super-family, whose membrane localization provides access to these hydrophobic diterpenes, particularly given the apparent exchange of such metabolites between the plastid and endoplasmic reticulum where CYPs are located (possibly via hemi-fusion of the outer leaflet of the outer membrane of each of these organelles).63 Regardless of how access is achieved, the CYPs also make essential contributions to (di)terpenoid biosynthesis.64,65 Phylogenetic relationships within the CYP super-family can be inferred to some extent in the associated nomenclature as, by definition, the numbered families share >40% and lettered subfamilies >55% amino acid sequence identity, with individual members assigned numbers.66 As an example of such nomenclature the ent-kaurene oxidase involved in phytohormone biosynthesis from A. thaliana is CYP701A3.67 However, given the increasing amounts of sequence data some merging of (sub)families has inevitably occurred, which has been addressed with assignment of tribes to describe such latter realized phylogenetic relationships.68,69While the TPSs can generate oxygenated products zalexins .62 The rent-kaurene oxidases, which seems to be necessary for phytohormone biosynthesis throughout all land plants,1 with only individual members from rice and maize found to exhibit alternative activity.70–72 Nevertheless, it should be noted with the subsequently acting CYP88 family (at least for gibberellin phytohormone biosynthesis occurring in vascular plants), where almost all characterized members act as ent-kaurenoic acid oxidases, it has been reported that a few members operate in more specialized triterpenoid biosynthesis,73 hinting at the possibility that others may be involved in more specialized diterpenoid metabolism. Regardless, more specialized diterpenoid biosynthesis seems to largely rely on use of the same (sub)families operating in other such metabolism. For example, members of the expansive CYP71D subfamily operate in not only diterpenoid but other types of terpenoid, and even flavonoid, biosynthesis.74 Nevertheless, there is some correlation between CYP (sub)family and metabolic function, at least within the various plant lineages.Unlike TPSs, there is very little direct derivation of CYPs from those involved in phytohormone biosynthesis to more specialized diterpenoid metabolism. Indeed, the only known examples stem from the CYP701 family, where almost all characterized members act as 75 as well as CYP725A subfamily in Taxol biosynthesis,76,77 has long been appreciated.74 Also apparent at the time of the last review1 were roles in diterpenoid biosynthesis played by members of the CYP71Z, CYP76M, CYP99A and CYP701A subfamilies in rice,78 CYP76AH subfamily in the Lamiaceae,79,80 and CYP71D subfamily in tobacco.81 Since that time, it has been shown that members of the CYP71Z,7,8,82,83 as well as CYP701A,71 subfamilies operate in more specialized diterpenoid biosynthesis throughout the Poaceae plant family.31 Some evidence also has been provided suggesting broader use of the CYP76M subfamily, along with possibly the CYP76L subfamily, in Poaceae diterpenoid biosynthesis as well.84,85 Both of these CYP76 subfamilies, as well as the CYP71Z and CYP99A subfamilies, are unique to this plant family, suggesting these may have evolved for such metabolic roles. However, rather than a common origin the CYP701A subfamily members operating in more specialized diterpenoid metabolism in rice and maize appear to have independently diverged from the ancestral role in phytohormone biosynthesis, providing an example of parallel evolution. In Lamiaceae, since the time of the last review additional roles for CYP76AH,86 and roles for the CYP71AU,87 CYP71BE,88 CYP71D,89 CYP76AK90 and CYP76BK91 subfamilies in diterpenoid biosynthesis have been reported. Again, given that the CYP76AH, CYP76BK and CYP76AK subfamilies are unique to this plant family, these may have evolved for this purpose. By contrast, the CYP71AU and CYP71BE subfamilies are found throughout eudicots, with very few characterized members, so any broader roles in diterpenoid biosynthesis are uncertain. In addition, roles for the CYP71D as well as CYP726A subfamilies have been reported in macrocyclic diterpenoid biosynthesis in the Euphorbiaceae plant family.92–95 The CYP71D subfamily also has been found to operate in tomato diterpenoid biosynthesis.9 Notably, it is now evident that the CYP71D subfamily is intermixed with the CYP71BE subfamily, and also encompasses the CYP726 family along with CYP71AV, CYP71AU and CYP71AY subfamilies as distinct clades . This tribe is fairly closely related to the CYP71Z subfamily and CYP99 family (which is a distinct clade within the CYP71 family), hinting at the possibility that these evolved for roles in (di)terpenoid biosynthesis within angiosperms , which is catalyzed by CYP82G in Arabidopsis,97 but CYP92C in maize (Zea mays),98 provides another example of parallel evolution within the CYP superfamily in distinct lineages, here potentially monocot versus eudicot .92,95 Although these differ in absolute stereochemistry, that just applies to the cyclopropyl moiety, with the macrocycle actually targeted for hydroxylation otherwise exactly analogous, such that this is again suggestive of parallel evolution in monocots and eudicots  as an early step in phytocassane and oryzalexin biosynthesis,70 but as a later step in momilactone biosynthesis,84,103 although it also can react  at an earlier step as well.72 The latter type of promiscuity has been found with many CYPs and indicates that the relevant biosynthetic processes may operate as metabolic grids rather than linear pathways. By contrast, in certain cases it has been found that previous modification can block activity. For example, in rice oryzalexin biosynthesis CYP701A8 catalyzes C3α-hydroxylation of ent-sandaracopimaradiene,70 but does not act on the 7β-hydroxy derivative.62 In other cases, previous modification alters regiospecificity – e.g., CYP76M6 and CYP76M8 both catalyze 7β-hydroxylation of ent-sandaracopimaradiene, but only CYP76M8 does so with the C3α-hydroxy derivative, forming oryzalexin D, while CYP76M6 instead catalyzes C9-hydroxylation, forming oryzalexin E ,89 and that CYP99A & CYP76M subfamily members cooperatively form a hemi-aldehyde as a precursor to the lactone ring characterizing the rice momilactones.84,103 These examples emphasize the importance of CYP activity to diterpenoid biosynthesis  in gibberellin biosynthesis and carbon–carbon double bond formation in tanshinone biosynthesis (Relatively little is known about the subsequently acting tailoring enzymes involved in (di)terpenoid biosynthesis. These are generally soluble enzymes that act on the now soluble metabolites produced by TPSs and CYPs, but can continue to add oxygen to increase polarity and hydrogen-bonding capacity. For example, the final steps in gibberellin phytohormone biosynthesis, as well as several regulatory/catabolic reactions, are catalyzed by 2-oxoglutarate-dependent dioxygenases (2ODDs).ynthesis .via oxidation of aldehydes in their gem-diol form to a carboxylic acid, some families of which have been specifically expanded in plants.117 Roles for SDRs in more specialized diterpenoid metabolism has been reported since the last review, which did not cover such enzymes.1 In particular, SDR activity relevant to biosynthesis of the rice oryzalides118 and momilactones,84,103 as well as macrocyclic diterpenoids in Euphorbiaceae.93,119 Notably, in the Euphorbiaceae one of the identified SDRs catalyzes rearrangement (further cyclization) of the hydrocarbon backbone terpenoid biosynthesis.Another enzymatic super-family whose activity can lead to the addition of oxygen is the short-chain alcohol reductases/dehydrogenase (SDRs), albeit only backbone ,93 while76,77 Several UDP-dependent glycosyltransferases have been identified from stevioside biosynthesis as well.120,121 More recently, two aceyltransferases have been identified for forskolin biosynthesis in Coleus forskohlii.86 In each case, these functional groups are critical for the desired biological activity, highlighting the important role these enzymes can play in diterpenoid biosynthesis.76,77,122Regardless of origin, the presence of hydroxy groups provides a facile target for addition of larger functional groups. Only a few such transferases are known in diterpenoid metabolism. Early work identified several types of transferases involved in Taxol biosynthesis.5123–125 Here the discussion will be largely limited to those associated with diterpenoid metabolism. At the time of the last review just two such BGCs were known.1 Both were found in rice, are involved in producing labdane-related diterpenoids and were discovered based on the early sequencing of the rice genome.126,127 In particular, as this not only enabled biochemical investigation of its arsenal of sequentially acting class II diterpene cyclases (CPSs) and class I synthases (KSLs),128,129 but also recognition of their functional clustering.130–132 The accompanying CYPs and SDRs were latter shown to operate on the products of the co-clustered CPS and KSL(s),62,70,104,133–136 meeting the formal plant BGC definition – i.e., at least three genes of distinct evolutionary origin contributing to a shared metabolic pathway.124 Since that time another diterpenoid BGC has been identified in rice,7,8 and the three rice diterpenoid BGCs shown to exhibit varying degrees of conservation across the level of species7 and genus,85 as well as the Poaceae plant family more broadly.137,138 In addition, a diterpenoid BGC conserved within the Lamiaceae32 and another conserved within the Euphorbiaceae92,94,95 along with one from a bryophyte,96 also have since been reported.The presence of biosynthetic gene clusters (BGCs)s and their implications for metabolic evolution within plants is a topic of some interest.134 Moreover, the first evidence that negative selection pressure against interruption of particular biosynthetic pathways might be involved in BGC assembly was provided from genetic studies with the BGC on rice chromosome 4 (Os4BGC) associated with production of the momilactones, where a knock-out line for the relevant OsKSL4 was found to exhibit reduced seed germination rates.139 This is coupled with the expected positive selection pressure for production of the final bioactive natural product,140 in the case of Os4BGC the momilactones that not only act as phytoalexins and allelochemicals,139,141,142 but may also play a role in regulating stomatal opening in certain genetic backgrounds .143 Together this supports co-inheritance as the primary driving force for assembly of at least the Os4BGC.Arguably the best understood diterpenoid BGCs are those from rice, which were among the first to be identified and have been extensively investigated, providing a wide overview of BGC function and evolution even more generally . For exa84 Intriguingly, genetic dissection of these two rice BGCs further demonstrated that loss of the Os2BGC, and even more specifically the two  CYP76M(7/8) genes involved in momilactone biosynthesis, led to a deleterious (lesion mimic) phenotype, which was dependent on the presence of the Os4BGC, again consistent with negative consequences for interruption of momilactone biosynthesis.105 The lack of recruitment of the relevant gene to the Os4BGC may be due to tandem CYP76M gene duplication within the Os2BGC, which has been suggested to have an earlier origin within the Oryza genus.85 In particular, the original CYP76M subfamily member presumably originally operated in biosynthesis of the phytocassanes with which this BGC is most closely associated – e.g., this contains the upstream OsCPS2 and OsKSL7 ref. 84 there simply may not have been sufficient time and/or selective pressure for the transfer of CYP76M8 from the c2BGC to the c4BGC. A similar argument might be made for the lack of recruitment of CYP701A8, but it should be noted that its location in a tandem array of CYP701A orthologs, including the requisite kaurene oxidase for gibberellin phytohormone biosynthesis, ensures its co-inheritance. The multiple roles CYP701A8 plays in various biosynthetic processes further complicates the selective pressures on this gene.However, elucidation of the momilactone biosynthetic pathway further revealed that it is not entirely encoded by the Os4BGC, as genes from the Os2BGC , as well as a separate CYP701A tandem gene array, are also required.84 However, this is even more widespread in the Os2BGC, which actually contains four closely related CYP76M subfamily members , along with two from the CYP71Z subfamily, and three KSLs ,144 although not others ,132 yet appear to have been recruited latter in the Os2BGC assembly process and are clearly a result of subsequent tandem gene duplication.85 In these latter cases, it is unclear what drove the sweep of the BGC containing the  redundant duplicated genes throughout the population (or if these simply accompanied other desirable loci selected for during domestication).Similar gene duplication appears to have occurred in the Os4BGC, where apparently redundant CYP99A(2 & 3) subfamily members are present see , along wree KSLs . While m7,8 This composition does not meet the formal BGC definition, as the CYP71Z subfamily members appear to have arisen from local gene duplication. However, the CYP71Z subfamily members have diverged to catalyze distinct reactions in the relevant biosynthetic pathway, providing insight into the selective pressure for this gene duplication event. The Os7BGC also is noteworthy for two other features. First, in its basis from ent-casbene, resulting from direct reaction with GGPP by the encoded TPS, which draws an interesting parallel with the Euphorbia diterpenoid BGC, as this similarly encodes casbene synthases, and both BGCs contain CYPs carrying out analogous hydroxylation reactions as well . However, despite these biochemical similarities the two BGCs are not otherwise homologous , indicating their independent assembly. Consistent with the co-inheritance model, the macrocyclic casbene has been reported to exert phytotoxic membrane disruption, which is not expected to vary with the limited configurational difference between enantiomers, suggesting analogous negative selection pressure against casbene synthases in the absence of downstream acting CYPs.8 Second, production of the diterpenoid phytoalexin (ent-10-oxodepressin) encoded by the Os7BGC exhibits a limited distribution, almost exclusively found in the japonica subspecies (ssp.) of rice, with only one example found in the >10 examined lines of ssp. indica.7 Given the usually distinct growth conditions for these two major subspecies, it seems likely that the observed distribution of the Os7BGC might reflect a selective advantage for production of ent-10-oxodepressin in the usual conditions for ssp. japonica but not indica. This is consistent with recently advanced hypothesis for BGC assembly,123 as such concentrated genetic architectures are expected to arise when a locally adaptative trait is evolving within a wider population and there is migration between local populations. However, it should be noted that ent-10-oxodepressin has only been reported to act as a phytoalexin against the same fungal blast pathogen (Magnaporthe oryzae)8 and bacterial leaf blight (Xanthomonas oryzae)7 targeted by the other rice diterpenoid phytoalexins,141,142 whose production relies on the two other more widely distributed BGCs.The most recently identified rice diterpenoid BGC, located on chromosome 7 (Os7BGC), contains a single TPS along with at least two closely related members of the CYP71Z subfamily see .7,8 ThisOryza genus that similarly produce momilactones,85 but also in barnyard grass 145 and wheat,138 as well as even more widely throughout the grasses.146 Given the evolutionary divergence between rice and especially barnyard grass, as these are separated between the two major clades in the Poaceae plant family, this BGC appears to provide an example of lateral gene transfer.137,146 However, while nothing is known about the metabolic pathway encoded by the BGC from barnyard grass, it has been shown that at least the CPS and KSL from the relevant wheat BGC exhibits divergent function.138 Accordingly, this BGC does not appear to have evolved for momilactone production, and not only the identity but also function of the resulting labdane-related diterpenoid remains unclear in the other genera where it is found. On the other hand, it must be noted that the bryophyte C. plumiforme has been found to not only produce momilactones but also contain an associated BGC, which clearly independently evolved from that found in the Poaceae despite proceeding via functional analogous biosynthetic transformations.96 Given the parallels to what has been observed in rice, potential phytotoxicity of the relevant intermediates has been suggested, which would again be consistent with a primary role for co-inheritance in driving assembly of this BGC as well.137By contrast, the Os4BGC exhibits a dramatically broader distribution, with homologous BGCs found in not only other species from the 6ent-kaurenoic acid for use in phytohormone biosynthesis. Indeed, as discussed above this provided the origin of the plant TPS family and, hence, more specialized terpenoid metabolism throughout the spermatophytes (seed plants). Highlighted here is a recent report that provided further insight into the early evolutionary history of this key enzymatic family, particularly as this included predictive use of motifs hypothesized to be specific to the ent-CPP and ent-kaurene synthases conserved for phytohormone biosynthesis, which were identified despite the repeated derivation of these enzymes to more specialized diterpenoid metabolism in a plant lineage specific manner. Related insights into at least class II diterpene cyclase enzymatic structure–function relationships provide additional predictions, verification of which will enable more confident bioinformatic suggestions for biochemical activity of such enzymes more broadly. With regards to the subsequently acting CYPs it appears that different subfamilies have been recruited to diterpenoid biosynthesis in distinct plant lineages as well, again of potential use in investigating such metabolism. Relatively little is known about even further downstream tailoring enzymes, offering a fertile field for future investigation – e.g., the installation of nitrogen in diterpenoid alkaloids remains completely opaque. Finally, the importance of more specialized diterpenoid metabolism is highlighted by the assembly, as well as subsequent lateral gene transfer, of associated BGCs, investigation of which appears to support co-inheritance as the primary driving force, although this is another area ripe for further studies.As described here, the origins of plant diterpenoid metabolism can be traced back not just to the ubiquitous need for GGPP for phytosynthetic pigments but more specifically the early production of 7There are no conflicts to declare.


After adjusting for confounding factors, the logistic regression model revealed that the associated factors were dizziness and loss of balance experience , self-awareness of abnormal voice , subjective hearing discomfort , and tinnitus . The inversely associated factor for dry eye was bilateral hearing loss . The hearing threshold was lower in the dry eye group than in the non-dry eye group (p < 0.05). Conclusions: Tinnitus was associated with dry eye and bilateral hearing loss was inversely associated with dry eye. These results suggest that hypersensitivity of the senses and nerves, which is neuropathic hyperesthesia, is one of the main mechanisms of dry eye. Treatment of neuropathy may help in treating dry eye associated with dizziness or tinnitus.This study aimed to evaluate the association between dry eye and inner ear diseases in a Korean population. Methods: Data from the Korean National Health and Nutrition Survey , a national cross-sectional health examination and survey, were collected by the Korea Centers for Disease Control and Prevention. The association between dry eye and inner ear disease was determined using the chi-square test and logistic regression analysis. The individuals were divided into two age groups (<60 and ≥60 years). Results: In total, 17,542 individuals ( Dry eye is a common disease, with a prevalence of 5–50% ,2. Dry eNeuropathic mechanisms include hypersensitivity of the peripheral nerve to normal sensations, experiencing non-existent sensations, and perception of sensations to be suppressed in the central nervous system ,14. TinnThe Korean National Health and Nutrition Examination Survey (KNHANES) was approved by the Institutional Review Board (IRB) of the Korean Centers for Disease Control and Prevention, and all participants provided written informed consent. This study adhered to the tenets of the Declaration of Helsinki and was approved from IRB approval by the institutional review board of Hallym University Kangnam Sacred Heart Hospital.For this cross-sectional, population-based study, we used data from the Korean National Health and Nutrition Survey V (2010–2012), a series of cross-sectional surveys of nationally representative samples of the civilian Korean population, conducted annually to assess the health and nutritional status of the South Korean population. To obtain representative samples, KNHANES uses a stratified, multistage, cluster probability sampling design according to geographical area, age, and sex. For the health interview survey, a trained interviewer asked questions directly to individuals aged ≥19 years old. This study included 17,542 adults  aged ≥19 years who met the eligibility criteria, completed a questionnaire regarding independent risk factors, and underwent slit-lamp examinations. Dry eye was evaluated by ophthalmologist. The subjects were asked whether they were diagnosed with dry eye by an ophthalmologist as like the interview about whether thyroid disorder or heart diseases was diagnosed. Interview was conducted by ophthalmologists. Risk factor analysis using this type of dry eye diagnosis by KHANES has been reported in a lot of studies ,21,22,23Pure-tone audiometric testing was conducted using an SA 203 audiometer . All audiometric tests were performed in a soundproof booth inside a mobile bus reserved for the KNHANES under the supervision of an otolaryngologist. The lowest level at which the participant responded to 50% of the pure tone was set as the threshold. The tested frequency ranges were 0.5, 1, 2, 3, 4, and 6 kHz. Hearing loss was categorized as unilateral or bilateral. Unilateral hearing loss was defined as hearing loss at a threshold of ≥25 dBHL in the ear with worse hearing, and bilateral hearing loss as hearing loss at ≥25 dBHL in the ear with better hearing.p-values less than 0.05 were considered statistically significant. The chi-square test was used to compare discrete variables between the groups. The percentage (%) differences were calculated as the absolute value of the change in value divided by the average of the two numbers, all multiplied by 100. To estimate the odds ratios (ORs) of dry eye and potential factors, we conducted logistic regression analyses using a generalized linear model for a complex survey design. The ORs and 95% confidence intervals (CIs) were calculated in the following ways: confounder adjustment for age and sex.Statistical analyses were performed using SPSS Version 27.0 , and two-sided p < 0.001), self-awareness of abnormal voice (p < 0.001), subjective hearing discomfort (p = 0.002), tinnitus (p < 0.001), tympanic membrane of the left ear (p = 0.030), and bilateral hearing loss (p < 0.001) in dry eye.The characteristics of the study population are shown in In the logistic regression analysis between dry eye and potential risk factors adjusted for age, sex, dizziness, loss of balance, self-awareness of abnormal voices, subjective hearing discomfort, tinnitus, and bilateral hearing loss , the risDry eye is mainly caused by decreased tear volume and increased evaporation . Risk faDiseases of the inner ear, including dizziness, loss of balance, self-awareness of abnormal voice, and tinnitus, may contribute to the pathogenesis with dry eye. Dizziness and loss of balance indicate disturbances or inflammation of the inner ear, such as labyrinthitis or vestibular neuritis ,32. NeurThe possible mechanisms of the association between dry eye and tinnitus can be suggested as follows. First, both dry eye and tinnitus may have common causes, such as neuropathy or neuroinflammation. Neuropathic causes are important in dry eye ,44,45, aConversely, bilateral hearing loss was inversely associated with dry eye. The causes of bilateral hearing loss include age, noise exposure, heredity, and medication . The neuThe limitations of this study are that this study was conducted in Korean adults older than 19 years and dry eye diagnosis was assessed by interviews without objective examination of ocular surface and tear film. However, dry eye was evaluated by ophthalmologists. There have been a lot of studies reporting risk factor analysis using this type of dry eye diagnosis by KHANES ,21,22,23Tinnitus is associated with dry eye, whereas bilateral hearing loss is inversely associated with dry eye. The hearing threshold was lower in the dry eye group. These results suggest that hypersensitivity of the senses and nerves, which is a neuropathic hyperesthesia, is the primary mechanism of dry eye. Treatment of neuropathy may help treat dry eye syndrome associated with dizziness or tinnitus.


Helichrysum stoechas (L.) Moench is a medicinal plant traditionally used in the Iberian Peninsula to treat digestive disorders, but its effects on gastrointestinal motility have not been scientifically demonstrated. The aim of this work was to evaluate the antispasmodic effect of a polyphenolic extract of H. stoechas (HSM), its mechanism of action and its antioxidant activity. Isometric myography studies were performed in rat ileum, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) levels were measured in rat jejunum. HSM reduced the integrated mechanical activity of spontaneous contractions. In Ca2+-free medium, HSM reduced the concentration–response curve of CaCl2 similarly to verapamil. Pre-incubation with the extract blocked the contraction induced by Bay K8644, KCl and carbachol. L-NAME, ODQ, Rp-8-Br-PET-cGMPS, KT-5823, apamin, TRAM-34 and charybdotoxin reduced the relaxant effect of the extract on spontaneous contractions. MDA+4-HDA levels in LPS-treated tissue were reduced by the extract, showing antioxidant activity. In conclusion, HSM showed antispasmodic activity through inhibition of Ca2+ influx, activation of the NO/PKG/cGMP pathway and opening of Ca2+-activated K+ channels. The results suggest that H. stoechas could help in the prevention or treatment of FGIDs.Functional gastrointestinal diseases (FGID) are worldwide prevalent conditions. Pharmacological treatments can be ineffective, leading the population to turn to herbal or traditional remedies.  Digestive disorders with symptoms without a clear etiology, as abdominal pain, motility alteration or nausea, are included as functional gastrointestinal diseases (FGID), affecting a large part of the population ,2. IrritCAM is all practices and products that are not considered part of modern medicine. CAM includes not only herbs and other plant treatments but also non-botanical supplements and mind–body therapies . AccordiHelichrysum arenarium, for instance, has been approved by the WHO and the EMA for the treatment of digestive problems such as fullness and bloating -1H-pyrazole, a selective inhibitor of intermediate conductance Ca2+-activated K+ channels (IKCa) (1 µM); charybdotoxin, a specific inhibitor of intermediate- and large-conductance Ca2+-activated K+ channels ; glibenclamide, an inhibitor of ATP-sensitive K+ channels (KATP) ; BaCl2, an inhibitor of the inward rectifier K+ channel (KIR) ; and quinine, an inhibitor of voltage-sensitive K+ channels .H. stoechas extract on the AUC from the ileum spontaneous contractions . However, glibenclamide, BaCl2 and quinine, although they reduced this effect slightly (by approximately 15%), did not reach statistical significance , respectively. However, the fact that glibenclamide, BaCl2 and quinine did not modify the H. stoechas extract-induced relaxation showed that ATP-sensitive, inward rectifier or voltage-sensitive K+ channels are not involved. Thus, these findings suggest that the spasmolytic activity of H. stoechas is mediated through the opening of Ca2+-activated K+ channels. Similar results were obtained with essential oil of Chrysactinia mexicana in rabbit ileum oxadiazolo quinoxalin-1-one (ODQ) and Rp-8-Br-PET-cGMPS were purchased from Tocris . Bay K8644 was dissolved in ethanol. Apamin was diluted in acetic acid. Gliblencamide, TRAM-34 and ODQ were prepared in dimethyl sulfoxide (DMSO). All other chemicals were dissolved in distilled water.Acetylcholine (ACh), carbamoylcholine chloride (carbachol), Bay K8644, apamin (AP), charybdotoxin (ChTx), glibenclamide (Glib), quinine (Q), verapamil (V), H-89 dihydrochloride hydrate, KT-5823, (+)-(R)-trans-4- (1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and barium chloride dihydrate , calcium-free Krebs  1) or Ca2+-free high K+ Krebs ([K+]o = 50 mM). The buffers were adjusted to pH 7.4. The compounds of the buffers were obtained from Sigma.Tissues were incubated in Krebs buffer  from Escherichia coli O111:B4 was obtained from Sigma.Male Wistar rats weighting 200–250 g were purchased from Janvier, LeGenest St. Isle, France. Animals were fed ad libitum with standard feed and free access to water.The experimental protocols were approved by the Ethics Committee from University of Zaragoza under Project License PI66/17 (18 January 2018). Animal care and use of animals were conducted in accordance with the Spanish Policy for Animal Protection RD 53/2013, RD1386/2018 and RD118/2021, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes.2 and 5% CO2. Mechanical activity was amplified with a range of 2 mV, recorded and digitalized using a data acquisition system , Cibertec, Madrid, Spain). An initial tension of 1 g was applied to the preparations to achieve spontaneous contractions. The segments were allowed to equilibrate in Krebs solution for 60 min before use by changing the bath buffer every 20 min.After cervical dislocation, ileum was carefully removed, placed in ice-cold Krebs buffer, and cleaned of fat and adherent connective tissue. The ileum was cut into longitudinal smooth muscle segments (10 mm long) and each segment was individually connected to an isometric force transducer  for tension measurement. The organ bath, with 5 mL Krebs buffer, was maintained at 37 °C and gassed with 95% OH. stoechas extract, methanol (solvent of the extract) or verapamil, increasing concentrations of these compounds were added every 10 min and cumulative concentration–response curves were performed. The spontaneous contractions of the ileum recorded in Krebs solution before agents were considered as the control. The relaxant effect of H. stoechas was calculated as the percentage change from the control period (Krebs).After the stabilization period, the spontaneous contractions of longitudinal smooth muscle from ileum were recorded. To study the effect of 2+ influx on the H. stoechas extract-evoked relaxation of longitudinal smooth muscle, after incubation with Krebs buffer, the segments were incubated with Ca2+-free Krebs solutions for 20 min and then with Ca2+-free high-K+ buffer. Subsequently, the segments were pre-incubated with methanol, H. stoechas extract (0.3 and 1 mg/mL) or verapamil (1 µM) for 15 min and cumulative concentration–response curves for CaCl2 (10−5–10−2 M) were performed by adding each CaCl2 concentration every 5 min. The responses to CaCl2 obtained in the presence of methanol served as control.To examine the role of CaH. stoechas extract on L-type Ca2+ channels was evaluated. Thus, ileum segments were incubated 15 min with H. stoechas extract (0.3 and 1 mg/mL) and verapamil (10−6 M) before the addition of Bay K8644. The contractile response of Bay K8644 obtained in presence of H. stoechas extract or verapamil was compared with the response obtained by Bay K8644 alone .Furthermore, the effect of the H. stoechas extract (1 mg/mL) on the effects induced by the contractile agents KCl and CCh. The tissue was incubated for 15 min with the extract or the solvent prior to the addition of KCl (80 mM) or CCh (10−6 M). Contractions to KCl or CCh in the presence or absence of extract were compared to the control period.A similar protocol was used to examine the effects of H. stoechas extract (1 mg/mL) in spontaneous motility, segments were incubated 20 min before the extract with L-NAME (10 µM), an inhibitor of NO synthase (NOS); ODQ (1 µM), a potent and selective inhibitor of soluble guanylyl cyclase; Rp-8-Br-PET-cGMPs (1 µM), an inhibitor of cGKI and cGKII; KT-5823 (1 µM), a selective inhibitor of PKG, H-89 (200 nM), an inhibitor of the PKA; and 2,5-dideoxiadenosina , an inhibitor of the soluble adenylyl cyclase.To investigate the participation of AMP/protein kinase A (PKA) and nitric oxide (NO)/GMP/protein kinase G (PKG) pathways on the relaxant response evoked by + channels were involved in the response of the H. stoechas extract (1 mg/mL), 20 min before addition of extract, ileum segments were pre-incubated with apamin, a selective small-conductance Ca2+- and voltage-activated K+ channel (SKCa) inhibitor ; TRAM-34 (1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole, a selective inhibitor of intermediate conductance Ca2+-activated K+ channels (IKCa) (1 µM); charybdotoxin, a specific inhibitor of intermediate- and large-conductance Ca2+-activated K+ channels ; glibenclamide, an inhibitor of ATP-sensitive K+ channel (KATP)  and BaCl2, an inhibitor of the inward rectifier K+ channel (KIR), ; and quinine, an inhibitor of voltage-sensitive K+ channels .To assess whether KH. stoechas extract (1 mg/mL), two combinations of inhibitors were also incubated: TRAM-34+AP and L-NAME+TRAM-34+AP, and their effects on the H. stoechas-induced relaxation compared with those evoked by each inhibitor alone.To study the possible simultaneous role of several pathways in the effects of the 2+-independent Rho-kinase pathway on H. stoechas extract (1 mg/mL) effect was studied with the Rho-kinase inhibitor Y-27632 (10–6 M). In this series of experiments, the response to each substance in the presence of H. stoechas extract was compared with the response obtained with the spontaneous contractions (control).Using the same protocol, the role of the CaSegments that did not show spontaneous activity were discarded. Thus, each segment served as its own control. Each experimental protocol was performed on ileum segments of longitudinal smooth muscle from 6–8 animals.H. stoechas extract (0.1 or 1 mg/mL), lipopolysaccharide  and H. stoechas+LPS (each at the two referred concentrations). After 1 h of incubation with H. stoechas extract or methanol, LPS or its solvent was added. After 2 h, the pieces of jejunum were frozen in dry ice for MDA+4-HDA analysis. The tissues were homogenized in ice-cold Tris buffer  and centrifuged at 3000× g for 10 min at 4 °C. In the assay, MDA+4HDA reacts with N-methyl-2-phenylindole to yield a chromophore with maximal absorbance at 586 nm; 1,1,3,3- tetramethoxypropane was used as a standard. Results were expressed as nmol MDA+4-HDA per milligram of tissue.The tissue concentrations of MDA and 4-HDA (MDA+4-HDA) were used as an index of lipid peroxidation. After cervical dislocation, jejunum was extracted. Jejunum samples were incubated at room temperature in Krebs buffer divided into the following experimental groups: MeOH (as control), 1) minus the AUC of the spontaneous contractions 3 min before adding the substance (control) (AUC0) (AUC = AUC1 − AUC0). Data were calculated as the percentage with respect to the mean value of the control period and are expressed as mean ± SEM. Normal distribution of the samples was assessed by the Shapiro–Wilk test. Statistical significance was analyzed using the Kruskal–Wallis test (non-parametric) followed by post hoc Dunn’s test. p values < 0.05 were considered statistically significant. The concentration of compound that inhibited 50% of the maximal contraction (EC50) was expressed as a geometric mean with 95% confidence intervals (IC) and calculated for the concentration–response curve. Statistical analyses and figures were carried out using GraphPad Prism 6.Amplitude and frequency of spontaneous contractions were calculated as the average of peak-to-peak differences and as the number of contractions per minute, respectively, during a 5 min recording period. The area under the curve (AUC) represents the integrated mechanical activity per second (g/s) and normalized to weight (g) of wet tissue. The AUC was calculated as the AUC during the first 3 min of response to the studied substance (AUC


In November 2013, the first edition of evidence-based guidelines for treatment of gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) was published in Japan. However, whether medical practitioners have adopted the first-line regimens recommended for metastatic GEP-NEN in clinical practice is not yet known. The purpose of this study was to identify which first-line systemic therapy regimens have been selected and the proportion of cases that are adherent to the guidelines . We combined hospital-based cancer registry data and insurance claims-equivalent data for patients with GEP-NEN treated between January 2013 and December 2014 and extracted those with metastatic GEP-NEN who received systemic therapy. The proportions that were adherent with the guideline were calculated according to tumor classification (neuroendocrine tumor [NET] or neuroendocrine carcinoma [NEC]), primary site , and hospital volume . The study included 109 patients with GEP-NET and 424 with GEP-NEC. Overall, guideline-adherent treatment was provided in only 54.8% of cases . The recommended therapy for GEP-NET was used in 16.5% of patients with GEP-NEC, and 21.5% received fluoropyrimidine-containing chemotherapy. This report is the first to describe real-world selection of first-line regimens for metastatic GEP-NEN. About half of all these patients received systemic therapy that was not recommended in the guidelines. In the US, the annual incidence of GEP-NEN has been reported to be 3.56 per 100,000 population, with increasing trends in both incidence and prevalence3; in Japan, the annual adjusted incidence of GEP-NEN is reportedly 3.53 per 100,0005.Neuroendocrine neoplasm (NEN) is a rare heterogeneous tumor arising from neuroendocrine cells in the diffuse endocrine system. Although NENs can occur throughout the body, a gastroenteropancreatic (GEP) origin is most common. According to the World Health Organization classification7 and the National Comprehensive Cancer Network (NCCN)8 to promote standardization of clinical practice based on evidence obtained from clinical research. In November 2013, the Japan Neuroendocrine Tumor Society (JNETS) also published clinical guidelines for GEP-NEN for the same purpose. These guidelines recommend use of a somatostatin analog (octreotide or lanreotide), a molecular targeted agent (everolimus or sunitinib), and streptozocin for metastatic GEP-NET and a platinum-containing regimen (platinum plus etoposide or irinotecan) for metastatic GEP-NEC.Despite the rarity of GEP-NEN, clinical guidelines have been published by the European Neuroendocrine Tumor Society (ENETS)9 and patient insurance claims-equivalent data.However, whether the first-line regimens recommended in the guidelines for patients with metastatic GEP-NEN have been adopted in clinical practice in Japan remains unclear. Therefore, the purpose of this study was to clarify the real-world selection of first-line systemic therapy for metastatic GEP-NEN using both hospital-based cancer registry10. This project used patient insurance claims-equivalent data that had been linked to data from hospital-based registries since 2011. All cancer care hospitals designated by the Ministry of Health, Labour and Welfare were invited to participate in the project, as were non-designated cancer care hospitals that played similar roles and had submitted their hospital-based registry data to the National Cancer Center for its annual statistical report. Designated cancer care hospitals play a central role in cancer care across communities in Japan11. As a condition for receiving this designation, these hospitals are required to provide standard treatment, including surgery, chemotherapy, and radiation, for cancer patients.This study is a secondary analysis of health insurance claims data linked with the hospital-based cancer registry of the project examining quality assessments and improvementsThe data from the hospital-based cancer registries included the following information on GEP-NEN patients: (1) clinical profiles, such as birth date, sex, tumor sites, and histology codes according to the International Classification of Diseases for Oncology, third edition; (2) clinical tumor-node-metastasis (TNM) staging based on the Union for International Cancer Control (UICC); (3) diagnosis month; and (4) details of first-line systemic therapy. The insurance claims-equivalent data contained the details of care for each patient, such as all the procedures and prescriptions provided to the patient with the corresponding dates in a manner like that used for fee-for-service insurance claims. We have referred to this data as “claims-equivalent” because the data points themselves are not used for reimbursement but are instead collected to monitor the effects of changes to reimbursement rules on care utilization. These two data sources were linked and anonymized according to hospital before data submission to the National Cancer Center.In this study, data from the linked databases that were submitted to the National Cancer Center in 2013 and 2014 were used. Overall, 309 hospitals joined the project, with 240 joining in 2012  and 297 joining in 2013 . Information on 858,565 cancers  was collected. In this study using a secondary data, informed consent was obtained in the form of opt-out on the website.From the linked data, we extracted information on patients diagnosed as having metastatic GEP-NET  and GEP-NEC .First, we identified the systemic therapy regimen that was administered on schedule and closest to the month of diagnosis with GEP-NET or GEP-NEC; this regimen was defined as the first-line systemic therapy regimen. If another anti-cancer drug was administered within 14 days of the first dose, the treatment was considered to be a combination systemic therapy, and the combined therapy was defined as the first-line systemic therapy regimen.7, the NCCN guidelines8, and the JNET guidelines12, we defined octreotide (approved in 2011)13, lanreotide (approved in 2017)14, everolimus (approved in 2016)16 and streptozocin (approved in 2014)17 for metastatic gastrointestinal (GI)-NETs; octoreotide (approved in 2011)13, lanreotide (approved in 2017)14, everolimus (approved in 2011) 16, sunitinib (approved in 2012)18, and streptozocin (approved in 2014)17 for metastatic pancreatic (P)-NETs; and etoposide plus cisplatin/carboplatin and irinotecan plus cisplatin/carboplatin20 for metastatic GEP-NECs as the first-line standard systemic therapy regimens recommended in the guidelines . The proportion of cases with guideline adherence was defined as the number of patients who received systemic therapies recommended in the guidelines . All the tests evaluating significance were two-tailed, with the alpha value was at 0.05. All statistical analyses were performed using STATA version 14.2 software.This study was conducted in accordance with the Declaration of Helsinki, and this study was approved by the institutional review boards of the National Cancer Center, Japan (2013-081).Figure p = 0.065). When calculated according to year, the guideline adherence proportions were 58.8% (10/17) in 2013 and 57.7% (15/26) in 2014 . When calculated according to year, the guideline adherence proportions were 60.0% (15/25) in 2013 and 65.9% (27/41) in 2014 . When calculated according to year, the guideline adherence proportions were 48.5% (65/134) in 2013 and 61.3% (103/163) in 2014 . Calculating by year, the guideline adherence proportions were 36.4% (20/55) in 2013 and 51.4% (37/72) in 2014 , and 14.5% of the metastatic GI-NEC patients received treatment for metastatic gastrointestinal cancer . Similarly, 25.2% received treatment for P-NET, and 7.1% received treatment for pancreatic cancer . These non-adherent treatments might create very serious problems, especially in middle- or low-volume hospitals, as they could lead to a survival disadvantage arising from inappropriate treatment. Especially, prognosis of NEC remains poor20, accurate diagnosis and treatments are needed for metastatic NEC patients. Considering our results such as about 60% patients with GEP-NEN received systemic therapies in middle- or low-volume hospitals, standardization of treatments and centralization of patients might be needed for providing guideline-adhered treatment to advanced NEC patients.In contrast, GEP-NEC is generally a more aggressive tumor than GEP-NETAt present, the appropriate standardization of first-line systemic therapy for metastatic GEP-NEN does not yet seem to have been achieved in Japan. To ensure that metastatic GEP-NEN patients can receive appropriate palliative systemic therapy, the development and dissemination of the guidelines on GEP-NEN or educational activities regarding treatments for GEP-NEN patients might be solutions. In contrast, when the guideline adherence proportion for GEP-NEC from before the announcement of the Japanese guidelines on the Internet in November 2013 was compared with that after the announcement in 2014, the proportion increased significantly, suggesting a possible impact of the guidelines on treatment selection.5. The reason might be the difference in disease prognosis between GEP-NET and GEP-NEC; for example, GEP-NEC was an aggressive disease and was detected as an advanced stage. Therefore, the frequency of metastatic GEP-NEC patients received systemic therapy might be high.The number of GEP-NEC patients who received first-line systemic therapy was numerically more than that of GEP-NEN patients in this study and past study showed same tendency, too29, might enable a more appropriate dissemination of evidence-based medicine for metastatic GEP-NEN patients. Therefore, further research that includes detailed data on informative patient characteristics, such as disease status, anti-tumor activity of treatment, and survival outcome  is needed.This study had several limitations. First, the current study did not investigate the association between the guideline adherence proportion and treatment outcome because the linked data did not include survival data, such as the progression-free survival and overall survival periods. Secondly, because we could not exclude patients with multiple cancers, the guideline adherence proportions could have been underestimated or overestimated. An analysis using individual patient data might be useful for evaluating guideline-adherent drug therapies for tumors with a relatively good prognosis. Third, the hospital-based registries did not include locally assessed data for the Ki-67 index and differentiation, and there were only data for 2013 and 2014, which made in difficult to make evaluations based on the WHO classification or in other years. Moreover, the accuracy of local assessments of differentiation and the Ki-67 index were unclear. Therefore, it is possible that cases of NET-G3 were included in our NEC group. However, the standard treatments for NET-G3 were not established based on the results of clinical trials, and further investigations for NET-G3 are needed to decide guideline-recommended treatments. Fourth, the hospital-based registries did not include data on the date of pathological diagnosis. Therefore, some patients might have received palliative chemotherapy before diagnosis of GEP-NEN because of aggressive disease, leading to underestimation of the guideline adherence proportions. Finally, the second edition of the Japanese guidelines for GEP-NEN, published in 2019In summary, this is the first report on real-world adherence with the Japanese guidelines for first-line systemic therapy regimens for metastatic GEP-NEN. In this study, about half of the patients with metastatic GEP-NEN received systemic therapy that was not recommended in the guidelines. Appropriate centralization of metastatic GEP-NEN patients and promotion of standardized treatments are warranted. Further studies, such as improving the guideline adherence proportions or evaluating the contribution of guideline-based treatments to survival benefit, are needed.


COQ6 variants; the cardinal phenotypes are steroid-resistant nephrotic syndrome (SRNS), which inevitably progresses to kidney failure, and sensorineural hearing loss (SNHL). Here, we describe the phenotypes and genotypes of 12 children with COQ10D6 from 11 unrelated Korean families and quantitatively explore the beneficial effects of CoQ10 replacement therapy on SNHL. A diagnosis of SRNS generally precedes SNHL documentation. COQ10D6 is associated with progressive SNHL. Four causative COQ6 variants were identified in either homozygotes or compound heterozygotes: c.189_191delGAA, c.484C>T, c.686A>C, and c.782C>T. The response rate (no further hearing loss or improvement) was 42.9%; CoQ10 replacement therapy may thus limit and even improve hearing loss. Notably, the audiological benefit appeared to be genotype-specific, suggesting a genotype–phenotype correlation. The results of cochlear implantation were generally favorable, and the effects were sustained over time. Our results thus propose the beneficial effects of CoQ10 replacement therapy on hearing loss. Our work with COQ10D6 patients is a good example of personalized, genetically tailored, audiological rehabilitation of patients with syndromic deafness.Primary coenzyme Q10 (CoQ10) deficiency refers to a group of mitochondrial cytopathies caused by genetic defects in CoQ10 biosynthesis. Primary coenzyme Q10 deficiency-6 (COQ10D6) is an autosomal recessive disorder attributable to biallelic  PDSS1, PDSS2, COQ2, COQ4, COQ5, COQ6, COQ7, COQ8A/ADCK3, COQ8B/ADCK4, and COQ9 . ItCOQ8B affected protein stability and CoQ10 levels [COQ6 variants, COQ8B Arg174 alleviated the severity of the renal phenotype compared to that of COQ8B His174, suggesting that the COQ8B polymorphism may modify the human phenotype [COQ8B polymorphism status of our cohort when possible. Six patients were His174/Arg174 heterozygotes, 3 His174/His174 homozygotes, and 1 an Arg174/Arg174 homozygote (COQ8B SNP). All had SRNS; there were no significant differences in the age of onset  or the age of onset of hearing loss  by COQ8B polymorphism status. Specifically, in a sibling case, the younger sister (patient 8-2) with COQ8B His174/Arg174 exhibited a later age of SRNS onset and maintained normal kidney function to the age of 5.1 years, whereas the older brother (patient 8-1) with COQ8B His174/His174 progressed to kidney failure at the age of 1.3 years. In line with this, patient 8-2 evidenced better language development than patient 8-1, although the hearing thresholds were similar. The findings are in line with those of previous studies; COQ8B Arg174 may ameliorate the phenotype in terms of the severity of kidney symptoms and the extent of language development.A previous study that used a yeast model showed that a polymorphism  in TMPRSS3 variants [Notably, the results of CI were generally favorable in pediatric patients with COQ10D6, as in those with other forms of mitochondrial deafness , 23. Varvariants , 29, desvariants , 31. AltCOQ6 biallelic variants and the establishment of animal models harboring the human genotypes are essential to uncover the audiological characteristics and pathogenic mechanism of hearing loss caused by COQ10D6.This study had certain limitations that should be addressed in the future. In particular, the retrospective nature of the work may limit the generalizability of the results. Although we enrolled one of the largest numbers of pediatric patients with COQ10D6 reported to date, the number remains too small to allow us to draw firm conclusions, including any potential genotype–phenotype correlation. In addition, confounders were not completely assessed; this may have biased the results. Moreover, the lack of a control group  may weaken our findings. As our data is not based upon the case-control study, the results of this study cannot conclude the effect of CoQ10 therapy on COQ6 variants. We therefore suggest that future studies with larger cohorts, together with prospective case-control design, should be directed toward elucidating the effect of CoQ10 therapy on CoQ6 variants. Further studies on families segregating We have, for the first time, quantitatively analyzed the effects of CoQ10 supplementation on hearing loss in pediatric patients with COQ10D6. Almost half of the patients responded well, proposing the beneficial effects of CoQ10 replacement therapy on hearing loss for cases with COQ10D6. Importantly, the benefit appeared to be specific to the genotype, suggesting a genotype–phenotype correlation. Cochlear implantation significantly improved speech perception over time in patients with COQ10D6. Our work with COQ10D6 patients is a good example of personalized, genetically tailored, audiological rehabilitation of subjects with rare syndromic deafness.


Community Pharmacists (CPs) play an important role in patient counseling regarding the use of topical corticosteroids (TCS). We assessed CP’s self-reported counseling practices regarding TCS and patients’ reported counseling experiences.A previously developed questionnaire was adapted to the Malaysian context. A random sample of 364 registered CPs practicing in three states, Selangor, Kuala Lumpur, and Putrajaya were invited for an online survey. The questionnaire for CPs explored their perceived patient knowledge about topical corticosteroid (TCS) use, their counseling practices, and perceived barriers to counseling. Thirty patients were also selected from five pharmacies i.e., six consecutive patients who consulted each CP were invited to participate in the patient survey by completing a checklist about their experiences regarding the counseling received.A majority (> 90%) of the CPs mostly explained to the patients that the medication was TCS and the frequency and duration of application but only 10% correctly identified scenarios needing medical referral. Only about half of the CPs always explained about side effects, strength, efficacy, and storage of TCS. The two main barriers were patients’ negative perception of TCS (65.4%) and pharmacists’ lack of time for counseling (49.7%). Counseling practice score was associated with CPs’ age , pharmacists’ recommendation on TCS use , and time spent on counseling . Patients mentioned they were counselled on the frequency and duration of application of TCS, and potential adverse effects. Most were not counselled on action to take when an adverse event occurs and the storage and use of leftover medication.CPs counseling practices to their patient about the use of TCS requires improvement. Continuing education and hands-on training are needed for CPs regarding counseling about TCS use.The online version contains supplementary material available at 10.1186/s12875-022-01871-z. Community pharmacists (CPs), also known as retail pharmacists, play a significant role in primary health care delivery. They provide services such as supplying medication, providing medication reviews, medical and lifestyle counseling. Studies have demonstrated that utilizing CPs for the treatment of minor ailments results in optimizing health resources and reducing congestion at primary health facilities . ServiceThe first schedule to the poisons list classifies medicines into part 1, part 2, and exempt categories; type 1 is further subdivided into groups A, B, C, and D . TopicalCurrent clinical practice guidelines recommend the use of TCS as an adjunct with other treatments such as emollients, antibiotics, or antifungal topical medications, as well as systemic therapy with antibiotics, antihistamines, or oral steroids where appropriate . ImpropeCPs have the knowledge to advise patients and caregivers on optimum TCS use, and the provision of optimum and good quality counseling by CPs is associated with better treatment outcomes . The keyA cross-sectional online survey was conducted among community pharmacists practicing in three Malaysian states . Community pharmacists holding Type A license certification and currently working in community pharmacies were involved. Type A license is issued by the pharmacy enforcement at the state level and allows a pharmacist to import, store and deal generally with all poisons by wholesale and retail or by wholesale only or by retail only .The sample size for CPs was determined using the formula for the finite population. The expected proportion was 0.5. The proportion of CPs who had correct knowledge and practiced correct counseling techniques was not available from the literature. For a confidence limit of 95% and 5% allowable error, the calculated minimum sample size was 303. Taking into consideration an anticipated non-response rate of 20%, the final sample size was 364.A list of 1,207 CPs holding licenses from Selangor and 601 from Kuala Lumpur and Putrajaya was obtained from the Official Portal of the Pharmaceutical Services Programme . Names aThe questionnaire for CPs was adapted with permission from the original questionnaire developed by Kang et al. . The queFor multiple linear regression analyses., the independent variables were socio-demographic characteristics as age, gender, work location, work status, years of experience as a community pharmacist, and education level. Dependent variables were CP’s perception regarding patients’ knowledge about TCS measured using eight “yes/ no” type questions. The score for CPs perceived patient’s knowledge of TCS was obtained by adding the responses to eight items. The response options ‘yes’ or ‘no’ were coded as ‘1’ and ‘0’ respectively. The score on CPs counselling practices on TCS use was measured using an 11-item, 3-point Likert scale . The sum of scores represents the counselling practice scores (maximum score of 22).Due to the Coronavirus (COVID-19) pandemic, an online questionnaire was distributed from September to December 2021. Selected pharmacists were contacted using the email addresses or premise phone numbers listed on the pharmaceutical services website or obtained following a Google search, or via social media platforms such as Facebook or LinkedIn. After establishing contact with participants, the information sheet and questionnaire were delivered through email, or a link supplied via WhatsApp, Facebook, or LinkedIn messages. Participants who completed the questionnaire received RM5 (approx. USD 1.25) e-vouchers as a token of appreciation.CPs who participated in the online survey were requested to recruit patients who came to purchase TCS. Five of the CPs agreed to recruit patients for the sub study among the patients. From each of the five selected pharmacies, six consecutive consenting patients were selected. The questionnaire for patients was extracted and modified from Question 12 in the original Kang et al. questionnaire . The quePatients visiting the selected community pharmacy to purchase a TCS were invited to answer a self-administered anonymous online survey if they were able to read English or Malay language. The pharmacist explained the study details to the patients and obtained their consent. The patients were instructed to scan a QR code using their smartphones to access the study information sheet followed by an online survey about their perceptions regarding counselling received from the CPs about the use of topical corticosteroids. The participants were instructed to complete the survey after leaving the pharmacy premises.IBM SPSS (version 25) was used to analyse the data. Descriptive statistics were calculated, and the distribution of scores were checked for normality. Bivariate comparisons of CPs counselling practice score on TCS use were done with demographic factors of CPs, time taken for preparation and conduct of counselling on TCS use, perceived barriers to counselling and delivery time, training about adverse drug events (ADE) and the action taken if ADE to TCS was experienced by the patient. The bivariate comparisons that were significant at p < 0.25 were included in the multivariate analyses. CPs perceived patient knowledge of TCS scores, and TCS counselling practice scores were the outcome variables. Multiple linear regression analysis was conducted to identify the factors associated with the CPs counselling practice score. Adjusted odds ratios and the 95% confidence interval (CI) were estimated (p < 0.05).The International Medical University Joint Committee for Research and Ethics approved this research (Project ID number: MSPH I/2021(04)) on the 14th of February 2021. Informed consent was obtained from the CPs participating in the survey and the procedures were followed as per the declaration of Helsinki.The questionnaire were sent to 364 participants, out of whom 215 completed the questionnaire. The response rate was 70% and all responses were complete. Table About 97% of the CPs correctly identified “14 days or less” as the maximum duration for TCS treatment and 85.6% of the CPs correctly ranked three TCS in decreasing order of their potency. About 1 in 10 CPss10.2%) accurately identified scenarios that necessitate medical referrals. About 98% of CPs perceived that “pharmacist’s explanation” and 90.2% perceived “internet” as patient’s main source of information on TCS. Respondents rated steroid-only TCS as the most sold compared to other steroid-combination products, followed by steroid-antibiotics, steroid-antifungals, steroid-keratolytic, and steroid-other medicines combinations. Pharmacists ranked medication misuse and medication characteristics, such as potency of TCS, as the most common cause of adverse drug events; followed by patient characteristics, (patient’s age), and medication overuse. Dry skin and skin irritation were the most reported adverse drug events, followed by skin discolouration and skin infections. When patients contacted community pharmacists about adverse drug events caused by topical corticosteroids, 79.5% recommended them to discontinue the medication and advised them to see a doctor. Additionally, 76.7% of respondents indicated they would recommend treatment re-trial following a medication review and patient re-education. Only 27.0% of participants would report the event to the National Pharmaceutical Regulatory Agency (NPRA).Table Table A total of 30 patient responses were obtained in the sub study among the patients to verify the counseling practices reported by the CPs. Their mean age was 43.3 years (SD = 10.9). Of the 30 patients, 19 63.3%) were females, 15 were of Malay ethnicity while 11 (36.7%) were Chinese. Sixteen16 respondents (53.3%) were prescribed a steroid only preparation while 12 were prescribed a combination of a steroid and antimicrobial. Twenty (66.7%) purchased TCS for their own use. The content of counselling received by patients from a CP was also surveyed (Table 3.3% wereThe CPs working in Klang valley were surveyed online to explore the counselling they provided to their patient seeking to purchase TCS and the factors associated with the counseling practice. CPs most often provided information that the medication was a TCS, methods of application, frequency and duration of its use which was in conformity with patient reported counseling experience. Counselling practice scores were shown to be significantly associated with age, education level of the CP and time spent preparing and delivering counselling. In the current study, CPs had sufficient knowledge to conduct patient counselling and perceived themselves to be information providers for patients. Congruent to prior research by Kang et al., there was a significant association between knowledge of the patient as perceived by the CPs and counselling practice scores . AdditioThe age of CPs had a significant association with their counselling practice scores. Counselling practice scores decreased with increasing age. This may be anticipated, since younger pharmacists are typically recent graduates who have been exposed to a patient-centred pharmacy curriculum emphasising effective communication skills. As a result, they would have developed greater knowledge and skills to counsel patients comprehensively regarding the treatment and management of minor ailments . The phaIn this study only 27% of CPs mentioned that they would report an ADE/ADR. ADR reporting is influenced by several factors. In Korea it was seen that knowledge of spontaneous reporting, prior experience of ADRs and less concern about barriers to reporting were associated with higher reporting rates . A studyParticipating pharmacists also reported a lack of time for counselling as a barrier to providing counselling. Research postulated that the more time a pharmacist had to advise patients on the use of TCS, the more positive were the pharmacists’ views towards information provision .Most participating pharmacists reported experiencing barriers to counselling. The biggest barrier was patients’ negative perceptions of TCS, followed by the lack of time for counselling. This result is consistent with studies conducted among CPs in Korea [113]. Steroid phobia has been extensively researched in dermatological conditions, and is prevalent among both patients and caregivers . The corIn this study, counselling by CPs often included information on the “expected effectiveness”, “potency”, “dosage”, and “frequency of application” regardless of their perception of patient comprehension. If the CP considered that a patient may be unfamiliar with information such as “it is a topical corticosteroid”, “action to take in case of an adverse drug event”, “duration of application”, and “storage and use of remaining medication following treatment completion”, it was provided. These findings are almost identical to those of Kang et al., who hypothesised that counselling information is impacted by pharmacists’ assumptions about patients’ knowledge rather than real patient needs .Poor knowledge about the use of TCS in atopic dermatitis was noted among Australian pharmacists and these appear to be modifiable through targeted education provided by a dermatologist . Better To the best of our knowledge, this is the first study to explore patients’ experience receiving counselling from a CP on the use of TCS in Malaysia. The results show that patients’ experiences about counseling provided by CPs and CPs practices on counseling for TCS agreed with the information provided and the quality of counselling. Most patients indicated they received counselling on the potency, dosage, and frequency of the TCS purchased. This was consistent with the pharmacists’ self-reported behaviours of information provision. These results differ from a previous study by Teixeira et al. showing inconsistencies between pharmacists’ self-reported behaviour and patients’ claims where only 1 in 4 patients was informed of the dosage and 1 in 3 were provided with information about the duration of use of the medication prescribed . PharmacThis study also has several limitations. First, this study was conducted among CPs in Selangor, Kuala Lumpur, and Putrajaya only. Hence, the results may not be generalizable to all CPs in Malaysia. Larger nationwide studies on community pharmacists from urban, suburban, and rural places of practice would be beneficial. Second, this study did not evaluate the clinical outcomes of patients, to examine the influence of pharmacists’ counselling in the treatment of dermatological conditions. The third limitation was the small number of patients is non-representative of all patients receiving counselling about TCS. In a sub study 30 patients who received counseling from five CPs were surveyed to verify the TCS counseling reported by the CPs. Counselling experiences reported by the patients selected by CPs themselves is likely to be affected by the Hawthorne effect. This was a result of the strict social distancing measures during the Coronavirus pandemic. Lastly, since this was a self-administered questionnaire, social desirability and self-reported biases may impact responses. However, internal consistency of data collected and the parallels in results with earlier studies are significant, suggest that the findings are reliable.The CPs mainly counsel the patients about the frequency and duration of application of TCS. Patients sub study confirmed the CP reported counselling practices. Age of CPs, counselling preparation and delivery time were significantly associated with counselling practice. Continuing education and hands-on training and a greater collaboration between different healthcare providers are needed to improve counselling about TCS provided by the CPs.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs’ lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.Little is known about how co-infections and genotype dynamics affect The online version contains supplementary material available at 10.1186/s13567-022-01061-w. Mycoplasma hyopneumoniae is one of the primary agents involved in porcine respiratory disease complex (PRDC) . It increased with both M. hyorhinis  and A. pleuropneumoniae co-infection (0.65 ± 0.40 ln copies) and varied significantly in time depending on the group  , but they both had a significantly higher number of copies than positive pigs from Group C . However, the number of copies in Groups A and B decreased significantly from T0 to T1 (padj = 0.002 and padj = 0.01), while the average number of copies in positive pigs from Group C did not change over time (padj = 0.95). As a consequence, at T1 there was no longer a difference in the number of copies among any of the groups . Detailed results of the DSLM post-hoc comparisons are shown in Table The number of copies of p < 0.05)  . A breakdown of prevalence by time and experimental group is shown in Table n = 287) ranged from 0 to 22 . Ninety-two pigs showed no lesions, 76 pigs showed mild lesions, 67 pigs showed severe lesions, and 52 pigs showed very severe lesions as shown in Table M. hyopneumoniae infection , with pigs infected at least once having 40 times the odds of showing very severe lesions. Indeed, of the 19 animals that were never infected by M. hyopneumoniae, 17 showed no lesions and 2 showed mild lesions; none showed severe or very severe scores. Neither the experimental group nor any of the other respiratory infections included in the model had a significant effect on lung lesions . Considering only pigs with a history of M. hyopneumoniae infection, lung lesions were affected only by M. hyopneumoniae . Pigs with MX infections had significantly higher odds of showing severe lesion scores than pigs with SN infections . Once again, neither the experimental group nor the number of co-infections had any effect on the severity of lung lesions .The raw total lung lesion scores in pigs examined at T2  were less likely to be positive for M. hyopneumoniae.The three different groups of pigs sampled in each farm were defined according to farm localization and clinical conditions, leading to different exposures to M. hyopneumoniae load varied in time but with a different pattern depending on the group. The highest PCR number of copies was detected at T0 in Groups A and B and at T1 in Group C. Because the highest burden of M. hyopneumoniae has been proven to occur at 28 days post-infection [On the contrary to the infection status, the nfection , it can nfection , these fM. hyopneumoniae as a gate opener for secondary bacterial infections, especially P. multocida and A. pleuropneumoniae, is well known [M. hyopneumoniae and M. hyorhinis, with the latter more frequently observed in the presence of the former. Mycoplasma hyopneumoniae is one of the main pathogens involved in swine pneumonia [M. hyorhinis generally has slight importance with mild or no clinical respiratory signs [M. hyopneumoniae and other respiratory pathogens, with P. multocida even showing a slightly negative association with M. hyopneumoniae infection. The prevalence of P. multocida was indeed higher in pigs free from M. hyopneumoniae, which may suggest an unfavourable interaction between these two bacterial infections. However, a possible explanation for this observation is the strict selection criteria of the enrolled farms, which included pigs with signs of EP but without the typical signs of a complicated infection  [M. hyopneumoniae infections; therefore, the presence of P. multocida was presumably not due to secondary irruption. Furthermore, because P. multocida was isolated on only a few farms, we cannot exclude the possibility that this weak negative association was due to the limited sample size.The role of ll known , 19. In ry signs . No posiM. hyopneumoniae and A. pleuropneumoniae, co-infection by these two bacteria was found to be more common when M. hyopneumoniae showed a higher number of copies in the PCR assay. Similarly, infections with M. hyorhinis were more frequent with a higher M. hyopneumoniae load. Despite the fact that the exact mechanisms of co-infections in respiratory diseases are not well known [M. hyopneumoniae to be well established in the respiratory tract to potentiate secondary pathogens [M. hyopneumoniae could be beneficial to avoid A. pleuropneumoniae and M. hyorhinis co-infections.Although no association was detected between the presence of ll known , a possiathogens . ConsequThe prevalence of several respiratory pathogens seemed to vary with time and by group, but the often-limited number of infected pigs coupled with the non-random selection of farms did not allow for a more detailed analysis of the other infection dynamics. Furthermore, the vaccination strategies (including the type of vaccine used) were not considered as variables in this study and should be investigated in the future.Ascaris suum was not considered, this should be considered a limitation of the research.The respiratory pathogens considered in this study are the most common agents detected in pig farms in the study area ; therefoActinobacillus pleuropneumoniae is a major agent of chronic pleurisy [pleurisy . HoweverM. hyopneumoniae VNTR types, the prevalence of M. hyopneumoniae MX infections was stable through time in Groups A and B, whereas it increased significantly from T0 to T1 in Group C. Considering the aforementioned trend of the M. hyopneumoniae load that suggested the onset of the outbreak at T0 in Groups A and B and at T1 in Group C, the MX infections appeared to occur mainly in pigs later and farthest from where the outbreak of EP started.Concerning the dynamics of M. hyopneumoniae complicates the interpretation of these findings, the currently available data did not allow us to assess the actual mechanism that is occurring. However, a possible interpretation is that when an EP outbreak occurs, the VNTR types involved remain the same throughout the pathogenic evolution within each pig. The subsequent phase of the outbreak with extensive shedding of M. hyopneumoniae instead induces the circulation of high loads of different VNTR types, increasing the risk of pigs being exposed to multiple VNTR types. The data show a complex dynamic of different genotype infections with an evolution of the VNTR types along the different stages of the outbreak. Therefore, the timing of sampling could greatly affect the likelihood of detecting all genotypes hosted by a pig. However, these results should be interpreted with caution because although TBS are considered a very sensitive type of sample for intra vitam diagnosis [Since the endemic presence of iagnosis , VNTR tyiagnosis , and theM. hyopneumoniae in live animals [A. pleuropneumoniae and G. parasuis are more reliably cultured from lung tissue [TBS analysis was our primary choice because it is the most sensitive method for the diagnosis of  animals . Howeverg tissue , 33, andM. hyopneumoniae-positive pigs having an MX infection increased significantly with PRRSV co-infection. The presence of active PRRSV circulation is considered an indirect index of biosecurity [M. hyopneumoniae VNTR types [M. hyopneumoniae genotypes. However, these results should be interpreted with caution because of the very limited presence of PRRSV in our sample, and further investigations are surely needed to draw more substantial conclusions.Additionally, we found that the probability of security , and lowTR types . Thus, iTR types  and mighM. hyopneumoniae among lung lesion-associated bacteria is well known [M. hyopneumoniae. However, the severity of lesions was not related to the group, suggesting that the time point at which pigs become infected during the outbreak is not important; the critical point is rather the infection by M. hyopneumoniae itself. A practical implication of this is that in vivo samplings during an EP outbreak could allow for early identification of pigs that will likely develop lung sequelae.The high prevalence of ll known , and in M. hyopneumoniae and other respiratory co-infections during EP outbreaks. Previous studies have revealed the ability of M. hyorhinis to exacerbate M. hyopneumoniae lung lesions [M. hyopneumoniae in TBS were significantly associated with the presence of M. hyorhinis, but this co-infection was not associated with a worsening of lesion severity. Furthermore, despite the well-known ability of A. pleuropneumoniae and P. multocida to cause lung lesions [M. hyopneumoniae has a dominant role in determining their magnitude.Studying co-infections is very complex and challenging, and the evolution of co-infections in pigs during the fattening period is a largely unexplored field . The pre lesions . In the  lesions , no relaA. pleuropneumoniae infection is a risk factor for pleuritis sequelae [M. hyopneumoniae, which has the same tendency to become chronic, could follow a similar evolution. Notably, however, the clinical evaluation of pigs in the present study was carried out only at the beginning of the outbreak (T0) and after slaughtering (T2) by lung lesion scoring. An additional evaluation of the clinical status at T1 could indeed help to shed further light on the outbreak evolution.The longer finishing period adopted in Italy surely influences the dynamics of infection . Indeed,sequelae . LikewisM. hyopneumoniae or the presence of multiple VNTR types and lung lesion scores is controversial [The relationship between the detection of specific strains of oversial , 38, 39.oversial .M. hyopneumoniae was found to play a central role in respiratory outbreaks in fattening pigs, while co-infections seemed to play a more marginal role with no relevant effect on the severity of lung lesions. The presence of M. hyopneumoniae infections of multiple VNTR types during the finishing period led to more severe lung lesions in this study. Therefore, improving farm management and biosecurity and in turn limiting the circulation of multiple genotypes could potentially lead to a better lung lesion status.In conclusion, Additional file 1. Allele calling table. Each VNTR type is identified as a combination of different VNTR locus. The number of repeats of each locus is determined by the number of base pairs (bp) for that locus.