TNFRSF9, IDO1 and EBI3. Functional annotation revealed the enriched categories including positive regulation of immune system process and regulation of leukocyte activation. These findings demonstrated DNA methylation changes occurring in immune responses of PBMCs to poly I:C stimulation and a subset of genes potentially regulated by DNA methylation in the immune responses. The PBMC DNA methylome provides an epigenetic overview of this physiological system in response to viral infection, and we expect it to constitute a valuable resource for future epigenetic epidemiology studies in pigs.DNA methylation changes play essential roles in regulating the activities of genes involved in immune responses. Understanding of variable DNA methylation linked to immune responses may contribute to identifying biologically promising epigenetic markers for pathogenesis of diseases. Here, we generated genome-wide DNA methylation and transcriptomic profiles of six pairs of polyinosinic-polycytidylic acid-treated pig peripheral blood mononuclear cell (PBMC) samples and corresponding controls using methylated DNA immunoprecipitation sequencing and RNA sequencing. Comparative methylome analyses identified 5,827 differentially methylated regions and 615 genes showing differential expression between the two groups. Integrative analyses revealed inverse associations between DNA methylation around transcriptional start site and gene expression levels. Furthermore, 70 differentially methylated and expressed genes were identified such as  It has received intense attention due to their functions in regulating chromatin structure and gene expression, and their potential use as a biomarker for disease status2. In particular, DNA methylation is considered as the most stable and experimentally accessible biomarker candidate among different types of epigenetic marks. Characterization of DNA methylome is of importance for understanding the functional mechanisms of DNA methylation in human disease studies3. DNA methylation signatures have been found to be predictive of a series of diseases, including melanoma2, Wilms tumor4 and major depressive disorder5, which will be of great value in both clinical diagnostics and therapeutics. Furthermore, DNA methylation changes can regulate the transcriptional profiles of innate and memory lymphocytes and thus shape their function. It has been reported that DNA methylation alterations in activating molecules such as KSR1 and NCF4 are associated with human natural killer cell activation6. DNA methylation can direct transcription of the genes encoding cytokines, effector molecules and their receptors such as IFNγ, IL2 and CCR6 in memory T cells9. The survival of CD4+ memory T cells has been found to be affected by DNA methylation status of the cell death-related gene NOXA10. Dynamic changes of demethylation and remethylation occur at the programmed cell death-related gene PDCD1 which encodes a key regulator of cell proliferation and exhaustion during naive to effector to functional memory CD8+ T cell differentiation post viral infection11.Epigenetic alterations are a regular and natural occurrence that can result in heritable changes in gene expression without changing the underlying DNA sequence while being influenced by genetic changes and environmental cues12. Recently it has also been shown that global methylation of PBMCs has the potential to provide disease-specific epigenetic signatures14. Moreover, DNA methylation was found to be strongly associated with PBMC responsitivity to Toll-like receptor (TLR) ligands15. The TLR3 agonist polyinosinic-polycytidylic acid (poly I:C), an artificial surrogate for double-stranded RNA (dsRNA) virus, has been extensively used to simulate viral infection16. As such kind of external pathogenic stimuli can conduce to epigenetic changes, understanding of the variable DNA methylation linked to immune and inflammatory responses of PBMCs may contribute to identifying biologically promising epigenetic markers for pathogenesis of viral diseases.Peripheral blood mononuclear cells (PBMCs) are clinically relevant cells that display a variety of both innate and adaptive immune functions and have well-established roles in surveillance for infectious threats. PBMCs methylome at single base-pair resolution demonstrated it to be rich in biological information in humans18. Epigenetic analysis of porcine dendritic cells treated by lipopolysaccharide highlighted the involvement of epigenetic mechanisms in innate immune response19. In addition, pig PBMCs has been frequently used as an experimental model to investigate immune responses to vaccination, bacterial and viral infection22. As an appealing biomedical model with high similarities to humans in anatomy, genetics and pathophysiology, the immune system of pigs not only has a full set of innate and adaptive immune effectors, but also closely resembles humans for >80% of analyzed immune parameters, which enables the effective uses of pigs in modeling human immune responses and microbial infectious diseases24. Epigenetic regulatory mechanisms are of particular importance to establish a proper pattern of gene expression in immune cells in response to internal or external environmental variations25. However, changes in epigenetic profile of pig PBMCs induced by pathogenic stimuli remain largely unknown so far. Studies of epigenetic responses involved in immune reactions of pig PBMCs upon pathogens may shed light on the epigenetic dynamics during both animal and human immunological responses.As one of the important biomedical models for human diseases, methylation patterns of diverse pig tissues have been reportedin vitro model system to analyze changes in methylome profiling of PBMCs in response to poly I:C stimulation and explore potential roles of DNA methylation in this process. The methylated DNA immunoprecipitation sequencing (MeDIP-seq) was utilized to provide broad coverage of methylomic landscape of poly I:C-treated samples and controls. Integrative analysis of genome-wide DNA methylation and gene expression data was further performed to identify associations between epigenomic and transcriptomic responses of PBMCs to viral infection. The comprehensive analysis of PBMC methylome herein will advance our understanding of epigenetic contributions to immune responses to viral infection, and aid in the identification of potential epigenetic biomarkers associated with responsiveness to viruses.In this study, using the pig as a model we established an 9 paired-end reads with the length of 100 bp from 12 samples, of which 1.11 × 109 high quality reads were mapped to the pig genome (Sscrofa10.2), resulting in approximately 9.25 × 107 mapped reads per sample  of the approximately 28.0 million CpG sites in the pig genome were covered by at least one read  than in the control group , gene body, and 2.2 kb region downstream of the transcription termination site (TTS). We observed a clear trend for DNA methylation levels to decrease sharply at TSS and to increase dramatically towards the gene bodies Fig. . This isoup Fig. , which m32. We therefore analyzed the methylation status of CGIs and CGI shores, and observed that the DNA methylation level increased sharply towards the CGIs , which suggests CGIs may contain regulatory sites of gene expression in responses of PBMCs to poly I:C stimulation. The DNA methylation changes in these genomic features presumably reflected the ability of pathogens to trigger epigenetic responses of cells involved in the immune system33.In methylome studies, CpG islands (CGIs) are a notable genomic element of interest because of the role in controlling gene expressionGIs Fig. . Of note34, DMRs were defined as 1 kb windows with significant difference in normalized read counts between the two groups. We identified 5,827 DMRs, of which 2,322 (39.8%) were hypermethylated and 3,505 (60.2%) hypomethylated in the poly I:C-treated group. A circle diagram of DMRs along chromosomes revealed that distributions of DMRs tend to be enriched in genomic regions with higher CpGo/e ratio (~0.32) than the genomic mean value  that attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB35, forkhead box P1 (FOXP1) that participates in the transcriptional regulatory network of B lymphopoiesis36.We subsequently mapped all DMRs to their nearest genomic features and found that methylation alterations were unevenly distributed across the genome. The majority of the 5,827 DMRs were identified in intergenic and intronic regions Fig. , which cAs the differences in the gender and breed of individuals used in this study, we also revealed the potential DNA methylation differences derived from gender and pig breed differences. A total of 6,152 DMRs  were detected between the different pig breeds, and 2,208 DMRs  were identified between the individuals with different gender Tables  and S7.37. To investigate the biological significance of the differentially expressed genes, we undertook functional annotation analysis using DAVID bioinformatics resources. A subset of immune/defense response-related categories were significantly clustered, such as ‘response to wounding’ , ‘inflammatory response’ , and ‘defense response’ . Further pathway analysis revealed that the genes were significantly enriched in six disease resistance-related pathways. The cytokine-cytokine receptor interaction pathway  was the most representative pathway, followed by graft-versus-host disease , type I diabetes mellitus , allograft rejection , asthma , and chemokine signaling pathway   Table .Figure 5CCL2, IL-6, IL-10, PPARG, TNFRSF9, TNF-α, AIG1, ADAM28, FCRL3, NCF4, and PACSIN1 to detect their expression patterns using qRT-PCR. The expression pattern of all the genes tested matched that was observed in the RNA-seq analysis results, with a correlation coefficient of 0.94 (P = 1.72E-05), confirming the high reliability and accuracy of RNA-seq data in this study . To test the relationship between promoter methylation and CpG frequency, we classified all promoters into three types based on the CpG representation, i.e., low CpG , intermediate CpG  and high CpG content promoters   Figure . We obse) Figure , which cin vitro highlighted the roles of differentially expressed genes in response to pathogenic stimuli40. To detect whether the differentially expressed genes identified in this study were associated with differential DNA methylation, we performed integrative analyses of genes identified in the DNA methylation and gene expression analyses. The results showed that among the list of differentially expressed genes, 70 genes were discovered to be differentially methylated between the two groups (Table P = 1.89E-03). The analysis allowed the identification of a subset of antiviral response-related genes including TNFRSF9 that is expressed on activated T and natural killer cells41, IDO1 that can suppress viral replication42 and EBI3 that induces generation of regulatory T cells43, of which the genomic locations and degrees of the methylation and expression differences are shown in Figure P = 2.75E-03) and ‘pattern binding’ , and ‘lysosome’   Table .ex vivo stimulation, and we expected it to constitute a subset of resources for future epigenomic studies.Through a genome-wide comparative methylome analysis of pig PBMCs, we revealed the associations of epigenetic alterations with the responses of PBMCs to immune stimulation by the viral pathogen. We characterized differentially methylated regions that reflect the epigenetic changes potentially induced by poly I:C stimulation. Further integrated analysis of MeDIP-seq and RNA-seq data allowed the identification of immune response-related genes that may be driven by DNA methylation. The PBMC DNA methylome profiling provides an epigenetic overview of this physiological system in response to 44. Our methylome analysis of poly I:C-treated PBMCs demonstrated distinct distribution patterns of differential DNA methylation among genomic features. In agreement with previous studies showing different distributions of methylation changes in genomic features46, these suggest the potential of DNA methylation profile in reflecting the unique epigenetic information of immune responses and diseases. Consistent with the clear stratification of immune disease by DNA methylation signature37, our work further substantiated the general validity of the associations between DNA methylation and immune responses by unsupervised clustering analysis using DMRs. Some factors such as differences associated with sex and breed could impact on the DNA methylation17, and our findings also displayed sex and breed related DNA methylation differences which may result in that the individuals of Landrace and Dapulian could not be distinguished in the untreated group. In addition, individuals from the two breeds were separated in the poly I:C-treated group. Previous studies have shown the discrepancies in the ability of the two pig breeds in resistance to pathogenic stimulation47 and the close link between DNA methylation status and functions of immune cells44, which may account for the separation of pig breed in the poly I:C-treated group.It is known that pathogens can affect diverse epigenetic factors such as DNA methylation, histone modifications, noncoding RNAs40. The epigenetic modulations such as histone modification and DNA methylation can be manipulated by pathogens to influence the host’s gene expression programs associated with defense genes48. Our results provided evidence that poly I:C stimulation can trigger changes in DNA methylation to alter the expression of genes related to immune responses. Previous reports have elucidated the potential underlying biology for preferentially inducing epigenetic changes in immune-related genes during pathogenic infections such as modulation of histone acetylation, accession to histone-interacting transcriptional regulators by mimicking the histone H3K4 sequence and encoding proteins that can interact with cellular DNA methyltransferases51. In addition, DNA methylation is considered as only one of the mechanisms for governing gene expression, and recent studies have documented a not necessary correlation between DNA methylation changes and gene expression variations46. The other regulators such as transcription factors are also involved in the regulation of gene expression, which may in part account for the small number of differentially methylated and expressed genes identified in our study.The host’s gene expression programs especially those linked to host defense genes undergo massive changes during pathogenic infectionsTNFRSF9 displayed negative relationships between promoter methylation and gene expression, which may be associated with the transcriptional repression of DNA methylation by blocking the binding of transcriptional activators or recruiting co-repressors52. However, a subset of genes exhibited differently from this pattern, which indicated that the changes in promoter methylation and gene expression do not seem to follow a simple logic in PBMCs in response to poly I:C stimulation. Emerging evidence has revealed that the classical epigenomic models that DNA methylation negatively regulates gene expression apply only to a portion of genes. No correlation and positive correlation between promoter methylation and gene expression have been identified, such as in mouse intestinal adenoma and human PBMCs45. In addition, DNA methylation is prevalent within gene bodies. It has been shown that gene body methylation is involved in alternative splicing53, phenotypic plasticity54, and repression of spurious initiation of transcription55. Differential DNA methylation in gene bodies was found to be positively correlated with the expression of some genes including the EBI3 gene, which is concordant with previous reports57. The molecular mechanisms may be that the methylation in gene body may promote transcriptional activity through hindering the initiation of intragenic promoters or by influencing repetitive DNAs activities located in the transcriptional units58. However, recent studies revealed that not all of the gene body methylation was positively correlated with expression59. In this study, we also observed a subset of genes showing positive or negative associations between gene body methylation and expression. A possible rationale is that the function of gene body DNA methylation in gene expression may vary with the targeted genomic elements in gene bodies, such as repetitive elements, enhancer, and alternative promoters. Therefore, more research is required to definitively characterize the function of DNA methylation in genomic features in regulating gene expression.Functions of DNA methylation vary with the distribution of methylation across the genomic elements. Here we found an overall inverse relationship between promoter methylation and gene expression levels. Among the genes showing differential promoter methylation, several genes such as In summary, we pioneered a comprehensive methylome analysis of pig PBMCs in response to poly I:C stimulation using MeDIP-seq. This study demonstrated clearly methylomic differences after stimulation with poly I:C, and provided an epigenetic overview of this physiological system in response to viral infection. Our findings highlighted the significance of epigenetic modifications that may trigger transcriptional changes in immune-related genes involved in responses of PBMCs to viral infection. Given the variable and complex processes of PBMCs responses to pathogenic infections, further endeavors will be pursued to analyze the genome-wide methylation kinetics of PBMCs at a variety of time points after infections in larger independent cohorts in future studies.To increase the biological diversity of the samples and identify the strongest and common signatures of methylation and gene expression, six clinically healthy pigs at 35 days old from two different breeds (Dapulian and Landrace) were used in this study Table . All ani61 at 37 °C and 5% CO2, and then were collected for nucleic acid extraction. Genomic DNA and total RNA were extracted using Qiagen DNeasy Tissue kit  and RNAiso Plus , respectively, and frozen at −80 °C until DNA methylation and RNA-seq analysis.PBMCs were separated from whole peripheral blood by a density gradient centrifugation method using Ficoll-Paque PLUS . Freshly isolated PBMCs were maintained in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal calf serum, and the cell viability was confirmed by trypan blue exclusion assays. The PBMC samples obtained from each piglet were classified into two groups that treated with poly I:C at a final concentration of 20 μg/mL or with vehicle . PBMC cells were maintained in culture for 24 hoursi) end repaire, (ii) phosphorylation of the 5′ prime ends, (iii) A-tailing of the 3′ ends, (iv) ligation of adapters, and were performed with the Paired-End DNA Sample Kit  following the manufacturer’s guidelines. Adaptor-ligated DNA was immunoprecipitated by highly specific anti-5-methylcytosine monoclonal antibody supplied in the Methylated DNA IP Kit . MeDIP efficiency was confirmed by quantitative PCR using SYBR green mastermix , using primers for positive and negative internal control DNA of non-human samples contained in the Magnetic Methylated DNA Immunoprecipitation Kit . The methylated fraction and 10% input DNA were purified with ZYMO DNA Clean & Concentrator-5 columns according to the manufacturer’s instructions and were amplified by adaptor-mediated PCR. Amplification quality and quantity were analyzed by Agilent 2100 Analyzer. Ultra-high-throughput 100 bp paired-end sequencing was performed using the Illumina HiSeq. 2000 following manufacturer’s protocols.We herein used MeDIP-seq to profile the methylomic landscape of PBMC samples derived from poly I:C-treated and control groups. To prepare for MeDIP-seq, 6–10 ug genomic DNA was sheared into random fragments ranging in size from 150 to 800 bp with the AIR™ DNA Fragmentation Kit (Bioo Scientific Corporation). Libraries were prepared according to the Illumina Paired-End protocol,  and having at least 70% of bases with PHRED quality score > = 10 were retained. Then we aligned the remaining reads to the pig reference genome (Sscrofa10.2) with Bowtie 263. The pig reference genome was downloaded from the University of California Santa Cruz Genome Bioinformatics Site . Finally, we utilized the Samtools64 to convert the SAM format file to BAM format and discarded the reads with MAPQ score < 10. To avoid sex-based methylation differences, we removed methylation data for the X and Y chromosomes. The final BAM format files were prepared for subsequent downstream analysis.Raw sequencing reads were firstly filtered using IlluQC.pl provided in the NGS QC Tookit63. Read counts for non-overlapping genomic windows of 1 kb were calculated using the MEDIPS package26. For each window, the unique read pairs  that overlap with the window of each sample were counted. The MEDIPS.couplingVector function was used to control for local CpG density for an estimate of actual DNA methylation level. Raw read counts were normalized using the estimateSizeFactors function in the DESeq package in R statistical program65. For each window, we carried out paired t test on the differences between poly I:C-treated and control groups using the normalized read counts. The resulting t statistic can be used to determine the significance66. Using the distribution of t statistic obtained from each window, we defined a threshold (|t| = 7) for determining DMRs. The windows with the absolute value of t statistic that was larger than 7 were taken as DMRs. The improved permutation method was utilized to evaluate the FDR in DMR calling67. We randomly shuffled the normalized read counts for each non-DMR window and repeated DMR analysis . This analysis indicated that the DMRs were identified with an FDR < 0.1 across the genome.For differential methylation analyses, after filtering the low quality reads, the MeDIP-seq data were aligned to the pig reference genome (Sscrofa10.2) using Bowtie 268, which are listed in Table ™ Kit . PCR reactions were then undertaken using the ZymoTaq™ PreMix  following the manufacturer’s protocols. The PCR products were purified and cloned into the pMD18-T vector . More than ten positive clones for each subject were randomly selected for sequencing. The final sequences were analyzed by the online software QUMA69.Bisulfite sequencing PCR primers were designed by the online MethPrimer programA total of 10 ug total RNA was treated with poly-T oligo attached magnetic beads to isolate Poly (A) mRNA. The captured mRNA was fragmented randomly into smaller pieces using RNA fragmentation buffer  following the manufacturer’s procedure. Then the cleaved RNA fragments were reverse-transcribed to construct the final cDNA library according to the protocols for the mRNA-Seq sample preparation kit , and the average insert size for the paired-end libraries was 300 bp ( ± 50 bp). After that, 100 bp paired-end sequencing was performed on the Illumina Hiseq. 2000 following the vendor’s recommended protocols.70. The script htseq-count included in HTSeq was used to count for each gene how many aligned reads overlapped its exons. The edgeR71 software was used for read counts normalization and differentially expressed gene determination using the procedure of a generalization of paired t-test for paired samples. We defined a threshold of FDR < 0.05 for determining the significance of expressed genes.We filtered out low quality RNA-seq reads that contain sequencing adaptor or sequencing primer or with base quality score lower than 20. The clean reads were then aligned to the pig reference genome using TopHat2® RT reagent Kit with gDNA Eraser  Co., Ltd) following the manufacturer’s instructions. Quantities of mRNA were then measured with qRT-PCR using a LightCycler® 480 Real-Time PCR System . The qRT-PCR assays were performed with a volume of 20 μL containing 10 μL SYBR Green Mixture, 7 μL deionized water, 1 μL template of cDNA, 1 μL of each primer. The thermal conditions were as follows: 95 °C for 5 min, 45 cycles of 95 °C for 10 sec, 60 °C for 10 sec, 72 °C for 10 sec. Primer sequences used for qRT-PCR assays are presented in Table ΔΔCt− method72.Total RNA was purified and reversely transcribed into cDNA using PrimerScriptP value less than 0.05 were considered to be statistically significant.The DAVID  web server (http://david.abcc.ncifcrf.gov/) was used to investigate the biological relevance of the differentially methylated genes. Since only a limited number of genes in the pig genome have been annotated, the identified genes were firstly mapped to their corresponding human orthologs, and then the gene lists were submitted into the databases for functional enrichment analysis. Annotation terms with a 2, promoters were defined as −2,200 bp to +500 bp of the transcription start site. The promoters were further classified into three groups based on the CpG representation as previously described37, i.e., low CpG (LCP), intermediate CpG (ICP) and high CpG content promoters (HCP). DNA regions greater than 200 bp with a C + G content equal to or greater than 50% and an observed CpG/expected CpG in excess of 0.6 were defined as CGIs73. The sequences up to 2 kb distant from CpG islands were termed as CpG island shores74.DMRs were annotated with Ensembl genes, transcripts, promoters, CpG islands (CGIs) and repeatmask. We obtained the genomic locations of these genomic features from the UCSC pig reference genome (Sscrofa10.2). As described in previous studyAdditional file 1Additional file 2


Positive end-expiratory pressure (PEEP) can increase intracranial pressure. Pneumoperitoneum and the Trendelenburg position are associated with an increased intracranial pressure. We investigated whether PEEP ventilation could additionally influence the sonographic optic nerve sheath diameter as a surrogate for intracranial pressure during pneumoperitoneum combined with the Trendelenburg position in patients undergoing robot-assisted laparoscopic prostatectomy.2O PEEP group . The sonographic optic nerve sheath diameter was measured prior to skin incision, 5 min and 30 min after pneumoperitoneum and the Trendelenburg position, and at the end of surgery. The study endpoint was the difference in the sonographic optic nerve sheath diameter 5 min after pneumoperitoneum and the Trendelenburg position between the ZEEP and PEEP groups.After anesthetic induction, 38 patients were randomly allocated to a low tidal volume ventilation (8 ml/kg) without PEEP group  or low tidal volume ventilation with 8 cmHOptic nerve sheath diameters 5 min after pneumoperitoneum and the Trendelenburg position did not significantly differ between the groups . Optic nerve sheath diameters 30 min after pneumoperitoneum and the Trendelenburg position also did not differ between the groups .2O PEEP application under low tidal volume ventilation does not induce an increase in the optic nerve sheath diameter during pneumoperitoneum combined with the steep Trendelenburg position, suggesting that there might be no detrimental effects of PEEP on the intracranial pressure during robot-assisted laparoscopic prostatectomy.An 8 cmHNCT02516566ClinicalTrial.gov  Robot-assisted laparoscopic prostatectomy requires a pneumoperitoneum and the steep Trendelenburg position to facilitate a surgical field. These specific conditions induce decreased pulmonary functional residual capacity and pulmonary compliance , which aAlthough the lung protective ventilation strategy during laparoscopic surgery is promising in its potential to improve outcomes in regards to pulmonary function, the application of PEEP as an important component of this strategy does not necessarily favor the function of other major organs. In particular, it has been proposed that PEEP results in increased intracranial pressure (ICP) by impeding cerebrospinal flow outflow and cerebral venous drainage . FurtherIn the present study, we sought to evaluate the effect of PEEP on the ONSD as a surrogate for ICP during pneumoperitoneum and the steep Trendelenburg position in patients who have undergone a robot-assisted laparoscopic prostatectomy.This monocentric, parallel-group, randomized controlled trial was conducted between September and October 2015. The study protocol was approved by the Institutional Review Board at Asan Medical Center (2015–0741) and written informed consent was obtained from each patient. This study was also registered with ClinicalTrial.gov (NCT02516566). Patients scheduled for robot-assisted laparoscopic prostatectomy were enrolled and evaluated. Patients with cerebrovascular disease, glaucoma, or who refused to participate were excluded. Patients who were younger than 20 years were also excluded.2 partial pressure (ETCO2) between 30 and 40 mmHg. The inspiratory-to-expiratory time ratio was set at 1:2. Anesthesia was maintained with end-tidal sevoflurane concentration of 2 vol% plus the continuous infusion of remifentanil. Remifentanil was infused in the range of 0.07–0.16 μg/kg/min which was titrated to maintain hemodynamic stability. A 50% oxygen was supplied using medical air.After applying routine hemodynamic monitoring  and attaching the cerebral oximeter sensors (INVOS 5100) to the right and left frontal areas, anesthesia was induced using a bolus intravenous injection of 5 mg/kg thiopental sodium followed by 0.6 mg/kg rocuronium. After tracheal intubation, patients were mechanically ventilated with a tidal volume of 8 ml/kg ideal body weight and a respiratory rate to maintain end-tidal CO2O PEEP (One investigator (W-JK) generated a randomization code using block randomization procedure with 1:1 allocation ratio. Random allocation was carried out with the user-developed Stata module . The participants were assigned to randomization codes kept in sequentially numbered opaque envelopes. After anesthetic induction, these envelops were opened by an investigator who controlled ventilator setting, and 38 patients were randomly allocated to one of two groups: the zero end-expiratory pressure (ZEEP) group (n = 19) which received mechanical ventilation with a tidal volume of 8 ml/kg of ideal body weight without PEEP, and the PEEP group (n = 19) which received mechanical ventilation with a tidal volume of 8 ml/kg of ideal body weight with 8 cmH2O PEEP . One invThe ONSD was measured by investigators trained in ocular sonography. Briefly, patients were placed in the supine position with their eyes closed, and a thick gel layer was applied to the closed upper eyelid. A 7.5 MHz linear probe was placed on the gel without excessive pressure and adjusted to the proper angle to display the optimal contrast between the retrobulbar echogenic fat tissue and the vertical hypoechoic band. An ultrasound beam was focused onto the retrobulbar area using the lowest possible acoustic power that could measure the ONSD. The ONSD was measured 3 mm behind the optic disc. Measurements were performed in the transverse and sagittal planes of both eyes, and the mean values of four measurements at each time point were used in the analysis. To determine intra-observer and inter-observer variability, a random sample of about 25% of the ONSD was submitted twice to the first investigator and once to a second investigator. The inter-observer variability was then calculated as the mean absolute difference between the two readings from the first and second investigator divided by their mean and expressed as a percentage. Similarly, the intra-observer variability was calculated as the mean absolute difference between the two readings from the first investigator divided by their mean and expressed as a percentage.2, arterial CO2 partial pressure, arterial O2 partial pressure (PaO2), hemoglobin concentration, and regional cerebral oxygen saturation (rSO2) using near infrared spectroscopy.When hemodynamically stable conditions were reached, measurements were taken as follows: 10 min after anesthetic induction in the supine position before random allocation (T0), 5 min after applying ventilation strategies according to a random allocation (T1), 5 min after establishing pneumoperitoneum (15 mmHg of insufflation pressure) and the steep Trendelenburg position (35° incline) (T2), 30 min after establishing pneumoperitoneum and the steep Trendelenburg position (T3), and at the end of surgery after the desufflation of pneumoperitoneum in the supine position (T4). At each predetermined time point, we measured the following variables; ONSD, mean arterial blood pressure, heart rate, tympanic body temperature, static pulmonary compliance , dynamic pulmonary compliance , ETCOThe study endpoint was the difference in ONSD measurements at T2 (5 min after establishing pneumoperitoneum and the Trendelenburg position) between the ZEEP and PEEP groups.2O PEEP  . Similar2 level in the PEEP group tended to increase compared with that in ZEEP group (P = 0.104) (2 level and static and dynamic pulmonary compliance in the PEEP group tended to increase compared with these measurements in the ZEEP group (At 5 min after applying ventilation strategies during the supine position without pneumoperitoneum (T1), the PaO= 0.104) . At T1, EP group .2O PEEP application under low tidal volume ventilation did not induce an increase in the ONSD during the specific conditions of pneumoperitoneum and the steep Trendelenburg position. In addition, the 8 cmH2O PEEP application resulted in an increase in the ONSD in the supine position without pneumoperitoneum.We found that an 8 cmH2O did not significantly influence ICP in patients with acute stroke [2O had no significant impact on ICP [2O did not increase the ICP in patients with a high ICP, whereas the same levels of PEEP increased the ICP in patients with a normal ICP [PEEP is a lung protective ventilation strategy component that has been known to reduce postoperative respiratory complications in patients who are at high risk for adverse events during abdominal surgeries including laparoscopic surgery ,3. PEEP e stroke . In anott on ICP . Interesrmal ICP .Robot-assisted laparoscopic prostatectomy requires a pneumoperitoneum and the steep Trendelenburg position. Although these specific conditions are necessary for a good surgical field, they can induce pathophysiologic changes in major organs including the central nervous system . However2O PEEP under low tidal volume ventilation did not induce an additional increase in ONSD which already increased during pneumoperitoneum and the Trendelenburg position, implying no further detrimental effects of PEEP on an increased ICP during robot-assisted laparoscopic prostatectomy. A PEEP application might not increase the perioperative risks for cerebral complications in patients without cerebral pathology undergoing laparoscopic surgery. Additionally, we found that an 8 cmH2O PEEP resulted in an increase in ONSD in the supine position without pneumoperitoneum. Our present results are in line with those of a previous study that reported an increased ICP level in patients with a normal ICP following a PEEP application, whereas the same level of PEEP did not alter the ICP in patients with a high ICP [Our current study was initiated from a concern about additional increases in the ICP following a PEEP application during pneumoperitoneum combined with the steep Trendelenburg position. Our findings have indicated however that an 8 cmHhigh ICP . Our finhigh ICP . In the high ICP . Our cur2 at 30–40 mmHg by controlling the respiratory rate may have also contributed to the result observed 30 minutes after the patients were placed in this position. Since leaving ETCO2 as it had been during laparoscopic surgery without any deliberate manipulation could raise ethical issues, it was adjusted during surgery.We further found that ONSD at 30 minutes after initiating pneumoperitoneum combined with the steep Trendelenburg position had not increase further compared with that at 5 minutes after this position change; rather it was less than ONSD at 5 minutes after this position change. This observation might suggest that cerebral blood flow was probably adjusted over time during surgery, and partially compensated by cerebrospinal fluid translocation . Mainten2O), which has been suggested previously by Futier et al. as part of a lung protective ventilation strategy [Our study had the following limitations. First, we did not measure the ICP directly, as this is not possible in non-neurosurgical patients due to ethical issues. Instead, we measured the sonographic ONSD that has been known to correlate with the directly measured ICP using ventriculostomy . Second,2O PEEP application under low tidal volume ventilation does not induce an ONSD increase during pneumoperitoneum combined with the steep Trendelenburg position. Our results suggest that PEEP under low tidal volume ventilation might be used with no additional risk of an unwanted increase in ICP in patients without cerebral pathology who are undergoing robot-assisted laparoscopic prostatectomy.In conclusion, an 8 cmHS1 CONSORT Checklist(DOC)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


A contributing factor to poor placental perfusion, leading to intrauterine growth restriction and preeclampsia, is the failure of invading extravillous trophoblast (EVT) cells to remodel the maternal uterine arteries during the first and second trimesters of pregnancy. Noninvasive assessment of EVT cells in ongoing pregnancies is possible beginning three weeks after conception, using trophoblast retrieval and isolation from the cervix (TRIC). Seven proteins were semi-quantified by immunofluorescence microscopy in EVT cells obtained between gestational weeks 6 and 20 from pregnancies with normal outcomes (N = 29) and those with intrauterine growth restriction or preeclampsia (N = 12). Significant differences were measured in expression of PAPPA, FLT1, ENG, AFP, PGF, and LGALS14, but not LGALS13 or the lineage marker KRT7. These findings provide for the first time direct evidence of pathology-associated protein dysregulation in EVT cells during early placentation. The TRIC platform provides a novel approach to acquire molecular signatures of EVT cells that can be correlated with pregnancy outcome. Serum biomarker screening and uterine artery Doppler ultrasound are currently the standard of care for predicting intrauterine growth restriction (IUGR) and preeclampsia (PE) before the onset of maternal diseaseHistological examination of placentas delivered by women with severe PE and IUGR suggests a prior disruption of EVT function in the first trimester that predisposes to uteroplacental insufficiency, characterized by reduced EVT invasion, inadequate remodeling of the spiral arteries, deferred elimination of endovascular trophoblastic plugs, and elevated EVT cell death4567810Although access to fetal cells for prenatal diagnosis is technically complex, and in some cases, such as amniocentesis or chorionic villous sampling, poses high risk16181920trophoblast retrieval and isolation from the cervix (TRIC) to efficiently separate EVT cells in endocervical specimens from the resident maternal cells, using a monoclonal antibody to HLA-GThe unique expression of human leukocyte antigen (HLA)-G on the surface of EVT cells, exclusive of adult tissuesWe hypothesize that the molecular signatures of EVT cells early in gestation reflect their disrupted function in at least a portion of pregnancies that later develop IUGR or PE. Galectin 13 (LGALS13), galectin 14 (LGALS14), pregnancy-associated plasma protein-A (PAPPA), and placental growth factor (PGF) are proteins secreted by the placenta into the maternal circulation that decrease with abnormal placentation, prior to the onset of IURG or PE262728293132333435Endocervical specimens (N = 200) were collected with informed consent from women receiving initial prenatal care after spontaneous conception or infertility treatment at Wayne State University and Detroit Medical Center facilities. This study was carried out in accordance with the Declaration of Helsinki and all consent forms and protocols were approved by the Institutional Review Board of Wayne State University. Inclusion criteria specified women between 18 and 45 years of age, 5 to 20 weeks of gestation, both primagravida and multiparous females. Patients with multiple gestations or experiencing active vaginal bleeding were excluded from the study. Medical records were reviewed and women diagnosed with PE and/or IUGR were identified and selected (N = 12) for further study. IUGR was defined as estimated fetal weight less than the 10th percentile for gestational ageEndocervical specimens were obtained, as previously describedThe TRIC procedure was completed by combining cervical cells with mouse anti-HLA-G antibody bound to goat anti-mouse IgG conjugated to 250 nm magnetic nanoparticles , and incubating overnight at 4 °C in 100 μl sterile PBS with mixing, as previously describedin situ hybridization with probes for the X and Y chromosomes reveals consistent XY or XX labeling of nuclei in agreement with the sex of babies delivered at the conclusion of pregnancy, verifying the fetal origin of cells isolated by TRICTRIC-isolated EVT cells were collected over a 12-month period and archived on slides. For ICC, small groups of approximately 50 cells were mounted on microscope slides by cytocentrifugation, inserting the end of a 100 μl (yellow) pipet tip into a Shandon Cytospin funnel to focus the cells within a 50-μm diameter area to facilitate imaging. The isolated cells were 97% positive for hCGB , whereasMicroscope slides containing either isolated HLA-G-positive cells or recovered HLA-G-depleted cells were examined, using a standardized and quality controlled immunofluorescence protocol, with antibodies raised against hCGB, KRT7 , LGALS13, LGALS14, PGF, PAPPA, AFP, ENG, or FLT-1 (R&D Systems). Antibodies were each titered to ensure a linear fluorescence signal with labeling, as previously reportedEach protein was quantified in individual EVT cells by immunofluorescence microscopy, using our published methodThe background values were averaged and subtracted from each fluorescent value. The background-subtracted values for each specimen were averaged to calculate the average fluorescent units (AFU). Each AFU value was divided by the average of the AFUs for the control cohort to generate the relative fluorescent unit (RFU) values. Slides were assessed together in a random fashion. KRT7, a trophoblast marker, was assessed in 17 of the specimens (9 controls and 8 adverse cases) to provide a technical and biological control for non-specific variances within the sample populations, assuming it was uniformly expressed in all EVT cellsU-test to evaluate statistical differences between groups, where p < 0.05 was considered significant.Statistical analyses were performed using JMP . Data were compared using the nonparametric Mann–Whitney Cluster analysis was performed and analyzed by BioNumerics , using a hierarchical clustering algorithm with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Recently, this technique has been used to produce clinically meaningful compact clustersTo calculate the accuracy of candidate protein markers in predicting adverse pregnancy outcomes, receiver operating characteristic (ROC) curves were employed and the areas under curves (AUCs) with 95% confidence intervals were calculated for each protein or combination of proteins.th percentile). Specimens were obtained between 6 and 20 weeks of gestation at similar gestational ages . The purity of isolated cells (hCGB-positive) was similar in both groups (medians = 97%). The ratio of recovered fetal cells to maternal cells was similar in adverse gestations (median = 65 × 10−5) compared to controls (median = 54 × 10−5).Seven candidate biomarkers for adverse pregnancy outcome and the trophoblast-specific intermediate filament protein KRT7 were analyzed by standardized immunofluorescence microscopy to derive RFU values, as detailed in the Materials and Methods section  and 2. LWe used a hierarchical clustering algorithm to determine whether the two pregnancy outcome groups  were distinguishable. In this model , ClusterReal-time, noninvasive assessment of EVT cells in ongoing pregnancies is now possible beginning at five weeks of gestation (three weeks after conception), using TRICThe assessment of EVT cells obtained by TRIC for known circulating biomarkers with potential correlation to pregnancy complications could fill the existing gap in knowledge about the contribution of dysregulated early EVT development to the underlying etiologic cellular and molecular mechanisms of IUGR and PE. Galectin-13 (LGALS13/PP13) and LGALS14 are among the placenta-specific galectins of mammals274142There is encouraging evidence for the utility of circulating sFLT1, sENG and PGF as biomarkers several weeks before clinical signs of PE appear44454648Clustering analysis demonstrated that, within the population of patients studied, 79% of the healthy pregnancies (N = 23) partitioned into a distinct group composed of 92% with uncomplicated outcomes . These f9First trimester screening of multiple predictors is currently uninformative even when combined in a multivariable modelSafe access to intact fetal cells throughout pregnancy is considered the “Holy Grail” of perinatal diagnosis. The molecular signature of EVT cells from the placenta can now be monitored noninvasively in real-time, using TRIC and downstream analyses as demonstrated here with semi-quantitative immunohistochemistry. Candidate biomarkers quantified in EVT cells captured through TRIC have successfully distinguished healthy from adverse pregnancies, introducing a new era of perinatal screening to manage fetal and maternal health.How to cite this article: Bolnick, J. M. et al. Altered Biomarkers in Trophoblast Cells Obtained Noninvasively Prior to Clinical Manifestation of Perinatal Disease. Sci. Rep.6, 32382; doi: 10.1038/srep32382 (2016).


In the present research, we investigate the communicative strategies of 20 month old human infants and great apes when requesting rewards from a human experimenter. Infants and apes both adapted their signals to the attentional state of the experimenter as well as to the location of the reward. Yet, while infants frequently positioned themselves in front of the experimenter and pointed towards a distant reward, apes either remained in the experimenter’s line of sight and pointed towards him or moved out of sight and pointed towards the reward. Further, when pointing towards a reward that was placed at a distance from the experimenter, only the infants, and not the apes, took the experimenter’s attentional state into account. These results demonstrate that prelinguistic human infants and nonhuman apes use different means when guiding others’ attention to a location; indicating that differing cognitive mechanisms may underlie their pointing gestures. While two month old human infants readily engage in dyadic face-to-face interactions, it is not until the age of 9–12 months before they start to communicate about objects and events. This inclusion of external entities into their interactions with others is first achieved through triadic gestures. Triadic gestures are gestures that reference external entities such as objects or events, for instance by showing, offering or pointing )As several studies have demonstrated, apes understand the actions of others in terms of intentional motives  and diffHowever, assigning mental states is not necessarily an all-or-nothing affair. Conceptualizing attention as a dynamic mental state that is modifiable and directable might be more challenging than understanding attention as something that can just be either present or absent . While S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 VideoTowards-same condition; experimenter on the right side; reward on the right side.(MP4)Click here for additional data file.S2 VideoTowards-different condition; experimenter on the left side; reward on the right side.(MP4)Click here for additional data file.S3 VideoAway-same condition; experimenter on the left side; reward on the left side.(MP4)Click here for additional data file.S4 VideoAway-different condition; experimenter on the right side; reward on the left side.(MP4)Click here for additional data file.


Bronchiolitis, often caused by respiratory syncytial virus (RSV), is the commonest cause of hospitalisation in infancy. Serum transaminases are sometimes raised in children with bronchiolitis. We tested the hypothesis that raised transaminases are associated with increased disease severity in children ventilated for bronchiolitis. Prospective observational cohort study of mechanically ventilated children with community-acquired RSV bronchiolitis. Alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured daily. Children with normal transaminases were compared with those with elevated levels. Over 11 consecutive winters, 556 children with RSV bronchiolitis were mechanically ventilated – 226 had comorbidities and therefore excluded; 313 of remaining 330 were under 2 years age; 305 had early transaminase measurements. 57/305 (19%) had elevated transaminase (AST and/or ALT) levels. For the first time we show that duration of ventilation and length of admission were both significantly longer, and paediatric index of mortality and C-reactive protein higher, in those with elevated AST levels on admission (but not those with elevated ALT levels). Furthermore, transaminase elevations were transient, generally having normalised by seven days following admission. RSV bronchiolitis was more severe in children with early elevated AST levels and could be used early in the illness as a predictor for disease severity. RSV is the most common viral respiratory cause of hospital admission and of death in children below 5 years of age, especially in infancy1. RSV bronchiolitis occurs in predictable annual seasons (epidemics) that globally generate surges in hospitalisation rates, which often strain hospital and paediatric intensive care unit (PICU) capacity nationally and regionally. Approximately 3–10% of infants hospitalised with RSV bronchiolitis develop respiratory failure and require admission to PICU. In the United Kingdom between 2004 and 2012, bronchiolitis accounted for up to 12% of PICU admissions each year, with the vast majority (93%) being infants under 1 year of age3.Respiratory syncytial virus (RSV) bronchiolitis causes significant morbidity and mortality worldwide every year4. Elevated transaminases have been described in a subset of children who require mechanical ventilation on PICU with RSV bronchiolitis7. Suggested causes for this phenomenon include: virus-induced hepatitis (including RSV itself), high viral load leading to spill-over of RSV from the lungs into the systemic circulation, hepatotoxic medication, hypoxic hepatitis, hepatic congestion or ischaemia secondary to right heart failure/strain. With the advent of new anti-viral drugs for treatment of viral lower respiratory tract infections (LRTI), it will become important to differentiate infection-related transaminitis from medication-related hepatotoxicity. It has been suggested in previous smaller studies that children with raised transaminases have increased disease severity8. Comorbidity in this patient group  can confound interpretation of cause and effect – as the underlying comorbidity could play a role in the right ventricular failure and/or pulmonary hypertension.Despite most RSV research having concentrated on the lungs and the mechanics of pulmonary immunopathology, RSV bronchiolitis is not merely a single organ disease (i.e. lung) but impacts on extrapulmonary organ systemsTo interrogate the hypothesis that children ventilated for RSV bronchiolitis with raised transaminases have increased severity of disease, we studied a novel homogenous cohort of children under 2 years of age over 11 RSV seasons/11-year period. To avoid non-RSV disease confounding interpretation, children with comorbidities were excluded from analysis.Aim of the study was to compare disease severity as judged by duration of ventilation, length of PICU admission, respiratory indices and mortality in young children mechanically ventilated for RSV bronchiolitis with and without elevated alanine transaminase (ALT) and aspartate transaminase (AST) levels.21-bed regional multidisciplinary tertiary PICU in a university-affiliated, multispecialty, children’s hospital, which is also the regional paediatric cardiac referral centre, with an annual PICU admission rate of around 1100 children.All children under 2 years age mechanically ventilated with community-acquired RSV bronchiolitis over 11 consecutive RSV seasons (October 2002–March 2013) who had had liver function tests performed within 48 hours of admission to PICU were included. Children with any comorbidity  and any nosocomial or healthcare-acquired RSV infection were excluded to remove non-RSV disease as a confounding factor for increased transaminase levels. The study was approved by the Clinical Audit Department – a division of the Directorate of Research and Clinical Development at the Alder Hey Children’s NHS Trust (reference number 5192). The Directorate ruled that institutional ethics review was not required as data were obtained from routine clinical investigations and records and patient details were not identifiable.9 were taken on admission. Samples were collected by specialist respiratory physiotherapists or PICU staff members. Over the 11-year period, a number of different tests were used to detect RSV. Thus RSV was identified by immunochromatographic assays: DirectigenTM RSV test (2003–2004) or NOWTM RSV test (2005–2013); or immunofluorescence: SimulFluorTM reagents; and/or multiplex RT-PCR: InvitrogenTM (2009–2013). Bacterial co-infection was defined as microbiologically proven bacterial infection – required bacteria colony counts >105 cfu/ml of diagnostic sample for each single species obtained from lower airways secretions or urine, or a positive blood culture.Diagnostic samples of nasopharyngeal aspirates (for RSV detection) and lower airway secretions  through endotracheal tube using sterile precautionsTM, Roche Diagnostic Ltd.) at the laboratories for Clinical Chemistry at the Alder Hey Children’s Hospital. Normal and elevated paediatric reference ranges were based on those published by Hick and Soldin10. In infants <3 months of age AST and ALT levels were elevated when above 73 IU/L and 44 IU/L respectively. In infants and children >3 months of age, elevated AST and ALT levels were defined as being above 58 IU/L and 36 IU/L and respectively. To enable the analysis of liver function tests in children of different ages, transaminase levels were graded based on the Division of Microbiology and Infectious Diseases (DMID) paediatric toxicity tables (November 2007)11. Thus, Grade 1 was defined as 1.1 - < 2.0 × upper limit of normal (ULN) for age; Grade 2 as 2.0 - < 3.0 × ULN; Grade 3 as 3.0–8.0 × ULN; Grade 4 as >8.0 ULN. Given the small numbers of children, data from those with Grade 3 and 4 toxicity were analysed graphically together.Most children had, as part of a daily routine biochemical profile, aspartate transaminase (AST) and alanine transaminase (ALT) levels determined, soon after admission to PICU and daily thereafter. Transaminase levels and C-reactive protein (CRP) were measured by photometric assays  were carried out on PICU. Lung protection strategies, such as permissive hypercapnia and restriction of excessive tidal volumes (volutrauma), were standard practice. Inhaled nitric oxide was utilized in severe lung disease when hypoxaemia had been refractory to prior ventilator strategies and/or modes. Muscle relaxants and steroids were not routinely used.12. Markers of severity utilized were: duration of ventilation; length of PICU admission; worst oxygen index (mean airways pressure X FiO2/PaO2) and ventilation index (respiratory rate X PaCO2 X peak inspiratory pressure/1000) within the first 72 hours of PICU admission13; PIM12 and mortality. Continuous data were analysed using the Mann-Whitney-U test and categorical data using Fisher’s exact test. Statistical calculations were performed with the Statistical Program for Social Science release 21.0 . A p-value < 0.05 was considered statistically significant. All p-values were two-tailed.Prospective observational cohort study. Results were expressed as a number out of the total study population: mean and standard deviation (SD) or standard error (SE), or median and inter-quartile ranges (IQR). The Paediatric Index of Mortality (PIM) and resulting predicted probability of death were calculated from variables extracted on admissionAll data are available from the corresponding author on reasonable request.no-comorbidity study group are shown in Table – too low to differentiate between groups.Over the study period of 11 RSV seasons, 556 children with RSV bronchiolitis were mechanically ventilated – 226 had comorbidities and were therefore excluded; 313 of the 330 with no comorbidity were under 2 years of age. 305/313 (97%) had transaminases measured on either Day 1 (293 (94%)) or Day 2 (269 (84%)). Subsequently, 245 (78%), 205 (65%), 150 (48%), 94 (30%), 64 (20%) children had transaminases measured on Days 3, 4, 5, 6, and 7 respectively. Children generally only had transaminases measured when intubated and ventilated. The decreasing numbers of tests performed over time reflects that these children were improving and being extubated. 57 (19%) of the study group with no comorbidities (n = 305) had early elevated transaminase levels. The characteristics of this 11 in age-group subgroups are presented in Table Grading of transaminase elevation according to DMID criteria (paediatric toxicity tables)Changes in serum AST and ALT concentrations (based on DMID grading) following admission to PICU are shown in Figs 12 score and CRP . Only PIM score was higher in those with elevated ALT levels. An AST/ALT ratio >2 was associated with prolonged ventilation, increased length of PICU admission and elevated CRP , had increased severity of disease – as judged or measured by duration of mechanical ventilation, length of PICU admission and PIM. Raised transaminase levels were not uncommon in this patient group, occurring in nearly a fifth (19%) of our study population. This is the first study on this topic using a large and homogenous study cohort with community-acquired RSV bronchiolitis that is not ‘contaminated’ by underlying comorbidity or nosocomial infection.11. Clinical impact appeared not to be associated with age-group or grade of transaminase elevation. The elevations in transaminases were transient with AST settling earlier than ALT, but both generally returning to normal ranges within a week eek Figs . This woDisease severity was significantly increased in the group with elevated AST levels on admission as measured by longer duration of ventilation and length of admission and higher PIM (risk of death on PICU admission) (p < 0.05), but not those with elevated ALT levels Table . Interes4. Extrapulmonary effects (like hepatitis or myocarditis) raise the question as to whether direct RSV effects (i.e. RSV infection of liver or myocardium) or indirect effects  are at play? RSV has been detected in the liver, myocardium, and cerebrospinal fluid4. However, most often it was RSV-RNA detected by RT-PCR, which is essentially cell-associated RSV genome and not necessarily viable RSV. It is yet to be conclusively demonstrated that RSV can escape the alveolar macrophages and replicate in distant organs. Our previous study discounted concomitant viruses causing hepatitis in this patient group6. It is unlikely that elevations in transaminases were due to drug-induced hepatotoxicity. This group of children are generally not prescribed medications in the community , as there are no specific treatments for bronchiolitis. Furthermore, no hepatotoxic medications were used following hospitalisation in both cohorts  and elevations in transaminases were transient, even at transaminase grade 3 or 4 toxicity levels 14 whereas our patients’ oxygen levels were continuously monitored and hypoxia was avoided .Despite RSV infecting the respiratory epithelial cells and predominantly causing lung disease, clinical impact peripheral to the lung parenchyma is described in RSV infectionels Figs  and 2, m18 – hence their specific exclusion for this study. Previously, transient raised cardiac troponin levels (resolved by admission day 4) demonstrating myocardial injury or strain in children ventilated for RSV bronchiolitis with structurally normal heart was shown in up to 40% of this patient group18. No significant left ventricular dysfunction was identified on echocardiography in this patient group ventilated for RSV bronchiolitis18. However, reduced right ventricular function assessed by echocardiography (Tei index) was found in 20%, but not associated with raised cardiac troponin levels7. Neither was there an established association between reduced right ventricular function and elevated transaminases, but sample size was small (n = 34)7. Hepatic congestion or ischaemia secondary to right heart strain consequential to severe lung parenchymal disease is a plausible explanation for the elevated AST in our study. More severe lung disease in the group with elevated AST was attested by longer duration of mechanical ventilation and increased length of PICU admission. Right ventricular dysfunction/strain has been shown in children ventilated for RSV bronchiolitis with structurally normal hearts7. Fortunately, raised transaminase levels were transient, settling within 4–5 days, thereby making substantial or lasting (hepatic) injury less likely in our young study population.Myocardial dysfunction is well described in children with severe RSV bronchiolitis, especially those with congenital heart disease19. The AST/ALT ratio (De Ritis ratio) is utilized as an indicator of the aetiology of hepatitis with a ratio <2 suggestive of (acute) viral hepatitis. AST/ALT ratios >2 are seen in newborns , rhabdomyolysis and muscle injury, fulminant hepatitis or alcoholic hepatitis19. In this study AST/ALT ratio >2 was associated with prolonged ventilation, increased length of PICU admission and elevated CRP  and AST  are also found in heart muscle, skeletal muscle and kidneyRP Table . It is pPredicted probability of death calculated from PIM scores was higher in children with elevated transaminase levels as compared to those with normal levels. This increased risk of death infers increased disease severity in the elevated transaminase group on admission and supports elevated transaminase levels as a potential additional marker of severity. Only 3 children died over the 11 RSV seasons – too low in any group for mortality to be utilised as a marker for severity.per se. Future studies should include patients with non-RSV lung disease. To establish whether elevated transaminase levels would predict severity of disease in non-ventilated children with RSV bronchiolitis will be more challenging. They are less likely to undergo routine blood sampling and reliable measurements of disease severity would need to be determined.Without a comparable non-RSV group, it is difficult to assess whether the association of elevated transaminases to severity of disease is a RSV-specific phenomenon or merely reflective of severity of lung parenchymal disease RSV bronchiolitis was more severe in children admitted with elevated AST levels. This study suggests that early elevated AST levels could be used as a predictor for disease severity in RSV bronchiolitis.


Arctic grayling develop an interlamellar cell mass (ILCM) in their gill filaments when exposed to a half-strength seawater saline exposure but can recover if a freshwater environment is re-established within 48 h post saline water exposure. Microscopy analysis reveals that ILCM development is associated with a salinity-induced immunologic response. Thymallus arcticus). Compared to more euryhaline salmonids, Arctic grayling have a reduced salinity tolerance and develop an elevated interlamellar cell mass (ILCM) in response to salinity exposure (17 ppt). In this study, we described the gill morphology and cell types characterizing the ICLM. Further, we investigated whether Arctic grayling could recover in freshwater following a short-term (<48 h) salinity exposure. Arctic grayling were exposed to 17 ppt saline water for 12, 24 and 48 h. Following the 24 and 48 h salinity exposure, Arctic grayling were returned to freshwater for 24 h to assess their ability to recover from, and reverse, the osmotic disturbances. Physiological serum [Na+], [Cl–] and total osmolality were significantly elevated and progressively increased at 12, 24 and 48 h salinity exposures. The 24 h post-exposure recovery period resulted in Arctic grayling serum ion concentrations and total osmolality returning to near normal levels. Similar recovery patterns were observed in the salinity-induced ILCM, which developed as early as 12 h of exposure to 17 ppt, and then reverted to control levels following 24 h in freshwater. Gill histology indicates an increased number of apically located mucous cells in the interlamellar space following salinity exposure of Arctic grayling. The scanning electron microscopy and transmission electron microscopy data show the presence of granule containing eosinophil-like cells infiltrating the ILCM suggesting a salinity-induced immune response by the Arctic grayling.Freshwater environments are at risk of increasing salinity due to multiple anthropogenic forces including current oil and gas extraction practices that result in large volumes of hypersaline water. Unintentional releases of hypersaline water into freshwater environments act as an osmoregulatory stressor to many aquatic organisms including native salmonids like the Arctic grayling ( Thymallus arcticus) is the only Thymallus species, of the monogeneric sub-family Thymallinae, that is native to North America. Historically, T. arcticus range extended west of Hudson Bay to Alaska, across the northern halves of British Columbia, Alberta, Saskatchewan and Manitoba, with populations found in Michigan and Montana  . Spills nkaα1b). Importantly, exposure to 17 ppt saline water resulted in gill remodelling and the appearance of an ILCM in the grayling that was not present in rainbow trout under the same treatments (+/Cl–) under hypertonic environments.With the exception of a few cases of individuals from Arctic coastal river populations being captured in brackish estuary waters (<18 ppt)  can recover and reduce the resultant ILCM. We further investigated the general gill morphology with a goal to identify the cell types present in both the normal gill epithelium and ILCM of Arctic grayling.Artemia and Mysis shrimp, every other day.Arctic grayling  were collected in July 2015 from a tributary found in the Athabasca River watershed in collaboration with Alberta Environment and Parks (AEP) using conventional angling methods (fly-fishing). The grayling were transferred in an Alberta fish transportation truck to the University of Alberta, where they were held in a bio-secure aquatics facility, in a 600 l circular tank supplied with flow-through dechlorinated Edmonton city tap water (10°C) and constantly aerated. Fish were acclimated for 3 months prior to experimentation. Arctic grayling were fed a mixture of Two identical systems  were used for the exposure experiments. Each system consisted of three (60 l) in-line tanks, a top header tank, an exposure tank (housed fish) and a sump tank, which re-circulated 180 l of water maintained at 10°C with an attached chiller unit. Both systems were initially supplied with dechlorinated Edmonton city tap water, which was aerated with air stones placed in each exposure tank. Dissolved oxygen levels were maintained above 10 mg/l. The saline system water was made up to 17 ppt (50% seawater) with the addition of a soluble salt mixture . This salinity concentration was chosen based on our previous research revealing sub-lethal effects at this salinity. When necessary, salinity levels were maintained by adding salts and/or freshwater following measurement using a handheld digital salinity probe .n = 5 for each treatment) were transferred to either 17 ppt saline water and held for 12, 24 and 48 h, or to the freshwater system for 24 h (control) before lethal sampling. For investigations into the Arctic grayling’s ability to recover following the 24 and 48 h acute saline exposure, fish were transferred back to a freshwater environment where they were allowed to recover for an additional 24 h before undergoing lethal sampling.Arctic grayling were fasted for four days prior to experimentation. Fish (3), weighed and measured (fork length), followed by immediate blood and tissue sampling. Blood was drawn from caudal puncture using non-heparinized syringes, injected into 1.5 ml Eppendorf tubes and centrifuged . Serum was separated and frozen at −80°C for future analysis. The second and third gill arches were excised from each side of the fish and processed for light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM).Fish were anesthetized with a lethal dose of MS-222 . Serum [Cl–] was measured via digital chloridometer . Total serum osmolarity analysis was performed using a freezing point depression osmometer . Statistical analysis was performed with one-way ANOVA, followed by Tukey’s multiple comparisons test (Graphpad Systems: Prism 6).Arctic grayling serum from each of the six treatments was thawed and analysed for total osmolarity (mOsm), sodium , serum [Cl–] and serum osmolality significantly (P < 0.05) and progressively increased when Arctic grayling were exposed to 12, 24 and 48 h of 17 ppt (50% seawater) water. At 48 h of exposure, Arctic grayling serum levels were significantly elevated to 204.54 ± 5.27 mM Na+, 184.40 ± 4.29 mM Cl– and 411.0 ± 5.0 mOsm (mean ± SE), an increase of 20%, 55% and 44%, respectively, from freshwater levels . Serum [Na+] also tended to decrease, albeit non-significantly, in the 24 h + recovery sample. A similar osmotic recovery pattern was observed in fish that were returned to freshwater for 24 h, following exposure to 17 ppt for 48 h. Significant differences were seen between samples from 48 h exposure and the 48 h + recovery samples, with serum [Na+] decreasing by 10%, serum [Cl–] decreasing by 21% and total serum osmolality decreasing by 18%. Although 24 h recovery in freshwater resulted in significant decreases from both the 24 and 48 h exposure periods, serum ion levels were still significantly elevated above those of fish held in control freshwater . The ILCM remained elevated at the 24 and 48 h time points. However, for both the 24 and 48 h exposures, after a 24 h recovery period in freshwater, there were significant reductions in ILCM height  .Histological analysis of hematoxylin and eosin stained gill filaments revealed an abundance of mucous-like cells apically located in the ILCM region of fish following salinity exposure compared to those under freshwater control conditions and those following 24 h recovery in freshwater Fig. A–C. FollMorphological comparisons of Arctic grayling branchial epithelia with that of the rainbow trout following 24 h exposure to 17 ppt salinity were performed using SEM and TEM. As described previously for the morphology of the trout gill A. RainboIn comparison to the structure of the rainbow trout gill, the Arctic grayling filament was composed of lamellae in similar branching orientation, however, they extended laterally to a lesser extent leaving more surface area on the leading and trailing edges Fig. A. Large Transmission electron micrographs of Arctic grayling gill sections revealed the presence of different cell types near the apical surface depending on the environmental salinity, as well as the various cell types present in the interlamellar cell mass. Figure Representative samples of control fish  showed both exposed mitochondria rich cells, mucous cells and some eosinophil-like cells (containing large granulocytes) at the apical surface of the filament in the interlamellar space Fig. A. Upon eThe present study was performed to understand the unique and important physiological responses to industrially relevant environmental disturbances in the sensitive native salmonid species, the Arctic grayling. Physiological data confirm the poor salinity tolerance and relatively stenohaline behaviour of this salmonid while novel morphological evidence effectively provides insight into the cell and tissue alterations occurring during these osmotic perturbations. Our study answers the significant ecological and conservation science-relevant question of whether grayling are capable of recovery if re-introduced into freshwater following a short-term environmental exposure to high salinity. In addition to characterizing the gill remodelling that occurs following exposure and recovery from acute elevated environmental salinity, this is also the first study to provide general branchial morphological characteristics of the Arctic grayling.As we reported earlier  which in this study was associated with a combination of effects resulting from decreased pH, metal toxicity and increased conductivity   raising stream conductivity from 200 μS/cm (<1 ppt) to over 35 000 μS/cm (>25 ppt) . High saalinity) . While tIt is extremely important to note volume, concentration and time of a spill when interpreting the imminent negative consequences to the environment and organisms. As our study demonstrates, if spills result in environmental salinity increasing to <17 ppt, Arctic grayling can potentially survive this perturbation if the salinity stress is removed within 48 h. It can be theorized that under environmental spill conditions an ‘acute transfer’ of freshwater organisms into salinity can indeed occur given a large sudden spill into a river, although a slower transition is more likely under the circumstances of a pipeline break or overland leak of smaller relative volume. In turn, the rapid recovery to freshwater could also be feasible given the leak was quickly fixed or flow was stopped. However, a protracted recovery to full freshwater is more likely, and a slower recovery of plasma ion levels as well as a reduction ILCM would reflect that transition.Our fresh waters are at a constant risk of increasing salinity due to anthropogenic forces including, road salt application, hydraulic fracturing and other oil and gas activities, and decreasing flows due to climate change. Salinity is known to have adverse consequences on the aquatic ecosystem. For this reason, increased regulations and higher standards need to be met and enforced regarding salinity effects on freshwater environments . However


BDNF val66met polymorphism) and the dopamine D2 receptor (DRD2/ANKK1 glu713lys polymorphism) in humans. A retrospective analysis was performed to determine whether these polymorphisms influence data from our two previous studies, which both demonstrated that a single bout of aerobic exercise prior to motor practice enhanced implicit motor learning. Here, our main finding was that the effect of acute aerobic exercise on motor learning was dependent on DRD2/ANKK1 genotype. Motor learning was enhanced when aerobic exercise was performed prior to skill practice in glu/glu homozygotes, but not lys allele carriers. In contrast, the BDNF val66met polymorphism did not impact the exercise effect. The results suggest that the dopamine D2 receptor may be involved in acute aerobic exercise effects on motor learning. Such genetic information could inform the development of individualized aerobic exercise strategies to promote motor learning.The objective of the current work was to evaluate whether the effects of acute aerobic exercise on motor learning were dependent on genetic variants impacting brain-derived neurotrophic factor ( These behavioural findings are complemented by work demonstrating acute aerobic exercise-induced alterations in intracortical10 and interhemispheric excitability10, as well as increased plasticity in response to non-invasive brain stimulation protocols12. While the behavioural findings have potential implications for sport and rehabilitation, it is notable that there was substantial inter-individual variability in the magnitude of motor performance and learning benefits elicited by exercise in our prior work5. Uncovering the sources of this variability may provide insights into the mechanisms underpinning these effects and potentially inform the individualization of exercise strategies.Recent work demonstrated that performance of a single bout of aerobic exercise performed in close temporal proximity to motor practice can facilitate motor skill acquisition13. Considerable work has focused on a single nucleotide polymorphism (SNP) on the brain-derived neurotrophic factor (BDNF) gene18. This SNP (rs6265) causes a valine-to-methionine substitution at codon 66  and is associated with impaired activity-dependent secretion of BDNF19, a crucial characteristic for the involvement of BDNF in long-term potentiation (LTP) mechanisms that underpin memory formation20. As BDNF met allele homozygotes (met/met) are relatively rare  , homozygous and heterozygous carriers are commonly grouped together as met allele carriers in a dominant genetic model. Previous candidate gene studies demonstrated significant associations between the presence of the BDNF met allele and reduced LTP-like plasticity in the primary motor cortex (M1)23. BDNF val66met genotype effects on motor behaviour have also been studied, but with somewhat more equivocal results. While one study suggested reduced motor performance and learning in met allele carriers16, others found no difference between BDNF val66met genotype on behavioural outcomes23. These discrepant findings have led to speculation that the effects of this SNP may interact with specific types of neuroplasticity21 and depend on the complexity of the skill being learned22. Taken together, these results also suggest that there may be other genetic factors at play in determining certain individuals’ susceptibility to beneficial effects of exercise on the motor system.Past studies suggest that genetic variation contributes to inter-individual variability in neuroplasticity and motor learning in humans25. A dopamine-related genetic variant that may be particularly important for motor learning is located near the DRD2 gene and within the ANKK1 gene26. This SNP (rs1800497) causes a glutamic acid to lysine substitution at position 713 (glu713lys) of the ANKK1 gene, with lys allele homozygotes (lys/lys) and heterozygotes (glu/lys) often grouped as lys allele carriers, due to a low frequency of lys/lys homozygotes in most populations  . Although the underlying mechanism is not well understood, human lys allele carriers exhibit reduced dopamine D2 receptor (D2R) availability and binding in the brain27. Consistent with previous work demonstrating the importance of dopaminergic signaling for motor learning25, a candidate gene study demonstrated that motor learning benefits elicited by administration of levo-dopa (L-dopa), a dopamine precursor, were attenuated in carriers of the DRD2/ANKK1 lys allele compared to glu/glu homozygotes26.Dopamine is another key orchestrator of neuroplasticity and memory28 and dopaminergic pathways29 in mediating aerobic exercise effects on brain function. Although neither molecule readily crosses the blood-brain barrier31, human studies reported positive relationships between increased systemic BDNF and motor learning32, and increased systemic BDNF and dopamine with declarative memory33 following acute aerobic exercise. Thus, it seems plausible that genetic variation impacting these molecules may influence acute aerobic exercise effects on motor learning. A small human study (n = 12) found that a 4-week aerobic exercise program enhanced declarative memory in BDNF val/val homozygotes and not met allele carriers, but did not consider dopaminergic gene variants34. Additionally, with reference to exercise effects on the motor system, past work showed that met carriers showed no significant difference in intracortical excitability after acute moderate-intensity aerobic exercise, compared to those possessing the dominant allele9. No prior work has considered genetic influences on acute aerobic exercise effects specifically on motor learning.Interestingly, animal studies demonstrated direct roles for BDNFBDNF gene val66met (rs6265) and DRD2/ANKK1 glu713lys (rs1800497) gene variants on data from our prior studies examining the effect of acute high-intensity interval cycling on motor learning5. We hypothesized that the positive effects of acute aerobic exercise on motor learning would be reduced in both BDNF gene met allele carriers, compared to val/val homozygotes, and DRD2/ANKK1 glu/glu homozygotes, compared to lys allele carriers.Presently, we conducted a retrospective analysis of the influence of the n = 16 in each)5 for a current study sample of 32 young, healthy participants with no known neurological disorders   . These high intensity intervals were interspersed with three sets of two-minute active rest periods, in which participants maintained a cadence of at least 70 rpm and a power output of 50 W. High-intensity exercise was employed based on prior work suggesting a dose-response relationship between aerobic exercise intensity and increases in neurochemicals thought to play a role in learning processes38, with one task involving continuous movements and termed the continuous tracking (CT) task39, and the other requiring discrete movements and termed the serial targeting (ST) task40. Both tasks are described in more detail elsewhere5.Both studies used variations of the Pew task3. Participants completed a 30 s trial for task familiarization prior to the rest period or exercise bout. Motor practice consisted of two blocks of 10 × 30 s trials, for a total of 10 minutes. The following day (24 ± 2 hours after motor practice), participants completed another block of the CT task (retention test)3. CT task performance was quantified by the root mean square error of the participants’ cursor relative to the target movements.Briefly, the CT task involved manipulating a small joystick with the non-dominant thumb to move a cursor to track the vertical path of a target moving at constant horizontal velocity across a screen Fig. 3. Partic5. ST task performance was quantified by the participants’ total response time (sum of reaction time and movement times) to complete a sequence of target movements.The ST task involved manipulation of a computer mouse with the non-dominant hand to move a cursor between a series of targets appearing one at a time on a computer screen Fig. . At the 41. In the CT task, the repeated movement sequence was presented to the participant 20 times throughout the practice period . The repeated sequence in the CT task lasted 10 s in duration and was flanked by 10 s of random target movements in each 30 s trial. In the ST task, the repeated movement sequence was presented to the participant 24 times throughout the practice period . The repeated sequence was six target elements long and flanked by a random set of seven targets. For both tasks, the movement sequences were reversed between conditions, such that the sequences differed but were of equivalent difficulty for the exercise versus rest conditions. Also, the order of presentation of conditions (rest and exercise) and movement sequences (regular or reversed) were pseudorandomized and balanced across the sample for both tasks. Recognition testing following experimental sessions indicated that participants did not gain explicit knowledge of the repeated sequence, as it could not be identified at a rate higher than chance for either task 5.Unknown to participants, in both tasks a repeated sequence of movements was practiced; this sequence was also present at the retention tests. The inclusion of the repeated sequences allows evaluation of sequence-specific implicit learning5, only data from this condition were considered in our present analyses. Measures of motor performance (root mean squared error for the CT task and response time [sum of reaction and movement times] for the ST task) were calculated for each sequence. Analyses reported in our prior work indicated no significant order effects or differences in initial performance between rest and exercise conditions5. For the CT task, performance on the first three trials of the first practice block (early practice), the last three trials of the last practice block (late practice), and the first three trials of the retention block was determined. The changes in performance from early to late practice (ACQ-∆) and from early practice to retention (RET-∆) were then calculated3. For the ST task, the same approach was used but with groups of four repeated sequences at each time point5. The slight difference in the number of trials used in the analyses for each task provided a similar proportion between tasks for the number of sequences comprising a time point to the number of sequences practiced . The datasets were combined by converting the raw change scores for each task to percent changes in performance5, where higher percent change scores indicated greater improvements. Difference scores between the exercise and rest condition were then calculated for each individual for both the ACQ-∆ and the RET-∆ variables, such that a higher difference indicated a benefit of exercise relative to rest.As our previous work suggested preferential effects of exercise on learning of the repeated sequenceBDNF val66met, DRD2/ANKK1 glu713lys) . In all tests, the covariates included were participant age, sex, ethnicity, cardiorespiratory fitness (V̇O2peak), and the motor task used (i.e. CT and ST tasks), due to our prior work suggesting a larger effect of exercise on CT, compared to ST task, change score5. Statistical significance was set at p < 0.05. Parameter estimates with bootstrapped 95% confidence intervals (1000 samples) and effect sizes  for the effects of Genotype and each covariate are reported. Effect size magnitudes were interpreted based on previously developed guidelines, such that η2partial values below 0.06, between 0.06 and 0.14, and above 0.14 were considered small, moderate and large, respectively42. All statistical tests were performed using the ‘lme4’ package from R statistical software  and SPSS software .Statistical testing used analyses of covariance (ANCOVAs). The fixed factor in all ANCOVAs was genotype (levels: SNP carrier and non-carrier), and a separate ANCOVA was run for each combination of dependent variable (difference in ACQ-∆ or RET-∆ between rest and exercise conditions) and assayed genotypes   and bootstrapped parameter estimates were also non-significant (p ≥ 0.27).ANCOVAs yielded no significant main effect of either 06) Fig. . All covBDNF val66met SNP did not significantly influence the effect of exercise on RET-∆  = 0.83, p = 0.37) and the calculated effect size was small   and bootstrapped parameter estimates were also non-significant (p ≥ 0.13).The 03) Fig. . ResultsDRD2/ANKK1 glu713lys SNP on the difference in RET-∆ between rest and exercise conditions  = 7.99, p = 0.01) . Moreover, the bootstrapped parameter estimate for the presence of the lys allele was also statistically significant , suggesting that the observed finding was valid for a population-level estimate. Figure DRD2/ANKK1 genotype. Considering raw data for each participant, 14 of the 17 (82%) glu/glu homozygotes demonstrated higher RET-Δ values under the exercise relative to the rest condition. In contrast, RET-Δ values were higher under the exercise relative to the rest condition in only 7 of the 15 (47%) lys allele carriers. Considering the covariates in the model, the Ethnicity  and Task  covariates demonstrated trends for significance with moderate effect size magnitudes.The main finding was a significant genotype effect for the 01) Fig. . The DRDDRD2/ANKK1 glu713lys SNP was associated with the extent to which acute aerobic exercise facilitated motor learning, but not skill acquisition. Given that prior animal work suggests a role of dopamine in neuroplasticity and motor learning24 and its upregulation by aerobic exercise44, there has been prior speculation that dopaminergic signaling plays a role in acute aerobic exercise effects on memory37. Nevertheless, results from human studies have been inconsistent. A human positron emission tomography (PET) study indicated no change in raclopride binding to dopamine in the basal ganglia following acute aerobic exercise, with the caveat that the PET scan has low sensitivity relative to in vivo microdialysis studies in animals45. Another study found no correlation between exercise-induced increases in systemic dopamine and improvements in motor learning32, but acknowledged that dopamine does not cross the blood-brain barrier in appreciable amounts and that systemic levels do not necessarily reflect cortical levels31. However, dopamine signaling pathways in the brain involve multiple receptor subtypes (D1-D5) that each yield specific outcomes46. As such, exercise effects on brain function may depend on changes in specific dopamine receptors, rather than changes in levels of dopamine itself47. Our current findings point to a specific role for the D2R.Our results show that the 2R expression in the basal ganglia may contribute to long-term aerobic exercise training effects on neuroplasticity and motor rehabilitation47. By examining a SNP known to impact expression of the D2R, the present study provides evidence that the D2R may also interact with acute aerobic exercise effects on motor learning. Notably, the DRD2/ANKK1 genotype effect was specific to the retention change score, rather than that associated with acquisition. Previous work suggested preferential interplay between acute high-intensity aerobic exercise and consolidation of motor memory36. Likewise, dopaminergic circuits involving the D2R are thought to be involved in the retention phase of motor skill learning48. Taken together, these results may suggest a specific role for the DRD2/ANKK1 SNP in moderating acute aerobic exercise effects on motor memory consolidation.Animal and human studies of Parkinson’s disease using PET imaging suggest that changes in dopamine D10. Paired-pulse TMS studies demonstrated decreased short-interval intracortical inhibition (SICI)9 and increased short-interval intracortical facilitation (SICF)10 immediately following acute aerobic exercise, leading to speculation that decreased γ-aminobutyric acid (GABA)-ergic inhibition post-exercise may contribute to facilitation of motor learning. A possible link between these studies and our current work lies in a human pharmacological study indicating that modulation of D2R binding influenced GABAergic inhibition in motor cortex; however, in this work a D2R agonist enhanced SICI and a D2R antagonist decreased SICI49. An alternative explanation emphasizes a role of glutamatergic neurotransmission. Acute aerobic exercise has also been shown to enhance intracortical facilitation (ICF)9, a paired-pulse TMS measure thought to rely on glutamatergic interneurons and possibly NMDA receptors51. Antagonism of the D2R receptor impairs glutamatergic-dependent synaptic potentiation and motor learning in an animal model52. Thus, if aerobic exercise positively impacts D2R expression and binding47, then it is plausible that the motor learning benefits observed here and in prior work are at least partly mediated by enhanced glutamatergic neurotransmission.Besides work investigating acute aerobic exercise effects on motor learning, other recent studies have also examined the impact of a single bout of acute aerobic exercise on M1 intracortical excitability2R expression) but not for lys allele carriers, while the opposite effect was observed for L-dopa administration26. Although speculative, these disparate findings may suggest differences in motor learning benefits realized from the endogenous physiological events elicited by acute aerobic exercise versus an exogenous stimulus such as administration of L-dopa. There is prior evidence to suggest that improved behavioural and neurophysiological outcomes with increased dopamine transmission follow an inverted U, such that an excess of dopamine transmission can be detrimental to behaviour and plasticity57. Indeed, the endogenous release and uptake of dopamine in response to exercise is potentially at a more inherently optimal level for the individual, as opposed to imposed, prescribed amounts. Alternatively, given differences in D2R expression and binding, it might be expected that glu/glu homozygotes are relatively closer to the optimal point of the inverted U than lys allele carriers when in a baseline state. The exogenous delivery of 100 mg of L-dopa in the prior work26 could have been an ideal stimulus for the lys allele carriers to promote motor learning, but too great of a stimulus for the glu/glu homozygotes, pushing them beyond the peak of the inverted U. In contrast, the endogenous stimulus of aerobic exercise may provide a smaller increase in the dopamine transmission that is ideal for glu/glu homozygotes, but too weak to benefit learning in lys allele carriers. If this is the case, then perhaps lys allele carriers could benefit from a greater volume of exercise than what was currently prescribed, such as an incremental exercise bout to exhaustion, which has been shown to facilitate increases in corticospinal excitability for non-exercise upper-limb muscle representations58. Another possibility that should also be considered is that the specific neural pathways involved in distinct memory subtypes may be influenced by the exogenous and endogenous stimuli differently and impact behaviour accordingly.Interestingly, we found that motor learning was benefited by acute aerobic exercise for glu/glu homozygotes  is a limitation of our study, but notably, is similar to related work . The small sample sizes in these studies and the current work are balanced by in-depth phenotyping of neurophysiological and behavioural traits. We also included a comprehensive analysis, including bootstrapping of parameter estimates and estimation of effect sizes  to communicate the strength of our main finding as clearly as possible. Finally, the inclusion of data from two different motor tasks is another limitation of our study; however, we controlled for the type of task in our statistical analyses by including it as a covariate.It is important to note that the genes studied in the current work are expressed across many different cell types involved in numerous biological processes. As such, inferences related to the mechanisms by which genetic variation influence the current results are speculative. Moreover, the effects of the genetic variants studied are likely dependent on complex interactions across the genome. The relatively small sample size (nDRD2/ANKK1 glu/glu homozygotes, but not lys allele carriers. The results suggest that the dopamine D2R may be involved in acute aerobic exercise effects on motor learning and could have implications for individualized prescription of acute aerobic exercise to promote motor learning. These findings provide an important preliminary step in elucidating the complex relationships between acute aerobic exercise, motor learning and the human genome.In the present study, acute aerobic exercise enhanced motor learning in Supplementary Dataset 1