Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically–relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile–derived metabolites in culture supernatants, including hexanoyl– and pentanoyl–amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300–fold increases in the expression of sporulation–associated genes were observed in FW media–grown cells, along with reductions in motility and toxin genes’ expression. Moreover, the expression of classical stress–response genes did not change, showing that C. difficile is well–adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility. Clostridium difficile is recognised as one of the major causes of health-care associated infections2 and exerts a negative and well–publicised impact on hospital morbidity and mortality rates1. C. difficile infection (CDI) can develop when broad spectrum antibiotics are deployed to treat underlying infections: they disrupt the body’s natural colonic microbiota thus allowing development of CDI if spores or cells of this multidrug–resistant pathogen are also present3. Colonisation of the host gastrointestinal tract depends on the germination of C. difficile spores, with subsequent growth of vegetative cells and the release of two large clostridial glycosylating toxins, toxin A and toxin B4. These toxins are responsible for the inflammation and epithelial tissue damage that results in rapid loss of fluid and consequent diarrhoea5. Clinical manifestations and severity of CDI vary from mild self-limiting diarrhoea to life-threatening pseudomembranous colitis and, in severe cases, death6.The Gram–positive spore–forming anaerobe C. difficile infection (CDI)6, the mechanisms underlying C. difficile expansion after microbiota disturbance are only just emerging. Both dietary and microbiota compositional changes have been demonstrated to lead to alterations in the colonic environment that favour or suppress certain enteric pathogens such as C. difficile11 and indeed C. difficile virulence has been linked to the ability to both effectively utilise nutrients in the dysbiotic gut environment12 and to sporulate13. During CDI, there is rapid expansion of the C. difficile vegetative cell population, with subsequent production of the two proven virulence factors, the toxins (A&B), and spores which serve as the transmissible elements15.While it is well established that gut microbiome disruption by antibiotics can lead to the development of C. difficile pathogenesis have been described16, studies with humans are, by contrast, limited to prospective or retrospective sampling and elucidation of C. difficile strain variants. While it could be argued that many of the experimental systems that exist to study C. difficile pathogenesis have rather limited relevance to the human gut, recent work with in vitro continuous flow bioreactors has elegantly demonstrated the increased competitive fitness of ribotype 027 C. difficile strains in a mixed microbiota model17 and shown that microbial communities representative of key features of the gut can be cultivated and manipulated successfully18. Such in vitro models have also been used to investigate and model antibiotic exposure19, intestinal biofilm development20 and genomic stability of C. difficile during simulated infection experiments21. In other systems that seek to investigate changes within the human gut, faecal water (FW), the aqueous phase of human faeces22, is an attractive means of linking changes in colonic contents with gut health outcomes27. FW has been used as a biologically–relevant challenge agent in a range of gut studies30 as it contains a variety of unbound, soluble components including bile acids, fatty acids, amino acid residues and derivatives, (poly)phenols, and short-chain fatty acids33. These metabolites are likely to modulate the function and composition of the microbiome. To allow physiologically–relevant modelling of C. difficile under controlled culture conditions, we have incorporated FW into growth media in order to better mimic the human gut environment. Using our novel approach we now demonstrate for the first time that expression of genes essential for pathogenesis are significantly differentially expressed in the human faecal water milieu.While a variety of model systems that are indispensable in the study of 32 from two male donors (age 40+/−2 years). We characterised the FW using LC–MSn and demonstrated that it contained components identified in previous investigations36. Some 30 FW components, many of which are known constituents of faeces (e.g. stercobilin and urobilinogen) were identified  sample was produced as previously describeded Table , while oC. difficile physiology in a way more reflective of the in vivo environment, we compared growth in faecal water/BHIS growth media (“FW media”) with the BHIS control. FW media was not detrimental to growth of C. difficile strain 630 over 6 h  were not subject to further metabolism or degradation by C. difficile and were largely unchanged between the start and end of the 6 h incubation , which could be due to the aldehyde, hexenal (C6H10O). In a similar manner, fragmentation of pentanoyl derivatives produced the M[−H] ion of the amino acid and a neutral loss of 84 amu due to the aldehyde, pentenal, . Other common fragmentations can be assigned , neutral loss of 56 (C4H8 = butene), neutral loss of 70 (C5H10 = pentene) and are consistent with such structures. Other neutral losses such as 44 and 46 amu, respectively, can be ascribed to CO2 and formic acid from the amino acid components. These putative hexanoyl and pentanoyl amino acid derivatives have not previously been identified in cultures of C. difficile but this microorganism is known to produce isocaproic acid  during growth and the accumulation of this C6 fatty acid has previously been used as a diagnostic for C. difficile in stool samples39. Investigations into biofuel production by Clostridia have shown that hexanoyl–coA is a key metabolite for the production of hexanol40 and the formation of these putative hexanoyl and pentanoyl amino-acid derivatives may be a consequence of growth of C. difficile in the amino acid–rich BHI media. Earlier work showing isocaproic acid accumulation39 used less sensitive GC–MS techniques that required sample derivatisation and thus would not have detected the amino acid derivatives we identified. Of all the metabolites whose abundance increased during growth of C. difficile (Table m/z 243) was higher in the BHI media than in FW media .We focused on metabolites whose abundance consistently increased during incubation, using the XCMS process with data checking to eliminate low abundance peaks, adducts and multiply charged ions   > 1.45) as a result of exposure to faecal water with 497 (56%) exhibiting increased expression and 392 (41%) being decreased  was in CD1065, which encodes a 146 amino acid ‘conserved hypothetical protein’. A number of investigations into C.difficile sporulation have indicated that CD1065, and indeed a number of genes encoded by CD1063A-CD1067, are strongly regulated by either σE or σK, with published data indicating that CD1065 is strongly induced by σE in the mother cell during sporulation43. The largest decreases in expression (>100–fold) were in a three–gene ATP binding cassette (ABC) transporter operon encoded by CD0873–0875. The largest fold–changes in gene expression were found in the categories of (i) transport, binding proteins and lipoproteins (up to 270–fold), (ii) sporulation (up to 300–fold), and (iii) genes encoding hypothetical proteins (up to 445–fold). Genes involved in specific metabolic pathways, RNA metabolism, protein modification, adaptation to atypical conditions and those categorised as miscellaneous exhibited fold changes of no more than 20–fold. The least extensively–changed genes were those involved with metabolism of phosphorus, sulphur, lipids, and coenzymes , σF, σE, σG, and σK (late)49. Thereafter, however, differences have been shown in the order, activation, and function of the sigma factors in C. difficile50. Genome–wide mutational analyses of sigma factor function in C. difficile have revealed that while their transcriptional and functional sequence  is broadly conserved with the B. subtilis model, there are differences in the C. difficile developmental programme51. Spores were not visible in cultures of sigma factor mutants, indicating their critical role in the various stages of sporulation and in the production of mature, heat-resistant spores42. Intriguingly, and in contrast to B. subtilis, the activity of σE was partially independent of σF; σG or σK did not require σE or σG, respectively and sigG transcription was not dependent on σF42. Taken together, the published data suggests minimal intercompartmental communication and a weaker connection between forespore and mother cell50, in addition to a looser association between gene expression and morphology in C. difficile51. While sporulation in C. difficile is more akin to B. subtilis than to other Clostridia, it at the same time represents a more ancestral, less tightly–controlled sporulation programme that facilitates a degree of population heterogeneity during infection52. The recent work of Browne et al.53 showed extensive sporulation ability within the human gut microbiota, with many taxa present in the spore form. Animal models of C. difficile infection have revealed that in mouse, C. difficile spores germinate by 6 h post–infection, leading to pathogenic lesions and heat–resistant spores – comprising up to 20% of the total cfu – being detectable 24–36 h post–challenge. Thus, while sporulation under infection conditions takes some time) with C. difficile strain 63015, the process happens as early as 6 h post–infection with the ribotype 027 strain, C. difficile R2029154. Temporal examination of C. difficile 630 gene expression during infection in mouse55 and pig ligated loop models56 has shown that many genes associated with host adaptation, all stages of sporulation and a diversity of genes encoding “hypothetical proteins” were expressed at increased levels in vivo, indicating their importance in the infection process and the requirement for extensive remodelling of the transcriptome during infection. We thus hypothesised that FW would induce sporulation in C. difficile and sporulation genes were indeed some of the most differentially expressed in FW medium, with increases up to 300–fold. Of 60 DE genes in our dataset that had a predicted or known role in sporulation, only two exhibited decreased expression . Of the remaining 58 genes, the expression of 22 of these was increased > 50–fold, indicating that FW is a potent inducer of sporulation genes in C. difficile strain 630. C. difficile sporulation has been extensively mapped, allowing definition of genes under control of Spo0A –the master regulator of sporulation–and the identification of potential links between sporulation and other phenotypes57. Phosphorylation of the Spo0A protein initiates a sigma factor cascade that, acting in both mother cell and forespore, influences expression of the sporulation–specific sigma factors σF , σE , σG  and σK that control expression of early (Bacillus stage II and III) and late (Bacillus stage V and VI) sporulation genes50. During sporulation, a septum results in asymmetric division of the bacterial cell and creates two unequally–sized compartments. The smaller–the forespore–develops into the spore, while the larger compartment prepares the forespore for dormancy51. Taken in the context of previous identification/analysis of sporulation–associated genes53 our data indicates that, at the point of harvest, FW–grown C. difficile 630 cells are physiologically at what in B. subtilis would be categorised as stage III of sporulation, i.e. the point at which engulfment of the forespore has occurred, but prior to cortex formation. Thus, genes including the spoIIIAA–spoIIAH operon , in addition to spoIIIJ , spoIIID (56–fold increased) and sigG (40–fold increased) exhibited considerably increased expression in FW–grown cells. Of the sporulation–associated sigma factors, σE exhibited the second–largest expressional increase (23–fold) in FW. The σE protein acts on a number of genes in the Clostridial sporulation cascade42 and we noted increased expression of σE–controlled genes including spoIIID, spoIVA (57–fold up), cspBA (22–fold up) and cspC (2.2–fold up). Furthermore, we observed increased expression of genes encoding certain spore coat proteins, including cotE . The peroxiredoxin and chitinase activities of CotE contribute to pathogenesis by facilitating degradation of gut mucus during infection59.In the model Gram positive organism oppABC  expression leads to earlier expression of sporulation–associated genes13 and the observation that oppABC expression was 50% lower in FW appears consistent with our other observations of FW–induced changes to the C. difficile transcriptome. Taken together, therefore, our gene expression data indicates that C. difficile cells are induced by FW components towards sporulation more rapidly than cells grown in BHIS media. The spores are the transmissible, resilient, and infectious form of the organism14 and thus our observation has clear implications for pathogenesis and transmission of the disease, in addition to being entirely consistent with observations by other researchers of extensive sporulation within the gut microbiota in vivo56.It has been demonstrated that decreased 45. By contrast, our current investigation revealed considerable changes in transporter gene expression. The PTS is the major bacterial carbohydrate assimilation system for hexoses, hexitols and disaccharides and consists of two general components–enzyme I (EI), and the histidine phosphocarrier protein (HPr)–in addition to sugar specific permeases (enzymes II) in the cell membrane. In FW media, expression of the gene encoding the EI component (CD2755) common to, and essential for, all phosphotransferase systems in the cell, was increased by 1.58–fold. In contrast, expression of the HPr kinase/phosphorylase (CD3409) that phosphorylates the cytoplasmic phosphocarrier protein Hpr at Ser42, and which also leads to activation of the LacI family carbon catabolite repressor, ccpA60, was 1.8–fold lower. Consistent with these observations, the gene encoding the IIABC component of the PTS system for uptake of beta-glucosides  was increased, as was the downstream gene bglA (CD0389) encoding 6-phospho-beta-glucosidase, reflective of the likely increased availability of such glucoside substrates61 in the FW media. In addition, expression of the sorbitol specific IIB component, srlEa, (CD0765) was increased as was expression of CD2269 encoding the fructose specific IIABC component, fruABC, as were genes encoding the IIA and IIB components of the glucose PTS transport system . Expression of the IIC and IID components of the mannose/fructose/sorbose transport system  were increased by 4– and 6.6–fold, respectively. Conversely, expression of PTS system components associated with uptake of xylosides  was lower in FW, while expression of the associated transcriptional regulator , which functions to reduce expression of genes for uptake and metabolism of xylose, was increased. These diverse perturbations in expression of carbohydrate transport–associated genes most likely underpin an adaptive response of C. difficile to additional carbon sources and other diet derived metabolites present in the FW. However, the PTS is also a signalling device which has been linked to chemotaxis and regulatory functions associated with C, N and P metabolism and to the virulence of C. difficile63. The complex interplay between a variety of cellular systems , controls toxin production. It is known, for example, that butyrate stimulates toxin production60 but, in FW–grown cells we noted lower expression of 12 genes associated with carbohydrate fermentation to butyrate. The likely reduction in metabolic flux towards butyrate is consistent with the 4–fold lower expression of tcdA (CD0663) observed in FW  sugar transport systems were largely unperturbed by heat stress64. The most downregulated gene (270–fold lower in FW) in our dataset was an ABC transporter ‘substrate-binding lipoprotein’ (CD0873), recently identified as an adhesin that enables C. difficile to bind Caco–2 cells65. Our data suggests that, in FW at the point of cell harvest and possibly at later stages in the infection cycle in a host, C. difficile exhibits reduced binding to epithelial cells, consistent with increased sporulation and lowered motility. This physiological state could thus facilitate evacuation of the bacterial population from the host. Ten different lantibiotic/multidrug ABC transporters were also DE. Four exhibited increased expression–CD0161 (4.73–fold); CD1349/50 ; CD2210/11 ; CD2406/7/8 . The precise function of such transporters has not yet been defined, and consequently genomic annotations are a “general function prediction” only. Nonetheless, within the intestine, C. difficile and other gut pathogens must contend with innate host defences including cationic antimicrobial peptides  produced by both host and indigenous microbiota66. McBride and Sonenshine67 have shown that proteins encoded by CD1349/CD1350 are involved in resistance to CAMPs, proposing the designation cprABC-cprK for CD1349 to CD1352. In FW–grown C. difficile cells, expression of CD1349 and CD1350  were increased by 5.3– and 2.9–fold, consistent with our hypothesis that increased levels of host or microbiota–derived antimicrobial peptides present in FW lead to increased expression of this specific mechanism.A number of ABC transporter–encoding genes were DE in FW, including some associated with transport of sugar phosphates, vitamins, oligopeptides, amino acids and also transporters associated with multidrug efflux mechanisms69, with flagella and type-IV pili comprising motility devices essential for the pathogenesis of certain bacteria71 including a variety of motile enteropathogens72. Flagellar biosynthesis is a highly–ordered process in which hierarchal control of gene expression ensures that synthesis of late–stage components is repressed until assembly of earlier components is complete73. Thus, only when the basal body and motor machinery is in place, do late–stage genes, including flagellin (fliC) become expressed. Expression of motility genes in C. difficile is regulated by a sigma–28 factor encoded by CD0266  whose expression was 1.5–fold decreased in FW–grown cells. El Meouche et al.74 (2013) demonstrated that sigD acts as a positive regulator of both flagella and toxin gene expression in C. difficile. Decreased expression of sigD could therefore be partially responsible for the reduced expression of motility genes. In FW–grown C. difficile, expression of genes located broadly in the F3 loci (CD0245–CD0271), including those encoding components of the basal body, motor, hook and rod, exhibited 1.5– to 2–fold decreased expression. Gene expression in the F1 locus (CD0226–CD0240) was reduced between 2– and 5.7–fold, as would be expected if these gene products were not required until assembly of the basal body was complete. In addition, expression of fliC (CD0239) was decreased by 4.4–fold to ~20% of the level in the control, an observation corroborated by our qRT-PCR data  expression was reduced by 5.7–fold and we noted likewise that expression of genes in the interflagellar F2 locus (CD0240–CD0244) decreased by just over three–fold in FW. Levels of transcript  locus. This increased expression of genes encoding a type–IV pilin, an associated type–II secretion system protein, and a pilus assembly ATPase (CD3294-6) suggests that pilus–driven motility may possibly be more important in a FW milieu and in certain stages of the infection process. Regardless of the role of TFP during infection, bacterial flagella are known to promote intestinal lesions via host inflammatory responses: 79 and with the exception of CD1726 and CD3522, were all predicted by SecretomeP80 to be non–classically secreted. Nine of the gene products had PsortB81–predicted locations in the cytoplasmic membrane and the majority of these 141 proteins possessed no conserved domains that might indicate their potential function. Nonetheless, literature and database interrogation allowed us to link many to a role in sporulation. The most downregulated conserved hypothetical protein–encoding gene was CD2344, which has been identified as a putative succinate transporter with a role in C. difficile gut colonisation12. We have also shown here that expression of a variety of other genes in the succinate to butanoate fermentation pathway–which lie transcriptionally downstream of CD2344 in the same operon structure82 –were decreased by ~4–fold in FW. A number of genes reported to be regulated by sporulation–specific sigma factors43, including σK (CD3580 & CD1065), σG (CD2808 & CD2375) and σE  were also DE in FW. Dembek83 reported that a large proportion of C. difficile spore transcripts encoded proteins of unknown function and proposed that these were indicative of the difference between the transcriptional programme of vegetative cells and spores. Three such genes exhibited increased expression in FW–CD3551A (71–fold), CD2374 (30–fold) and CD2229 (36–fold). In addition, DE genes including CD1929, CD1884, CD2657, CD2374 and CD2375 were also reported by Janoir et al.55 to be expressed at higher levels in stationary phase C. difficile 630 cells at 14 h and 38 h–i.e. where the sporulation process would be well–established.The largest number of DE genes (n = 141) were placed in the ‘Similar to unknown proteins’ category and 19 of these had expressional changes of > 20–fold in FW–grown cells. None of the proteins encoded by these genes had predicted signal peptidesin vitro. LC-MSn metabolomic analysis of FW allowed us to identify 30 individual components including urobilinogen, stercobilin and several cholic acid derivatives. Having established that the FW was–metabolomically–consistent with previous reports, we demonstrated that in the presence of FW, growth of C. difficile strain 630 was largely unaffected, save for an increase in cell length that our transcriptome data indicates is most likely a prelude to sporulation. A primary question was whether C. difficile strain 630 could utilise components of FW. Our analysis showed that while FW metabolites were not further metabolised during growth, a set of previously unknown C. difficile–derived hexanoyl– and pentanoyl– derivatives of amino acids were produced. These metabolites are not only novel biomarkers for the presence of this pathogen, but also reflect previously unrecognised metabolic capabilities within C. difficile strain 630. RNA sequencing showed clearly that the primary transcriptomic response of C. difficile strain 630 to FW was an acceleration of the sporulation cascade. FW–grown cells exhibited increases of up to 300–fold in the expression of sporulation–associated genes, with concomitant decreases in motility and toxin gene expression. These changes are reflective of the interplay between FW components and the expression of sensor kinase/response regulator systems, and transcription factors, many of which exhibited increased expression in FW–grown cells. Interestingly, none of the classical stress–response genes were differentially expressed, supporting the rationale that C. difficile adapts easily to a faecal milieu. The considerable modulation of a variety of transport systems is consistent with the addition of FW components to the growth media. Overall, therefore, our ex vivo FW model represents a new and unique means of assessing the response of C. difficile strain 630 to gut metabolites allowing us to describe, for the first time, the faecal milieu–associated physiological changes in this important pathogen.We set out to establish a new means of investigating gut pathogen biology All chemicals and reagents of Analar grade or better were purchased from Sigma-Aldrich  unless stated. Brain Heart Infusion (BHI) agar and broth and yeast extract were purchased from Oxoid . All molecular biology reagents were purchased from Invitrogen  save for qPCR reagents, which were obtained from Roche Diagnostics  and random primers, which were obtained from Promega . Lysing Matrix A tubes were from MP Biomedicals  and all glassware was cleaned with 1% Virkon  overnight prior to steeping in 2% Decon  for 4 h prior to use.et al.32. In brief, the faecal sample was weighed, mixed 1:1 wt/vol with ice cold 0.01 M phosphate buffered saline (PBS) then homogenised in a Seward 600 stomacher (2 × 2 min cycle). The resultant faecal slurry was centrifuged at 50,000 × g for 2 h at 4 °C in the 70.1Ti rotor of a Beckman L8-M centrifuge and the supernatant removed and filter–sterilised (0.22 μm filter) on ice before being aliquoted for storage at −70 °C.The Ulster University Research Ethics Committee exempted this study from review because donors were not involved in any intervention; the samples received were not collected by means of intervention and were used solely for preparation of a bacterial growth media. Written consent was obtained for provision of the donor faecal samples. Fresh faecal samples were provided by two apparently healthy individuals . Stool samples were collected from donors and stored at 4 °C for up to 2 h before processing. Faecal water was produced as described in Gill Clostridium difficile 630 (ATCC no. BAA-1382D-5) was grown under anaerobic conditions in a Don Whitley MACS MG500 anaerobic cabinet  using a single gas mix  of 80% N2, 10% CO2 and 10% H2, at 37 °C. Standard growth media was brain heart infusion broth supplemented with 5 g L−1 yeast extract and 1 g L−1 L-cysteine (BHIS). For media containing faecal water, 2–fold concentrated BHIS (50 mL) was prepared and a 50 mL aliquot of filter–sterilised FW added to this aseptically post–autoclaving, resulting in 1 × BHIS containing 50% FW (“FW media”). Control media was prepared from 2–fold concentrated BHIS to which was added an equal volume of sterile PBS. Three starter cultures of C. difficile 630 were set up in 20 mL of FW media in glass universal bottles and each was inoculated with a single colony of freshly grown C. difficile 630 from a BHIS agar plate. Starter cultures were incubated overnight and used to inoculate fresh media, in triplicate, at 5% (vol/vol). Growth was recorded hourly as attenuance at 650 nm (D650nm)44 against un–inoculated BHIS and FW media references. Multiple cell pellets were collected by centrifugation from all six cultures at mid–log phase of growth (D650nm = 0.6). Culture supernatants were removed to fresh tubes and both cell pellets and supernatants were placed briefly in liquid nitrogen before immediate transfer to −70 °C until required.45. RNA was checked for absence of DNA contamination by PCR with gyrA, rpsJ, and adk primers  glycogen and 1/10 vol. 3 M sodium acetate. Pellets were washed once with 80% ethanol, followed by 70% ethanol and after air-drying, pellets were resuspended in 58 µL of water. The standard protocol for use with Ribosome Depleted RNA was resumed for the remaining steps, except libraries were size selected using Agencourt AMPure XP beads at a 1.5 x ratio to retain the smaller sized fraction (~150 bp). The NEB Next Multiplex Oligos for Illumina kit (Primer set 1)  was used to generate barcoded multiplex libraries. Library QC was performed using bioanalyser HS kit  and libraries were quantified using qPCR . Libraries were pooled at desired concentrations, denatured and loaded for sequencing according to the manufacturer’s instructions. Sequencing was performed over 3 runs on the Illumina MiSeq sequencing platform to generate 2 × 75 bp reads.RNA sequencing (RNAseq) and initial bioinformatics analysis was performed at Deepseq . RNA samples were shipped to Deepseq on dry ice and upon receipt, total RNA quality was once more assessed using the Agilent RNA 6000 Nano Kit  on the Agilent 2100 Bioanalyzer. The total RNA concentration was measured using the Qubit RNA BR assay kit . A 1 µg amount of Total RNA was used for rRNA depletion using the Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) . Illumina stranded whole transcriptome sequencing libraries were prepared using NEBNext Ultra Directional RNA library prep kit for Illumina . The standard protocol for use with Ribosome Depleted RNA was followed except that, after second strand synthesis, the samples were precipitated with 1 µL (20 ng µLC. difficile strain 630 reference genome (http://www.ncbi.nlm.nih.gov/nuccore/115249003) with appropriate alignment software. The aligned files were then processed for tag counts per location mapped or normalised tag counts (RPKMs) and differential gene expression analysis. The DeepSeq Filtering Pipeline for Read Mapping was used to filter reads with low sequencing score, in addition to reads aligned to adaptor sequences. Reads from the sequencer were QC checked using FASTQC, then trimmed and filtered for low quality bases and adaptor sequences, and QC checked once more. Reads that passed this filter were mapped onto the reference genome in the context of known gene exon coordinates using the bwa mapping tool (http://bio-bwa.sourceforge.net/). Read alignments were recorded in a BAM formatted alignment file (named *.bam), and companion BAM index file (named *.bam.bai). Read alignments, both primary and unique, were then filtered further according to their mapping quality score (MAPQ). For gene expression, MAPQ20 uniquely aligned reads were used to generate counts per gene using ‘htseq-count’, which determines the number of uniquely aligned reads per gene (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html).For differential gene expression analysis the sequencing reads were mapped onto the annotated 85. DESeq models the distribution of the counts data in each sample and then compares the distributions to determine differentially expressed genes, with significantly differentially expressed genes having an adjusted p value < 0.05. The program implements a single analytical approach and when RNA–seq samples with biological replicates are available, as is the case here, DESeq analyses the variance between them in order to better model the expression values of individual genes within the group of replicates.These counts were used as the input for the DESeq program86 repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112422) with accession number GSE112422.The data discussed herein has been deposited in NCBI’s Gene Expression OmnibusC. difficile strain 630 genome (http://www.ncbi.nlm.nih.gov/nuccore/115249003) was used as a starting point for addition of the C. difficile strain 630 locus annotations88, in addition to protein name and Subtilist functional category90. This process was carried out essentially as in our previous work46 using the NCBI CDD database, BioCyc pathway tools and metacyc visual pathways software92 combined with literature searching to arrive at a functional role/categorisation and to identify predicted co–regulated genes and operon structures  and faecal water medium  growth conditions, in addition to p value, p-adjusted value and the ratio of FW/BHI base mean values, sorted by p-adjusted (padj) value from low to high. Some 1687 genes had p < 0.05, 1153 genes had padj < 0.05, reducing to 889 genes for which padj was < 0.001. The base mean values for these 889 genes were used to calculate log2 values for each FW/BHI ratio, from which was calculated the absolute fold–change for each gene. Subsequent analysis was undertaken with the statistically robust master list of 889 differentially expressed (DE) genes with pdaj < 0.001 and FC > 1.45. The NCBI 45 differential gene expression data was corroborated using qRT-PCR, on aliquots of the same RNA samples that were sent for sequencing. cDNA was prepared from 500 ng aliquots of the extracted RNA samples and 50 ng of random hexamer primer  with a SuperScript II Reverse Transcriptase kit . Successful reverse transcription and generation of cDNA was confirmed by PCR using rpsJ, gyrA or adk primers  using the information gained from standard curves previously constructed for each primer pair, thus ensuring PCR efficiencies were calculated accurately for each gene. All target gene RQs were normalised against the geometric mean of the reference gene RQs by dividing the former by the later (target/housekeeping) to generate normalised relative quantity (NRQ) values. Control sample NRQ values (BHIS) were subsequently used as a calibrator and corrected to 1 with all experimental NRQ values (FW media) being expressed as a relative expression ratio to the calibrator for each gene. Values were subsequently expressed as fold change ratios relative to the BHI control  was used to generate a Cm/z) in profile peak mode was followed by data–dependent MS/MS in centroid peak mode of the three most intense ions using a collision energy of 45 eV, activation Q of 0.25, trapping time 30 ms, and isolation width of 2 m/z. Full scan data were collected at a mass resolution of 30,000 . Scan speeds of 0.1 s and 0.4 s were applied in the LTQ and FT-MS respectively. The Automatic Gain Control was set to 1 × 105 and 5 × 105 for the LTQ and FT-MS, respectively. Prior to the analytical run, the LTQ and FT-MS were tuned to optimise conditions for the detection of ions in the mid detection range of m/z 80–2000, as well as being calibrated with the manufacturer’s recomended calibration mixture. ESI conditions were as follows: spray voltage −3.5 kV (ESI-); sheath gas 60; aux gas 30; capillary voltage −35 V (ESI−); tube lens voltage −100 V (ESI-); capillary temperature 380 °C. For the first three min of analysis, the eluent flow was directed to waste. All predicted formula data presented are accurate at <2 ppm.Culture media samples were frozen and transported to the Hutton Institute on dry ice where they were stored at −70 °C prior to analysis. After thawing on ice, samples (1 mL) were vortexed then transferred to 2 mL microcentrifuge tubes and centrifuged at 10,000 × g for 10 min at 5 °C in a refrigerated microfuge. A sub-sample (0.5 mL) was removed and placed in a 0.45 µm PTFE filter vial  prior to analysis using the LTQ-Orbitrap XL LC−MS system. Samples were analysed using a LC system consisting of a quaternary pump  and a PDA detector  coupled to an LTQ Orbitrap XL mass spectrometer . Duplicate 10 μL samples were injected in part-loop mode onto a 2 × 150 mm (4 μm) Synergy Hydro-RP 80Ä column fitted with a C18 4 × 2 mm Security Guard cartridge . Autosampler and column temperatures were maintained at 6 °C and 30 °C, respectively. The samples were analysed at a flow rate of 0.3 mL/min using a binary mobile phase of (A) 0.1% aqueous formic acid and (B) 0.1% formic acid in acetonitrile/water  with the following gradient: 0–4 min, 5% B; 4–22 min, 5–50% B; 22–32 min, 50–10 0% B. Following each analysis, the column was equilibrated for 5 min under starting solvent conditions. Mass detection was carried out using an LTQ Orbitrap XL mass spectrometer in negative ESI mode. Two scan events were employed; full-scan analysis (130–2000 m/z × time) for each per sample was converted (or deconvoluted) into a vector of peak responses, where a peak response is defined as the sum of intensities over a window of specified mass and time range (e.g. m/z = 102.1 ± 0.01 and time = 130 ± 10 s) using the freely available XCMS software (http://masspec.scripps.edu/xcms/xcms.php). A full description of the data deconvolution method performed within XC–MS is available93. In the current work, the band width setting was adjusted from 10 to 20 to accommodate the wider peak widths that result from HPLC as compared to UHPLC. The XC–MS deconvolution produced an MS Excel based X by Y matrix output as peak areas for detected peaks.The raw LC–MS data files were first converted into an MZML centroid format using the Proteowizard MSConvert software package. Each MZML based three-dimensional data matrix (intensity × https://umetrics.com) and principal components analysis (PCA) was carried out. PCA, using univariate scaling, clearly showed that the FW samples separated from the BHI-only samples on score 1, which explained 52% of the variation of the dataset. The beginning and end samples were clearly separated in scores 3 and 4 of the PCA, which explained 10% and 5%, respectively, of the variation  present) and the PutMedID set of workflows within the Taverna Workbench 1.7.2 software package94 was applied to predict putative metabolite identities using a library of known plant metabolites obtained from the Plant Metabolic Network PlantCyc database (http://www.plantcyc.org). In many cases, however, the putative identifications were not supported by subsequent examination (e. g.) of MS2 data. Therefore, further manual putative peak assignation was carried out by comparing the predicted molecular formulae and MS2 data with various databases and literature (Supplementary Data File The XCMS data selected each Images shown in Figs Images in Supplementary data file The Ulster University Research Ethics Committee exempted this study from review because donors were not involved in any intervention; the samples received were not collected by means of intervention and were used solely for preparation of a bacterial growth media. Written consent was obtained for provision of the donor faecal samples.Supplementary Data file 1Supplementary Dataset Table S3


Rheumatoid arthritis (RA) is associated with a 5 to 10 years reduction in life expectancy due to premature atherosclerosis. This reduction is the consequence of traditional cardiovascular risk factors (TCRF) as well as systemic inflammation. The aim of the present study was to describe the prevalence and factors associated with subclinical atherosclerosis in RA at the University Hospital of Kinshasa (UHK).Patients with a diagnosis of RA based on the 2010 ACR/EULAR criteria were included in this cross-sectional study from 1 June 2014 to 31 May 2015 at the UHK. RA disease activity was measured using the DAS28-ESR. Active RA was defined by a DAS 28 > 2.6. Severe RA was defined by the presence of extra-articular manifestation, joint erosions on X-rays or HAQ ≥0.5. An assessment of subclinical atherosclerosis was performed by the measurement of the carotid intima-media thickness (cIMT) using two-dimensional ultrasonography. Subclinical atherosclerosis was defined by a cIMT ≥0.9 mm. A diagnosis of atheroma plaque was retained when the cIMT was ≥1.5 mm. The association between subclinical atherosclerosis and potential risk factors was modeled using logistic regression analysis.p = 0.028), DAS28-ESR > 2.6 , severe RA , high blood pressure  and obesity  emerged as factors associated with subclinical atherosclerosis.We recruited 75 patients. The average age was 51.8 ± 14.6 years, with a sex ratio F/M of 4. The prevalence of subclinical atherosclerosis was 32%. In logistic regression being a woman of ≥55 years old (aOR 10.6, 95% CI [2.087–53.82], Subclinical atherosclerosis is common in RA patients attending the UHK. It appears to be associated with RA disease activity and severity apart from traditional cardiovascular risk factors. These results suggest that early management of subclinical atherosclerosis targeting remaining RA disease activity and cardiovascular risk factors could slow down progression to clinical cardiovascular disease. Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic condition of autoimmune origin that affects about 0.5 to 1% of adults worldwide . Beyond This high incidence of CV morbidity and mortality results from earlier and more severe atherosclerosis compared to the general population due in part to a higher prevalence of traditional cardiovascular risk factors  , 9. But In Democratic Republic of Congo (DRC), the relationship between atherosclerosis (ATS) and RA has not been evaluated yet. The aim of the present study was to describe the prevalence and associated factors of subclinical atherosclerosis in RA, at the University Hospital of Kinshasa (UHK).2).Consecutive RA patients were recruited between 1 June 2014 to 31 May 2015 at the rheumatology unit. Patients who suffered from another inflammatory rheumatic disease were not included in the present study. The following parameters were collected: socio-demographic data , delay between the disease onset and the first consultation, DAS 28-ESR, joint deformities, extra-articular manifestations , cardiovascular risk factors , blood pressure (mmHg), body weight (kg), abdominal perimeter (cm), heart rate, height (cm) and BMI (Kg / mThe diagnosis of RA was retained according to the 2010 ACR / EULAR criteria. RA disease activity was measured with the DAS28-ESR score (DAS 28 = [0. 56 x √ t28] + [0. 28 x √ sw28] + [0. 7 x Ln (ESR)] + [0.014 x (VAS-GH)]. The presence of extra-articular manifestations, joint erosions on X-ray and a HAQ ≥ 0.5 defined a severe RA. Blood pressure was measured 3 times, with measurements taken 2 min apart, using an OMRON Hem 700 IE® electronic blood pressure monitor. The patient had to be seated since at least 5 minutes and the average of the last two measurements was used for the analyses. The waist circumference (WC) was taken between the last rib and the iliac crest at the end of expiration with a tape measure. The size was taken using a SECA messband 206 cm. The weight was measured, using a KINLee DT01 scale, on a barefoot and slightly dressed patient. Pulse pressure (PP) was calculated as the difference between systolic blood pressure (SBP) and diastolic blood pressure (DBP).The carotid intima-media (cIMT) thickness was measured by a trained sonographer. This measurement was performed at 2 cm from the carotid bulb with a LOGIQ C5 Premium color doppler ultrasound. The ultrasound system had a linear high frequency probe of 7.5 to 12 MHz. This measurement was performed on the left and right sides of the patient laying down in the supine position, arms along the body and head in moderate extension.Upon completion of the clinical examination and ultrasonographic measurements of the cIMT, patients were referred to the UHK laboratory for blood sampling between 8 am and 9 am and after a 12 h fasting period. Fasting plasma glucose was measured using the enzymatic glucose oxidase method. Total cholesterol, triglycerides, HDL-c, uric acid were determined according to enzymatic methods with a semi-automatic device of Humalyser Primus brand. The LDL-c level was calculated using the formula of Friedewald : LDL-c =Hand X-rays were performed in all patients and read by a trained radiologist. Joint space narrowing, erosions and juxta-articular demineralization were looked for.Definitions of some concepts:Early RA: duration of disease ≤2 yearsEstablished RA: duration of disease > 2 yearssynovitis: swollen and/or painful joints2Obesity (WHO)  was defiHigh blood pressure (HBP) was defined as a blood pressure ≥ 140/90 mmHg or intake of antihypertensive therapy .Diabetes mellitus was defined as two fasting glucose levels ≥126 mg/dl or, regardless of blood glucose, intake of anti-diabetic treatment .Cigarette smoking: Smoking at least 1 cigarette/day for more than 5 years or having stopped smoking for less than 5 years .Alcoholism: defined as a regular intake of 2 or more drinks of beer per day, it being understood that a glass of beer is equivalent to 10 g of alcohol .High pulse pressure (PP): PP > 60 mmHg .Tachycardia: heart rate (HR) > 90 beats / min .Physical inactivity: not being engaged in minimal physical activity defined as the equivalent of a 30 min walk three times weekly .Metabolic syndrome: was defined according to NCEP-ATP III by the presence of at least 3 of the following criteria: BP ≥ 130/85 mmHg or intake of antihypertensive treatment, waist circumference > 102 cm (man) and > 88 cm (women), HDL-c < 40 mg / dl in man and < 50 mg / dl in women, triglycerides ≥150 mg / dl), fasting blood glucose ≥100 mg / dl or antidiabetic treatment .Active RA: DAS 28 > 2.6Severe RA: presence of extra-articular manifestation, X-rays erosions or HAQ ≥ 0,5High or abnormal ESR: > 16 mm / h1 in men, and > 22 mm / h1 in women.Significant inflammatory syndrome: ESR ≥ 60 mm/h1 .Highly positive rheumatoid factor: RF: > 60 IU / ml .High or abnormal CRP: CRP > 6 mg / lHyperuricemia: a level of uric acid> 70 mg / l in men and > 60 mg/l in women .Dyslipidemia was characterized by elevated total cholesterol ≥200 mg/dl, LDL-c ≥ 130 mg/dl, triglycerides ≥150 mg /dl, and HDL-c < 40 mg / dl in men and < 50 mg / dl in women .Subclinical atherosclerosis:cIMT ≥0.9 mm. The patient was classified as having an atheroma plaque in case of a cIMT ≥1.5 mm or in the presence of an established atheroma plaque .Data are presented as frequencies, average ± SD or medians with their extremes. Averages and median were compared using the Student t test and the nonparametric Wilcoxon/Mann Whitney tests respectively. The Pearson Chi-square or Fisher exact tests were applied to compare proportions. A multivariate regression analysis determined factors that were associated to subclinical atherosclerosis. Only variables significantly associated with subclinical atherosclerosis in univariate analysis were tested in multivariable analysis. The collinearity test was used to identify collinear variables .if two variables were collinear, only one had entered in the final model of logistic regression in multivariable analysis. Statistical analyses were performed with the SPSS software version 21.0.75 RA patients were enrolled during the study period, including 15 males (20%) and 60 females (80%). Their average age was 51.8 ± 14.6 years. Most patients presented an active, severe and long-lasting disease. The median duration of the disease was 3 years, ranging between 2.4 months and 12 years. Symptoms of synovitis and joint deformities were observed in less than half of patients. The most common extra-articular manifestation was the presence of rheumatoid nodules , severe RA, hypertension, diabetes mellitus, physical inactivity, obesity, hypertriglyceridemia and metabolic syndrome were the main factors associated with subclinical atherosclerosis Table . In multThe present study described the prevalence of subclinical atherosclerosis in Congolese RA patients and identified its determinants.The average age of participants was 51.8 ± 14.6 years. This is similar to the mean age reported in previous studies conducted in Kinshasa , 28, 29.The proportion of patients with rheumatoid factor was lower than those reported in Senegal , Togo 3 and espeThree out of 10 patients had subclinical atherosclerosis. This frequency is similar to those reported by Kassem et al. in 2010 (33.3%) , HannawiPatients with subclinical atherosclerosis had a significantly higher SBP and PP values than those without atherosclerosis. Indeed, the association between SBP, PP and subclinical atherosclerosis generally reflects the loss of the elasticity of the arterial wall of the aorta which becomes rigid, explaining the elevation of SBP as well as the decrease of DBP; and consequently the elevation of the PP .The present study did not show a significant association between subclinical atherosclerosis and the duration of the disease. These results corroborate those of Jonsson et al. , as wellBeing a woman ≥55 years old, disease activity level and severity of the disease, hypertension as well as obesity emerged as the main factors independently associated with subclinical atherosclerosis. The impact of age in the development and progression of atherosclerosis has been demonstrated in several studies , 44. AgeThe present study did not observe an association between CRP and subclinical atherosclerosis. This result must be considered with caution because of the small size of our sample. Apart from that, we have used a quantitative method of CRP with a cutoff of 6 mg/l (the most commonly available in our country). With this method CRP values and cutoffs are higher compared to the ultrasensitive method and may have underestimated the number of patients with high CRP level. So, it is possible that by using an ultrasensitive method, we might have found an association between CRP and subclinical atherosclerosis.The activity and severity of the disease have also been implicated in the occurrence of subclinical atherosclerosis. Systemic inflammation, which can precede the onset of joint symptoms in RA, may favor the development of atheromatous disease before joint manifestations. The pathogenesis of atherosclerosis is related to that of autoimmune joint disease , 48, 49.The present study has some limitations. First, the small sample size cannot provide enough power for the statistical tests used to detect possible associations. Second, the cross-sectional nature of this study excludes any possibility to establish a causal relationship. Third, the lack of measurement of ACPA in our study is also a limitation since ACPA may be related to the increasing of cardiovascular risk. These limitations may be solved by a case-control study or a longitudinal study on a large sample.However, this study has the merit of being the first to describe the prevalence of subclinical atherosclerosis in Congolese RA patients and to identify its associated factors. Moreover, it shows that the measurement of the cIMT may be used as a simple and reliable tool for detecting subclinical atherosclerosis, and identifying patients who are most likely to benefit from preventive measures.The current study showed that subclinical atherosclerosis is frequent (32%) among patients suffering from rheumatoid arthritis at the University Hospital of Kinshasa. It is associated with traditional cardiovascular risk factors and RA disease characteristics. Patients with subclinical atherosclerosis had a more active and severe disease than those without subclinical atherosclerosis. These results suggest that screening and early management of subclinical atherosclerosis and risk factors in patients with RA could slow down the progression to clinical cardiovascular disease.


This study aims to reveal the regulatory mechanism of lncRNAs–miRNAs–mRNAs network during the proliferative phase of liver regeneration (LR). High‐throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA–miRNA–mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases. It has a strong ability of regeneration after liver loss or toxic injury. Liver regeneration is a highly organized tissue growth process of restoring the original framework structure and tissue specific function after liver injury  rats weighing 210–250 g were provided by Laboratory Animal Center of Zhengzhou University . These rats were raised in a controlled temperature room 19–23℃ with a relative humidity 50–70% and illumination time 12 hr/day (8:00–20:00), and permitted to freely have water and food. A total of 36 rats were randomly divided into six groups with six rats per group: Five partial hepatectomy (PH) groups and one normal group (CG). The rats in PH group were subjected to 2/3 PH in accordance with the method of Xu C. et al. . They we2.2The mirVana miRNA Isolation Kit (Ambion) was used to extract total RNA and the TruSeq Stranded Total RNA with Ribo‐Zero Gold was used to construct the complementary DNA (cDNA) libraries. In brief, after total RNA extracted and ribosomal RNA digested, the RNA was broken into short fragments by the interrupt reagent. The first cDNA chain was synthesized using these short fragments as template and a random six‐base as primer. Then the second cDNA chain was synthesized using the first cDNA as template and the dTTP was replaced with dUTP. After repairing the end, jointing adenylate 3′ ends and sequence adapters, the second cDNA chain was digested by UNG (Uracil‐N‐Glycosylase) enzyme, and the first cDNA with different joints was retained. Agarose gel electrophoresis was used to select the fragment size. Finally, polymerase chain reaction (PCR) amplification was performed. After the constructed library passed the quality inspection with Agilent 2100 Bioanalyzer , Illumina sequencing platform (Hiseq X Ten) was used for sequencing.p value < 0.05 were used as the cut‐off criteria.Raw reads of fastq format were performed quality preprocessing by using Trimmomatic (0.36). After wiping off the adapter and low‐quality reads, the clean reads were obtained. The hisat2 (2.2.1.0) was use to align the clean reads with the rat reference genome. For unmapped reads, transcripts were reconstructed based on the probability model based on the comparison results of each sample by using StringTie (1.3.3b). Finally, the candidate lncRNAs were identified with software CPC (0.9‐r2), CNCI (1.0), PFAM (v30), and PLEK (1.2), which were used to predict the coding capacity of the transcripts. The expression abundance of lncRNAs and mRNAs were measured by FPKM. The differentially expressed mRNAs (DE mRNAs) and differentially expressed lncRNAs (DE lncRNAs) were detected by the negative binomial distribution test based on the DESeq package (1.18.0). The fold change ≥ 2 or fold change ≤ 0.5, and 2.3The mirVana miRNA Isolation Kit (Ambion) and TruSeq Small RNA Sample Prep Kits were used to extract total RNA and construct the cDNA libraries. The whole process was carried out in strict accordance with the reagent instructions. First, T4 ligase was used to ligate a 5′ adapter and a 3′ adapter to the RNA molecules. Then a SuperScript II Reverse Transcription Kit (Invitrogen) was used to reverse‐transcribed 5′ and 3′ adapter‐ligated RNA to cDNA and PCR amplification was performed. Finally, the cDNA product was purified by RNA Gel Electrophoresis and gel recovery. The size and purity of the sample were determined using an Agilent 2100 Bioanalyzer . The Illumina sequencing platform was used for sequencing analysis.p value was calculated by Audic–Claverie statistic. The fold change ≥ 2 or fold change ≤ 0.5, and p value < 0.05 were used as the cut‐off criteria.Raw reads of fastq format were processed consisting of removing adapter and low‐quality reads including sequences with quality score less than 20 and sequences with N base to obtain high‐quality clean reads. The Bowtie2 was use to map the clean reads to mature miRNAs in miRBase 21.0 database. These consistent sequences were considered as the known miRNAs. The expression level of miRNAs was measured by TPM. The 2.4http://genecodis.cnb.csic.es) and DAVID Bioinformatics Resources 6.8 ((https://david.ncifcrf.gov/). GO enrichment analysis included biological process (BP), molecular function (MF), and cellular component (CC). The p values have been obtained through hypergeometric analysis corrected by the false discovery rate (FDR) method. Both GO terms and KEGG pathways were considered to be significantly enriched with FDR < 0.05.To analysis the biological function of lncRNAs, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the DE mRNAs and predicted target genes of DE miRNA and DE lncRNAs using GeneCodis3 bioinformatics resources  to identify transcription factors (TF). Then a TF–miRNA–lncRNA regulatory network was constructed based on TF–miRNA and miRNA–lncRNAs interaction pairs and visualized by Cytoscape (v3.6.1) software.The mRNAs in the lncRNA–miRNA–mRNA interaction network was analyzed by AnimalTFDB database (33.1p < 0.05) at 12, 24, 30, 36, and 72 hr after PH , a total of 1,738 DE lncRNAs were identified, with 1,069 upregulated, 653 downregulated, and 16 up/downregulated . Five significant miRNAs, rno‐miR‐370‐3p (degree = 53), rno‐miR‐324‐3p (degree = 35), rno‐miR‐6315 (degree = 27), rno‐miR‐1956‐5p (degree = 25), and rno‐miR‐484 (degree = 25) had the most target mRNAs.In the next step, the DE lncRNAs regulated by DE miRNAs were analyzed by miRanda algorithm. Totally, 116 miRNA–lncRNA regulatory pairs were identified including 34 miRNAs and 108 lncRNAs . In the miRNA–lncRNA network, rno‐miR‐296‐3p (degree = 12), rno‐miR‐324‐3p (degree = 11), rno‐miR‐370‐3p (degree = 11), rno‐miR‐331‐3p (degree = 8), and rno‐miR‐6315 (degree = 8) had the most target lncRNAs.Based on the regulatory pairs of miRNA–mRNA and miRNA–lncRNA, a lncRNA–miRNA–mRNA network was constructed, consisted of 32 miRNAs, 107 lncRNAs, and 270 mRNAs  enrichment analysis of the 270 mRNAs was conducted to reveal their functions. The result showed that 86 BP terms, 19 MF terms, and 34 CC terms were enriched . The most enriched BP terms were multicellular organismal development, anatomical structure development, cellular component organization, nitrogen compound metabolic process, and cellular metabolic process. As for MF, the most enriched GO terms were protein binding, nucleotide binding, sequence‐specific DNA binding transcription factor activity, transferase activity, and nucleic acid binding. The most enrichment CC terms were intracellular, cell part, intracellular part, intracellular organelle, and membrane‐bounded organelle. Then Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the 270 mRNAs was performed to explore the signaling pathways involved. A total of nine significant pathways were enriched including insulin signaling pathway, pyrimidine metabolism, carbohydrate digestion and absorption, and pyruvate metabolism.3.5The lncRNAs–miRNA–mRNA network was consisted with 107 lncRNAs, 32 miRNAs, and 270 mRNAs. Of these miRNAs, five were reported to play an important role during LR, including miR‐21, miR‐127, miR‐34a, miR‐378, and miR‐125b, and they were regarded as the core miRNAs. The core lncRNAs were selected with a differently expression, and they were associated with the five miRNAs. Finally, nine core lncRNAs were correspondingly identified  were identified to be involved, and then a TF–miRNA–lncRNA regulatory network was constructed  were found to be involved in TF‐miRNAs regulation network during the proliferative phase of rat LR. Zinc‐finger protein ZBTB20, also named DPZF, HOF, and ZNF288, was a critical regulator of EGFR expression and hepatocyte proliferation in mouse liver regeneration (H. Zhang et al., Some limitations were existed in this study. LncRNAs have a variety of functions. However, only the role of lncRNAs as miRNA sponges was analyzed through building the regulatory network of lncRNA–miRNA–mRNA and lncRNA–miRNA–TF. In addition, key lncRNAs predicted by bioinformatics analysis were not experimentally verified during the proliferative phase of rat LR.5First, DE lncRNA, DE miRNA, and DE mRNA were analyzed by high‐throughput sequencing technology, and then the lncRNA–miRNA–mRNA regulatory network was constructed according to the regulation mechanism of lncRNAs. Finally, through literature review and lncRNA–miRNA regulatory pairs, nine key lncRNAs, and five key miRNAs were screened out, which may play an important role during the proliferative phase of rat LR. This study provided clues for revealing the mechanism of LR and offered new ideas for the treatment of liver‐associated diseasesAll experimental procedures in this study were approved by and performed according to the guidelines for the care and use of experimental animals that have been established by the Animal Care and Use Committee of the Ministry of Science of the People's Republic of China.The authors declare that there are no conflict of interests.Haijing Bai wrote the manuscript and analyzed data. Cunshuan Xu participated in its design and interpretation. Wei Jin, Jianlin Guo, Xueqiang Guo, and Cuifang Chang critically revised the manuscript, and all authors have read and given approval of the manuscript.The data that support the findings of this study are available from the corresponding author upon reasonable request.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


No study has been conducted to demonstrate the feasibility of an opioid-free anesthesia (OFA) protocol in cardiac surgery to improve patient care. The aim of the present study was to evaluate the effect of OFA on post-operative morphine consumption and the post-operative course.After retrospectively registering to clinicaltrial.gov (NCT03816592), we performed a retrospective matched cohort study (1:1) on cardiac surgery patients with cardiopulmonary bypass between 2018 and 2019. Patients were divided into two groups: OFA  or opioid anaesthesia (OA) (sufentanil). The main outcome was the total postoperative morphine consumption in the 48 h after surgery. Secondary outcomes were rescue analgesic use, a major adverse event composite endpoint, and ICU and hospital length of stay (LOS).n = 55; OA: n = 55). On inclusion, demographic and surgical data for the OFA and OA groups were comparable. The total morphine consumption was higher in the OA group than in the OFA group (15 (6–34) vs 5 mg (2–18), p = 0.001). The pain score during the first 48 post-operative hours did not differ between the two groups. Creatinine values did not differ on the first post-operative day (80 (IQR: 66–115) vs 77 mmol/l (IQR: 69–95), p = 0.284). Incidence of the composite endpoint was lower in the OFA group (25 patients (43%) vs 38 patients (68%), p = 0.021). The time to extubation and the ICU stays were shorter in the OFA group (3 (1–5) vs 5 (3–6) hours, p = 0.001 and 2 (1–3) vs 3 (2–5) days, p = 0.037).One hundred ten patients were matched (OFA: The use of OFA was associated with lower morphine consumption. OFA might be associated with shorter intubation time and ICU stays. Further randomized studies are needed to confirm these results.NCT03816592) on January 25, 2019.This study was retrospectively registered to ct2 (identifier:  Since the 1960s, the systematic administration of opioids has been considered one of the pillars of modern anaesthesia . HoweverOpioid-free anaesthesia (OFA) is a long-standing concept. It is based on the fact that a sympathetic reaction evidenced by hemodynamic changes in an anesthetised patient does not systematically reflect pain. In addition, a sleeping patient will not recall pain, while hormonal stress and sympathetic and inflammatory reactions can be controlled by therapeutic classes other than opioids , 5. TherThe main objective of the present study was to demonstrate that, compared with OA, OFA lowers postoperative morphine consumption. In addition, we evaluated the effect of OFA on operative hemodynamic stability, postoperative complications assessed by a composite criterion and the length of stay (LOS) in the ICU and in hospital.clinicaltrial.gov (NCT03816592). Our team started using an OFA strategy for cardiac surgery in 2017. Consequently, the number of patients receiving OFA has increased steadily since 2017. In practical terms, OFA administration was left to the discretion of the attending anesthetist regardless of a patient’s co-morbidities and surgical risk. The study was performed in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki. As the study was observational and used existing, routinely collected data, and in compliance with French law (loi Jardé n° 2012–300) informed consent was not required. We submitted the protocol study to the “Délégation à la Recherche et à l’Innovation (DRCI) », CHU de Dijon, Dijon, France, and followed the MR004 (méthodologie de référence 004) in accordance with the national commission for data protection (CNIL) guidelines. The present report was drafted in line with the STROBE statement [We performed a retrospective, open-label, matched (1:1), single-centre study in a tertiary university hospital  between 2018 and 2019. All patients were included within this time frame. The study was recorded on January 25, 2019 to The main inclusion criteria were as follows: age 18 or over, cardiac surgery with the use of cardiopulmonary bypass (CPB) (coronary artery bypass grafting (CABG), the surgical correction of valve disease , combined surgery , ascending aortic disease, and left ventricular assist device implantation). The exclusion criteria were: off-pump cardiac surgery, preoperative analgesic use, gabapentin use, antidepressant therapy and preoperative cognitive dysfunction.− 1), intravenous bolus of ketamine (0.3–0.5 mg kg− 1), intravenous bolus of lidocaine (1.5 mg kg− 1 bolus 15 min before the start of propofol) and propofol (0.4–2 mg kg− 1) until the loss-of-eyelash reflex. Lidocaine was continuously administered at 1.5 mg/kg/h until the end of surgery.Maintenance or withdrawal of preoperative medications adhered to international guidelines. Anaesthesia and cardiopulmonary bypass procedures were standardised for all patients. In the OA group, anaesthesia was induced with an intravenous bolus of ketamine (0.3–0.5 mg/kg), propofol (0.4–2 mg/kg) and sufentanil (0.5 ng/ml) until the loss-of-eyelash reflex. Sufentanil was continuously administered using Schneider’s target-controlled infusion model. All OA patients had loco-regional analgesia by serratus anterior plane block  or a continuous parasternal infusion of a local anaesthetic . None of the OA patients received intravenous lidocaine. In the OFA group, anaesthesia was induced with an intravenous bolus of dexamethasone (0.1 mg kg− 1), and neuromuscular blockade was ensured using intermittent bolus of cisatracurium to reach adequate muscle relaxation measured by peripheral nerve stimulation. Sufentanil and lidocaine were discontinued at the end of surgery. In both groups, anaesthesia was maintained by target-controlled infusions of propofol (started at 2–4 ng ml− 1). Sedation was titrated using the bispectral index  to obtain a value between 40 and 60. Arterial hypertension  was treated with esmolol in case of tachycardia (heart rate > 80 bpm) or urapidil/nicardipine if the heart rate dropped below 80 bpm.In both groups, tracheal intubation was facilitated with cisatracurium  was maintained at more than 65 mmHg by increasing the pump flow rate or, if required, by administering a bolus of phenylephrine (100 μg) or norepinephrine (5 μg). The CPB circuit was primed with 1500 ml of crystalloids  and 5000 ui of heparin. After systemic heparinization (300 ui kg− 1) to obtain a hemochron level of 400 s, median sternotomy or thoracotomy was performed and aortic and right auricular cannulations were started. Normoglycemia was maintained using intravenous insulin (intravenous bolus of 5–10 ui) if necessary. Patients with a haemoglobin value below 8 g dl− 1 received homologous red blood cell transfusions. Heparin was reversed with protamine at a 1:1 ratio.Cardiopulmonary bypass was conducted with a heart-lung machine  at a target blood flow of 2.4 l min− 1 h− 1). Tracheal extubation was done according to the French guidelines [At the end of surgery, all patients were sedated and the lungs were mechanically ventilated until haemodynamic stability and normothermia were obtained and blood loss was considered acceptable  and patient-controlled morphine analgesia. Before extubation, all patients received 1 g of acetaminophen. Immediately after, all patients received titrated intravenous morphine with a bolus of 2 to 3 mg until a score of 3 or less was obtained on the visual analogue pain scale. Patient-controlled analgesia morphine was then started as follows: 1 mg bolus, refractory period of 7 min, maximum dose of 20 mg every 4 h without continuous infusion. The use of complementary analgesics was left to the discretion of the attending physician. According to our institutional protocol, complementary analgesia comprised the use of ketoprofen (50 mg), tramadol (50 mg), and nefopam (20 mg). Pain was assessed every 4 h during the ICU stay with the visual analogue scale. Non-invasive ventilation was left to the discretion of attending physician. Common indications were high-risk patients, atelectasis, hypoxemia, hypercapnia and acute respiratory failure. Patients were discharged from ICU at the discretion of their attending physician. The patients’ electrocardiogram, pulse oxygen saturation and central venous blood pressure were continuously monitored. The scheduled blood tests included arterial/venous blood gas measurements on admission to the ICU, and then several times a day on request by the attending physician.The following variables were continuously recorded in the institutional database: age, gender, body weight, height, personal medical history, ASA score, EuroSCORE2, Euroscore, type of cardiac surgery, the preoperative left ventricular ejection fraction, the duration of CPB, the duration of aortic clamping, the need for intraoperative blood transfusion, norepinephrine, dobutamine, the use of a antihypertensive agent , the use of a short acting beta-blocker (esmolol), troponin values, creatinine value, time to extubation (hours), any occurrence of complications during the stay in the ICU or in the hospital, and the LOS in the ICU.− 1), creatinine (mmol l− 1), ICU LOS (days), and hospital LOS (days). The secondary composite endpoint was assessed during the hospital stay. All data was extracted from our institutional database and collected by a physician who was not involved in the care of the study patients.The primary endpoint was the cumulative dose of postoperative morphine in the first 48 h (in milligrams). The secondary endpoints were: analgesic rescue, a composite endpoint of major adverse events , fluid expansion (ml), total propofol dose (mg), antihypertensive agent use, vasoplegia syndrome, catecholamine use, troponin Ic .The trial was designed to investigate the potential superiority of OFA in terms of postoperative morphine consumption. According to the studies of Berthoud et al., we calculated that 55 patients per group would be sufficient to demonstrate a 10 mg difference in morphine consumption (with a mean consumption of 18 mg) with a power of 0.8 and an alpha risk of 0.05 , 21. Basn = 78), the median EuroSCORE2 was 1.6 (IQR: 0.89–3.01) and the median EuroScore was 6 (IQR: 4.9–8.3).Of the 931 patients operated during the study period, 110 were matched and analysed Fig. . In the p = 0.001 (Table p = 1 and 150 (IQR: 50–262) vs 125 mg (IQR: 50–263), p = 1)), nefopam  vs 20 mg (20–33), p = 0.317), and tramadol  vs 100 mg (IQR: 50–150), p = 1)) were used in equal measures. The pain scores were also similar in the two groups  vs 15 mg IQR: 6–34), 4, p = 0.p = 0.003) whereas more patients in the OA group were treated with ephedrine . The time to extubation was shorter in the OFA group (3 (IQR: 1–5) vs 5 h (IQR: 3–6), p = 0.001) (Table p = 0.021) along with a lower use of non-invasive ventilation . Creatinine values did not differ on the first post-operative day (80 (IQR: 66–115) vs 77 mmol/l (IQR: 69–95), p = 0.284). The incidence of cardiac and renal outcomes were similar. The OFA group spent less time in the ICU (2 (IQR: 1–3) vs 3 days (IQR: 2–5), p = 0.037) though the length of the hospital stay did not differ (8 (IQR: 7–15) vs 10 days (IQR: 8–14), p = 0.790).Operative data differed between groups in terms of vasopressor use, antihypertensive drug use, and total fluid infused Table . More paIn the present study, we found that OFA was associated with: (1) lower postoperative morphine consumption; (2) higher operative use of antihypertensive agents; (3) a decrease of orotracheal intubation time and the use of non-invasive respiratory support; and (4) shorter ICU stays.To date, two published case reports and one retrospective study have reported on the use of OFA study in thoracic surgery , 24, 25,Yet one important question remains: what is the analgesic/anaesthetic effect of OFA during surgery ? OFA is OFA may be associated with certain adverse effects such as a higher incidence of blood pressure events or adverse effects resulting from toxic plasma levels. We observed a trend towards higher use of norepinephrine and anti-hypertensive agents that may be the result of several factors: the increasing use of propofol, the half-life of urapidil/nicardipine (mostly used before CPB), and the vasoactive effect of lidocaine. Several studies have already underlined the growing incidence of hypertensive episodes and the higher use of anti-hypertensive agents , 7. Baka2 agent may have several advantages. First, the combination of dexmedetomidine and lidocaine was shown to provide better postoperative pain relief than the use of each agent individually [Though we did not use dexmedetomidine in our protocol because it is not available in our department, the uses of an alphavidually . Also, ividually . Moreovevidually , 32. AccWe demonstrated that OFA resulted in lower intubation time and use of non-invasive ventilation. The respiratory effects may be explained by the avoidance of opioids and better pain relief. In our experience, patients anesthetized with OFA have a shorter period of respiratory inhibition during surgery than patients anesthetized with opioids. Spontaneous breathing returns sooner following OFA and patients seem to become alert more quickly after orotracheal extubation. In our results, oxygen requirements and non-invasive ventilation was lower in the OFA group. These effects may be associated with the respiratory depression and cognitive dysfunction that are well-known effects of opioid sedation , 33.The present study had several limitations. Our study was a single retrospective study, which implies a certain number of design-related limitations. Despite protocol management for sedation and analgesia, bias may have been introduced by the attending physician and nursing staff. In addition, some patients received analgesia in addition to patient-controlled morphine analgesia, suggesting postoperative multimodal analgesia. Nevertheless, the protocol for postoperative intravenous analgesic use was similar in the two groups. Similar limitations should be mentioned for non-invasive ventilation and ICU stays, which were left to the discretion of the attending physician. Only controlled prospective randomized studies can confirm the present results. Moreover, further studies are needed to determine the optimal associations, dosages, and infusion protocols during cardiac surgery. We have included two types of surgery (sternotomy and thoracotomy) than could be not equivalent in term of pain. Because the matching was based on this aspect, the two groups did not differ in term of type of surgery. The relatively small number of patients in our population might also limit our study’s external validity. Nevertheless, we calculated a sample size based on morphine consumption, which has been demonstrated to be associated with OFA, and we included a mixed cardiac surgery population. We believe our results demonstrate the feasibility of OFA in several cardiac surgery subtypes. Our team currently uses OFA in cardiac surgery on a daily basis without any restrictions other than contraindications to lidocaine use.The present study demonstrated that OFA was associated with a decrease in postoperative morphine consumption. OFA might have beneficial effects of on the post-operative course of patients undergoing cardiac surgery with CBP. Further randomized studies are needed to confirm these results.


Adrenal vein sampling (AVS) represents the current diagnostic standard for subtype differentiation in primary aldosteronism (PA). However, AVS has its drawbacks. It is invasive, expensive, requires an experienced interventional radiologist and comes with radiation exposure. However, exact radiation exposure of patients undergoing AVS has never been examined.We retrospectively analyzed radiation exposure of 656 AVS performed between 1999 and 2017 at four university hospitals. The primary outcomes were dose area product (DAP) and fluoroscopy time (FT). Consecutively the effective dose (ED) was approximately calculated.2 (0.3–3181) and FT 18 min (0.3–184). The calculated ED was 6.4 mSv (0.1–636). Remarkably, values between participating centers highly varied: Median DAP ranged from 16 to 147 Gy*cm2, FT from 16 to 27 min, and ED from 3.2 to 29 mSv. As main reason for this variation, differences regarding AVS protocols between centers could be identified, such as number of sampling locations, frames per second and the use of digital subtraction angiographies.Median DAP was found to be 32.5 Gy*cmThis first systematic assessment of radiation exposure in AVS not only shows fairly high values for patients, but also states notable differences among the centers. Thus, we not only recommend taking into account the risk of radiation exposure, when referring patients to undergo AVS, but also to establish improved standard operating procedures to prevent unnecessary radiation exposure. Primary aldosteronism (PA) is the most common curable cause of secondary hypertension and characterized by the autonomous adrenocortical oversecretion of aldosterone leading to cardiovascular and metabolic complications , 3, 4, 5Another argument brought forward against AVS is the potentially high radiation dose, to which patients are submitted during the procedure. Even though there are proven risks linked to radiation exposure, e.g. an increased incidence of cancer, to our knowledge, there is no available data so far addressing this particular problem. We, therefore, aimed to retrospectively analyze radiation exposure of AVS in four different specialized centers across Germany.n = 53), Leipzig , Munich  and Würzburg  with documented information regarding dose area product , fluoroscopy time  and, if available, performing radiologist and success of cannulation based on cortisol measurements were included in the analysis, without specific exclusion criteria. We furthermore collected information on sampling locations and imaging modalities for each center. Data were obtained at each participating center and sent to the coordinating center after anonymization.We performed a retrospective analysis to assess radiation exposure caused by AVS. In all patients, informed consent for performance of AVS within clinical routine had been obtained in accordance with respective local regulations. The analysis was approved by the Ethics Committee of the University of Würzburg (AZ121/17). Overall, 658 AVS performed between 1999 and 2017 in Berlin  and effective dose . FT constitutes for the amount of time during the procedure, in which fluoroscopy is used. It poorly correlates with other dose indicators. DAP is defined as the product of the radiation dose to air multiplied by the area of X-ray field, as measured by an ionization chamber mounted on the X-ray collimator. DAP is used to assess the radiation exposure of irrigated tissues and forms the basis for calculation of ED. ED represents the stochastic risk related to ionizing radiation and was approximately estimated as previously described: ED (mSv) = DAP (Gy*cm²) × 0.2 (mSv/Gy*cm²) (Assessed radiation measures were FT (min), DAP (Gy*cm/Gy*cm²) .Across participating sites the following angiography systems were used: Artis Zee, Axiom Artis XA C, Axiom Artis BA, Multistar TOP, Polystar XA , Innova 4100  and ALLURA Xper FD System . For details, see U test. Normally distributed data are given as mean ± s.d., whereas not normally distributed parameters are shown as median (min–max). Kruskal–Wallis test and post hoc analysis (Dunn–Bonferroni) were used to compare DAP and FT grouped by number of performed AVS. P value <0.05 was considered statistically significant. SPSS version 24.0 (IBM) was used for statistical analysis.Comparison of not normally distributed continuous data was performed using Mann–Whitney n = 656). Median age of patients was 53 (16–85) years. Ten patients underwent AVS twice. Two centers  used rapid cortisol measurements during AVS to determine correct catheter localization. At center 3, bilateral simultaneous catheterization of adrenal veins was carried out in 35% of cases, whereas all other AVS procedures were performed as sequential catheterization. All AVS were performed without cosyntropin stimulation. Cannulation was successful in 80% of AVS with a large variability across participating centers P < 0.001).Overall, 658 procedures were assessed. Due to missing data for ED, DAP and FT, two patients were excluded from the study (2 (0.3–3181). However, DAP showed high variation among institutions but also within the respective centers, ranging from 16.2 Gy*cm2 (0.3–3181) up to 147 Gy*cm2 (1.1–1186) P < 0.001) P ≤ 0.001). n = 78) received a median ED of 3.4 mSv (0.1–237), patients >70 years (n = 44) were exposed to 15 mSv (1.2–178) (P < 0.001). Additionally, median ED was significantly lower in female patients .Median DAP across all centers was at 33 Gy*cmRegarding experience level of performing radiologists, radiation exposure decreased with increasing numbers of performed procedures successful cannulation = 16 min (0.3–184), FTunsuccessful cannulation = 28 min (4.4–116), DAPsuccessful cannulation = 25 Gy*cm2 (0.3–3181), DAPunsuccessful cannulation = 80 Gy*cm2 (1.1–1186), EDsuccessful cannulation = 5.0 mSv (0.1–636), EDunsuccessful cannulation = 16 mSv (0.2–237), all P < 0,001). Over the period of 18 years, there were significant differences in DAP, ED and FT with predominantly consistent values in more recent years. simultaneous = 27 Gy*cm2 (0.3–3181), DAPsequential = 14 Gy*cm2 (0.8–423), P < 0.001; EDsimultaneous = 5.4 mSv (0.1–636), EDsequential = 2.8 mSv (0.2–85), P < 0.001; FTsimultaneous = 18 min (0.3–184), FTsequential = 14 min (1.9–104), P = 0.001).DAP, ED and FT were also significantly lower in successful AVS (FTNumber of standard sampling locations varied between three (center 3) and six  . Highest median ED for one center was at 29 mSv, a value equivalent to 1470 chest X-rays  and 12 tP = 0.08)  . However = 0.08)  and longAnother main result of this study is the difference in radiation doses between participating institutions, which can be explained by the respective variation of imaging modalities. In contrast to all other centers, center 3 performed less samplings in fewer locations and, most importantly, did not use DSA for visualization of vascular structures at any point, but instead documented catheterization of adrenal veins by dynamic fluoroscopic sequences or using last image hold, allowing for the last image to be saved and displayed on the monitor after stopping fluoroscopy. The use of DSA causes approximately ten times higher radiation doses compared to fluoroscopic sequences, therefore being not only a plausible, but also an adjustable factor explaining differences in radiation exposure across participating centers. Furthermore, the use of 7.5, instead of 15 or even 30 frames per second, the patient’s distance from the X-ray tube and the detector, the correct use of collimators and the system settings used for performance of AVS account for more possible influencing parameters. Interestingly, bilateral simultaneous catheterization was associated with a significant increase in radiation exposure.Based on the results of this study, there is obvious room for improvement regarding technical performance of AVS. To minimize radiation exposure, we recommend adopting some adjustments to currently applied protocols in the future: Documentation of adrenal veins should be done using predominantly last image hold, therefore limiting DSA to a minimum. Additionally, it seems possible to reduce number of sample locations to both adrenal veins and the inferior vena cava without compromising the validity and quality of AVS results, also resulting in lower FT and DAP. The radiation field should be limited to the necessary minimum. These adjustments should not only be considered to reduce patient exposure, but also the operator dose, mainly caused and determined by patient scatter . Our datn = 39) and in 2009 compared to the following years can be attributed both to improved results over time in two centers and mainly by the absence of data from center 3 , therefore leading to higher values in general. However, it should be noted, that all participating centers were experienced in the performance of AVS.There are some limitations to this study: Data on BMI of patients, a factor known to influence DAP, was not recorded . HoweverIn conclusion, this first systematic assessment shows that AVS may be associated with fairly high radiation exposure of patients with PA with significant differences across participating centers. We therefore not only suggest taking this risk into account when referring patients to undergo AVS, but also recommend the establishment and application of a common protocol to reduce and prevent unnecessary exposure to radiation.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.This work was supported by the Deutsche Forschungsgemeinschaft (DFG)  to S Hahner, M Reincke and M Fassnacht and by the Else Kröner-Fresenius Stiftung in support of the German Conns Registry-Else-Kröner Hyperaldosteronism Registry (2013_A182 and 2015_A171) to Martin Reincke.


To predict the spontaneous closure of ventricular septal defect (VSD) and assist pediatrician to manage VSD children.Between January 2008 and December 2016, 132 children diagnosed with isolated VSD by echocardiography were enrolled. All participating children were followed‐up by echocardiography yearly until the closure of VSD or 6 years old. The clinical indicators and echocardiographic parameters of patients were collected. Statistically significant factors were used to establish a Logistic Regression model for predicting spontaneous closure of VSD. Receiver operating characteristic (ROC) analysis was used to assess the specificity and sensitivity of Logistic Regression model.Spontaneous closure occurred in 60% of all patients; 57% in perimembranous VSD (p‐VSD) and 64% in muscular VSD (m‐VSD) patients. Initial diagnosis age, defect size, aneurysms tissue of the ventricular membranous septum (ATVMS), pulmonary hypertension (PH), and left ventricular diastolic dimension (LVDD) were statistically significant. Defect size, ATVMS and LVDD were determined by the Logistic Regression model as representative factor. P‐VSD and m‐VSD model had areas under the ROC curves 0.854 and 0.898, respectively.We inferred that defect size, ATVMS and LVDD were characteristic and representative predictors for spontaneous closure of VSD. And we summarized the prognostic factors and recommended a follow‐up criteria to assist the pediatrician managing VSD children. Out of 132, 127 patients, 66 males, and 61 females with average age of 7.99 ± 8.52 months, were diagnosed either with p‐VSD or m‐VSD. Five children were excluded from the study during the follow‐up period due to surgical intervention (including 4 p‐VSD and 1 m‐VSD). This study did not include patients with inlet and outlet VSD. Because most patients were recommended for early surgical treatment, rather than follow‐up observation. All participating children underwent echocardiography yearly till the closure of VSD or 6 years old. All parents and guardians were provided written informed consent for the minors to participate in this study. The school ethics committee approved the study.2.2All echocardiographic examinations were performed using Philips iE33 to select S5‐1 (frequency 1.0‐5.0 MHz) or S12‐4 probe (frequency 4.0‐12.0 MHz). Clinical indicators  were collected at the beginning of our study. Since most patients have a first diagnosis age of less than 6 months, they were divided into two groups: less than 6 months and more than 6 months. Then, children patients underwent conventional echocardiography. They were placed in the conventional left lateral position, once asleep or quiet, and parasternal long‐axis, parasternal short‐axis, and apical four‐chamber views were obtained. The main purpose was to scan the ventricular septum completely, and combine with color Doppler echocardiography to determine the type and position of the defects. Defect size measured by 2D echocardiography. If the defect boundary was not clearly displayed, color flow mapping may be used. The diameter of defect was recorded as the maximal VSD diameter in multiple planes. Defect size was divided into three groups2.31), ATVMS (X2), and LVDD(X3) as independent variables.The model was generated with spontaneous closure as a dependent variable , defect size , and 3Evaluation of 127 children with isolated VSD by echocardiography showed that the frequency of p‐VSD was more common  than m‐VSD . These children patients (66 boys and 61girls), aged 17 days to 48 months (mean 7.99 ± 8.52 months), were followed from 4 months to 74 months (mean 35.20 ± 19.92 months). Spontaneous closure occurred in 76 patients (60%).3.1Spontaneous closure rate in patients with p‐VSD was 57% Figure . Males hP = 0.003), ATVMS (P = 0.005), and LVDD (P = 0.010) as statistically significant indicators that could discriminate spontaneous closure from open.Binary Logistic Regression analysis identified defect size (1 + 1.887X2 − 0.222X3. Likelihood ratio test generated χ2 = 70.187 (P < 0.001) indicating that the model is logical. Using this model, we observed that the correct rate of the prediction results of p‐VSD model was as high as 75.7%. The prediction of spontaneous closure of p‐VSD was assessed by area under the curve. Area under the curve was 0.854  which reflected effectiveness of the model in predicting the spontaneous closure of p‐VSD  = 7.143 − 1.438X3.2Spontaneous closure rate of 64% was observed among 53 patients with m‐VSD. As in p‐VSD, males showed a higher rate (66%) compared to females (63%) and 65% of single defects closed spontaneously compared to multiple defects (60%). Spontaneous closure was more frequent in age ≤ 6 months group (83%) vs than in age>6 months (39%) group. The small defects showed the highest frequency (80%) of spontaneous closure. Apical defects closed at a higher rate (67%) than mid‐ventricular (66%) and apical (67%). Similar to p‐VSD, PH was a negative factor for spontaneous closure. Three patients with PH were no closed. LVDD of spontaneous closure was smaller than no closure. Differences of results between patients with and without spontaneous closure of m‐VSD are summarized in Table P = 0.002) and LVDD (P = 0.004) statistically discriminated spontaneous closure from open.Binary Logistic Regression analysis showed that defect size (1 − 0.267X3. Likelihood ratio test generated χ2 = 38.264 (P < 0.001), indicating that the model is logical. Application of the Logistic Regression model revealed the frequency of spontaneous closure of m‐VSD was 83%. Area under the curve value was 0.898 , reflecting sensitivity and specificity of the model in predicting the spontaneous closure of m‐VSD  = 11.216 − 3.150X4With the improvement in sensitivity and clarity of 2D combined with color Doppler echocardiography in recent years,In this study, VSD children were followed up to 6 years old with echocardiography. We thoroughly studied the natural history of p‐VSD and m‐VSD. On the one hand, we found the prevalence of p‐VSD was more common  than m‐VSD . This confirmed the previous observation that the incidence of p‐VSD was higher than m‐VSD.Although high rate of spontaneous closure was satisfying, there was currently no simple and systematic way to predict spontaneous closure of VSD.Whether the position of m‐VSD affects the probability potential for spontaneous closure remains debatable. The results of this study indicated that the spontaneous closure of m‐VSD is independent of the position of the defect, which is in line with published reports.It is well known that spontaneous closure of VSD can occur at any age, but most commonly occurs in childhood.Patients with small VSD: These patients have the high possibility of spontaneous closure. If patients have ATVMS formation and no LVDD increased, the spontaneous closure may be more frequent. In patients with asymptomatic and no complications, surgery may be not recommended. It is reasonable to follow‐up regularly for every 1 to 2 years.Patients with medium VSD: These patients have the intermediate possibility of spontaneous closure. Similarly, ATVMS formation and no LVDD increased are good predictors. Once a year review is reasonable. Data should be obtained include change in defect size, presence or absence of complications, PH, and LV volume overload. Once presence, appropriate treatment strategy may be beneficial. And parents may be mentally as well as economically prepared.Patients with large VSD: These patients have low possibility of spontaneous closure. There is usually LV volume overload, PH, and even congestive heart failure and Eisenmenger syndrome in infancy or childhood. It is probably recommended that patients can be seen at a CHD center of at least yearly. Patients with complications and poor lesion should be considered for surgery.Clinical management of isolated VSD is imperative because long‐term persistence of defects may cause the occurrence of complications. Complications such as infective endocarditis, LV volume overload,5There were many limitations in this study. (a) The number of patients was not big enough, and the follow‐up time was not long enough. The validation of the model requires more cases and longer follow‐up. (b) This study did not combine other clinical examinations and laboratory indicators. Therefore, our information can be used to predict the possibility of spontaneous closure and decide treatment options, but not to provide an accurate diagnosis.6This study described the natural history and spontaneous closure rate of children with p‐VSD and m‐VSD. We inferred that defect size, ATVMS and LVDD were characteristic and representative predictors for spontaneous closure of VSD. A follow‐up criteria was recommended by summarizing this study, which may assist pediatricians in selecting the appropriate treatment strategy to manage VSD children.The authors declare no potential conflict of interests.