Trichoderma reesei is widely utilized in industry for cellulase production, but its xylanase activity must be improved to enhance the accessibility of lignocellulose to cellulases. Several transcription factors play important roles in this progress; however, nearly all the reported transcription factors typically target both cellulase and hemi-cellulase genes. Specific xylanase transcription factor would be useful to regulate xylanase activity directly.The filamentous fungus xyn1, xyn2, and xyn5), but not those of GH10 (xyn3) and GH30 (xyn4) xylanase genes, and thus regulate their transcription and expression directly. We also identified the binding consensus sequence of SxlR as 5′- CATCSGSWCWMSA-3′. The deletion of SxlR in T. reesei RUT-C30 to generate the mutant ∆sxlr strain resulted in higher xylanase activity as well as higher hydrolytic efficiency on pretreated rice straw.In this study, a novel zinc binuclear cluster transcription factor (jgi|Trire2|123881) was found to repress xylanase activity, but not cellulase activity, and was designated as SxlR . Further investigations using real-time PCR and an electrophoretic mobility shift assay demonstrated that SxlR might bind the promoters of GH11 xylanase genes  contains supplementary material, which is available to authorized users. Trichoderma, Aspergillus, and Penicillium. The biomass-degrading enzymes produced by these organisms also have applications in various fields of industry including food, fodder, paper, and textile industries. Trichoderma reesei is a well-known efficient producer of cellulase and hemi-cellulase, and is therefore widely employed by the enzyme industry for production of its own endogenous enzymes as well as production of heterogeneous proteins  and the sequence downstream of sxlr was inserted into the XhoI site by the same method mentioned above.For re-complementation of Agrobacterium-mediated transformation was conducted as described previously by Ma et al. [All vectors constructed were verified by sequencing. a et al. .FPAase activities were determined using cellulose filter paper as described previously . XylanasFor straw hydrolysis, different pretreated biomasses such as rice straw and a corn stover plus rice straw mixture (3:7 ratio)  were useT. reesei RUT-C30, Δsxlr, Osxlr, and Rsxlr were first prepared by growth on PDA plates at 28 °C and harvesting spores after 7 days. Spores were counted using a hemocytometer and 105 spores of each strain were inoculated onto PDA plates and MM plates containing 1% xylose, xylan, glucose, lactose, or Avicel. Double-layer Avicel plates were prepared by first casting an MM agarose bottom layer containing no carbon source, followed by an MM agarose top layer containing 1% Avicel; each strain was grown in triplicates. These plates were incubated at 28 °C before phenotypic examination.Spores of sxlr, Osxlr, and Rsxlr strain was collected. The samples were loaded into 12.5% PAGE gel, 70 min at 120 V were preferred for proteins separation. Coomassie brilliant blue R250  was used to color the gel for 1 h. Destainer  was added for another 2–4 h. Finally, the protein band was excised from the gel and send to Shanghai Applied Protein Technology Co. Ltd, protein analysis was conducted with a MALDI-TOF/TOF mass spectrometer 5800 Proteomics Analyzer , NCBI Trichoderma (59929 protein sequence) was selected as database.After 7 days’ culture with wheat bran and Avicel medium, the supernatant of RUT-C30, Δrpl6e  and sar1  as the reference genes, respectively.Mycelia were harvested after induction by wheat bran and Avicel for 4, 8, and 12 h, then transferred into TRIzol reagent  with standard protocol. Three biological replicates were prepared in the process. Real-time PCR was performed with primers listed in Additional file BamHI and XhoI double digestion to produce pGEX-4T-SxlR-B, then subsequently introduced into E. coli BL21 (DE3) for protein production. The recombinant protein was purified using a GST-Bind resin column  according to the supplier’s recommendations. SDS-PAGE was used to verify the purified protein.The DNA binding domain (residues 252–347) of SxlR (SxlR-B) was expressed by the pGEX system according to the manufacturer’s guidelines. First-strand cDNA was used as a template to amplify the fragment encompassing the DNA binding domain of SxlR using the primers indicated in Additional file T. reesei RUT-C30 were divided into six parts and each part was about 268 bp . The experiment was then performed as described previously by Chen et al. [xyn1 promoter, −890 to −882 position of xyn2 promoter and −1091 to −1083 position of xyn5 promoter. Motif 2 (5′-TGAWGAG-3′) was located in −1022 to −1016 position of xyn1 promoter, −957 to −951 position of xyn2 promoter and −1021 to −1015 position of xyn5 promoter. Motif 3 (5′-WTATAT-3′) was located in −998 to −993 position of xyn2 promoter and −1117 to −1112 position of xyn5 promoter. Motif 4 (5′-AATGSASAG-3′) was located in −1119 to −1111 position of xyn1 promoter, −890 to −882 position of xyn2 promoter and −1091 to −1083 position of xyn5 promoter. Motif 5 (5′-CATCSGSWCWMSA-3′) was located in −1110 to −1098 position of xyn1 promoter, −905 to −893 position of xyn2 promoter and −1053 to −1041 position of xyn5 promoter.For EMSA, the labeling of probes containing the promoter regions of specific genes was performed via PCR using the primers listed in Additional file n et al. . Motif 1Additional file 1. Putative specific xylanase transcription factors in T. reesei.Additional file 2.sxlr transformants verification. (A) PCR result for Rsxlr transformants identification. Three different transformants were chosen to verify. (B) Verification of random insertion in Rsxlr transformants. During the Agrobacterium-mediated transformation, the T-DNA will randomly insert into genome with LB and RB, so we can verify it with PCR. The expected PCR product length of 5′ and 3′ verification was 6.1 kb and 2.6 kb. The sxlr re-complementation vector was used as positive control. Three different transformants were chosen to verify. The sxlr transcription levels in sxlr transformants were normalized to the signal of β-actin (C) or rpl6e (D), a gene encoding a ribosomal protein. RNA was extracted after 12 h induction by wheat bran and Avicel. Error bars represent the standard deviation of three biological replicates.Additional file 3. Quantitative PCR analysis of cellulase and hemi-cellulase gene expression levels. The expression level of five xylanase genes in RUT-C30, Δsxlr, Osxlr and Rsxlr when wheat bran and Avicel were used as carbon sources. Expression levels were normalized to the signal of rpl6e, (A) or sar1 (B), a gene encoding a small GTPase. (C) The expression levels of cbh1, cbh2, egl1, xyr1, man1 and abf1 in RUT-C30 and Δsxlr. Expression levels were normalized to the signal of β-actin. RNA was extracted after 24, 48, and 72 h after induction by wheat bran and Avicel. Error bars represent the standard deviation of three biological replicates.Additional file 4. The description of xylanase genes promoter and EMSAs of SxlR binding to xyn3 and xyn4. (A) The nearly 1500-bp upstream regions (nucleotide position –1500 to –1) assumed to be the promoter regions of the xylanase-encoding genes from T. reesei RUT-C30 were divided into six parts and each part was about 268 bp. (B) DNA binding of SxlR to the xyn3 and xyn4 promoter regions. We used three concentrations of SxlR-B: 0, 0.5, and 1 μM; ~ 10 ng of Cy5-labeled probe was added to each reaction. For specific (S) and non-specific (NS) control experiment, 100-fold excess of unlabeled S and NS competitor DNA were added.Additional file 5. Truncation prmoter sequence of GH11 xylanase genes. (A) The description of DNA sequence truncation. Each sequence was divided into two parts. (B) EMSAs of SxlR binding to xyn2-P4, xyn1-P5 and xyn5-P5 truncation sequence, respectively. (C) Three putative SxlR binding consensus sequences derived by MEME. (D)The location of three putative SxlR binding consensus sequences in xyn2-P4-1, xyn1-P5-2 and xyn5-P5-2. Motif 1, 2 and 3 was labeled as red, blue and green, respectively. Dxyn2-P4-1, Dxyn1-P5-2 and Dxyn5-P5-2 was the sequence after truncation. (E) EMSAs of SxlR binding to Dxyn2-P4-1, Dxyn1-P5-2 and Dxyn5-P5-2, the SxlR-DNA complex was indicated by arrow. The amounts of purified SxlR binding domain  used were as indicated; ~ 10 ng of Cy5-labeled probe was added to each reaction.Additional file 6. Phylogenetic relationship between SxlR and putative orthologs. The phylogenetic tree was inferred using the neighbor-joining method. Evolutionary analyses were conducted in MEGA 5. The gene ID in NCBI is ETR97987.1 (T. reesei), XP_013961599.1 (T. virens), XP_013948752.1 (T. atroviride), XP_960943.2 (Neurospora crassa), EPS34484.1 , XP_681446.1 (Aspergillus nidulans), CAK40371.1 (A. niger) and KOC12472.1 (A. oryzae).Additional file 7. Oligonucleotides used in this study.


Patients receiving a kidney with at least one G allele for the CD46 promoter polymorphism rs2796267 (A/G) showed a lower rejection-free survival, though this became borderline significant after adjustment for potential confounders . A second CD46 promoter polymorphism , was also associated with a lower freedom from acute rejection in the presence of at least one G allele . Finally, the combined presence of both favorable genotypes of rs2796267 and rs147788946 had an additional protective effect both on acute rejection (p = 0.006) and graft survival (p = 0.03). These findings could help to identify patients who could benefit from intensified immunosuppressive therapy or novel complement inhibitory therapeutics.Complement regulating proteins, including CD46, CD55, and CD59, protect cells against self-damage. Because of their expression on the donor endothelium, they are hypothesized to be involved in accommodation. Polymorphisms in their promoter regions may affect their expression. The aim of this study was to investigate if donor polymorphisms in complement regulating proteins influence kidney transplant outcomes. We included 306 kidney transplantations between 2005 and 2010. Five polymorphisms in the promoters of CD46, CD55, and CD59 were genotyped. A CD59 promoter polymorphism (rs147788946) in donors was associated with a lower 1-year rejection-free survival  and a trend toward impaired 5-year graft survival ( Current immunologic matching of donor and recipient and risk stratification in kidney transplantation is largely based on AB0 blood group compatibility, HLA typing and the presence of donor-specific HLA antibodies, but does not include the potency of effector mechanisms such as the complement system –3. In kiMultiple studies on complement polymorphisms in kidney transplantation, including in C3, C4, and mannose-binding lectin, have been performed, but could not provide an indisputable association between these polymorphisms and acute rejection nor graft survival . We hypon = 38) or donor DNA was degraded and not suitable for genotyping anymore (n = 13), leaving 306 transplantations for this analysis. Clinical data were obtained from hospital records and the Dutch Organ Transplant Registry for which all patients provided written informed consent. Posttransplant follow-up data were available for all patients for at least 5 years. All patients provided consent for use of leftover sera samples. The primary outcomes in this study were 5-year death-censored graft survival and 1-year freedom from acute rejection. For acute rejection, we decided to look at 1-year freedom from acute rejection because the incidence of acute rejection is the highest within the first year and rejection after the first year is often related with nonadherence or overaggressive immunosuppressive tapering  for all transplantations between 2005 and September 2008 . For tra® version 2.7 (Applied Biosystems).Donor DNA was extracted from peripheral blood mononuclear cells or splenocytes at the time of transplantation for HLA typing purposes by using the MagnaPure Compact system according to the manufacturer’s instructions  and stored at 4°C. Because all donor DNA samples were stored with a donor code that was not traceable to an identifiable person, permission to use the leftover DNA samples for study purposes was granted by the Biobank Research Ethics Committee of the UMC Utrecht. Genomic DNA was amplified through polymerase chain reaction (PCR) by using specific primer-pairs for the promoters of CD46, CD55, and CD59 . Following enzymatic purification, the PCR products were sequenced by using sequence-primers and fluorescent capillary electrophoresis . Sequence data were analyzed with SeqScapep-values. A p-value of <0.05 was considered to be statistically significant.All data were analyzed with SAS Enterprise Guide 7.1  and R 3.2.2. Survival analyses were performed by constructing Kaplan–Meier curves and tested for significance with the log-rank test. No correction for multiple testing was performed because we selected the investigated polymorphisms beforehand based on literature and frequency within the general population instead of random testing of all identified polymorphisms within the CD46, CD55, and CD59 promoter regions . To adjuFive different polymorphisms in the promoters of CD46, CD55, and CD59 that are frequently present within the general population were sequenced. The observed genotype frequencies within our donor population are summarized in Table p = 0.08; Figure p = 0.02; Figure p = 0.07; Figure p = 0.03; Figure Kaplan–Meier survival analyses showed no associations between the polymorphisms in the promoters of the genes encoding for CD46 and CD55 and 5-year death-censored graft survival. For the CD59 promoter polymorphism, the survival curve hints at an impaired survival of kidneys with a SNP configuration without an adenine insertion (−/−), although this is not significant . For CD46 SNP A, patients receiving a kidney with at least one G allele tended to be more often retransplant patients and subsequently also had more often a PRA above 5% and induction therapy with an IL-2 receptor antagonist . After adjustment for donor type, PRA, retransplantation and induction therapy in a cox multiple regression model, there was still a trend toward a lower rejection-free survival  in patients receiving a kidney from a donor with at least one G allele for CD46 SNP A . Regarding 1-year rejection-free survival, a dose-dependent effect was observed, with a lower rejection-free survival in kidneys with the risk complotype (73%) compared to kidneys with an intermediate complotype .The combined presence of multiple complement polymorphisms, a complotype, may yield additional information. Therefore, we compared patients receiving a kidney with both protective variants of CD46 SNP A and CD59 SNP alongside kidneys with both risk variants and kidneys with one protective and one risk variant, the intermediate group. Recipient and transplant characteristics of all groups are summarized in Table This study demonstrated that two promoter polymorphisms in CD46 and one in CD59 in kidney donors correlate with a lower freedom from acute rejection within the first year posttransplantation. The survival analyses hint at a lower 5-year graft survival in patients receiving a kidney with the −/− configuration of the CD59 SNP, although this was not significant. The combined presence of both protective genotypes of CD46 SNP A and CD59 SNP has an additional preservative effect on freedom from acute rejection and 5-year graft survival. There was no association between the CD55 promoter polymorphisms and rejection-free nor graft survival.Mechanisms by which CD46 can protect against both cellular and antibody-mediated rejection include decreased production of complement C3a and C5a and inhibition of further complement activation and amplification. Locally produced C3a and C5a can bind to antigen-presenting cells (APCs) and T cells, thereby inhibiting T-cell apoptosis and enhancing T-cell proliferation and the production of co-stimulatory molecules and cytokines by APCs –27. CD46Donor CD59 protects the allograft against rejection by hindering the formation of the membrane attack complex. Membrane attack complex formation on the donor endothelium can results in lysis, whereas sublytic levels induce endothelial cell activation and altered proliferation –33, augmTo the best of our knowledge, this is the first study that assessed the impact of promoter polymorphisms in genes encoding for complement regulatory proteins in kidney donors on transplant outcomes. Park et al. studied the CD46 SNP A in kidney transplant recipients and reported an inverse association between this polymorphism and acute and late-onset acute rejection in kidney transplant recipients . Howeverp = 0.08). However, when taking death with functioning graft into account, no significant differences in 5-year graft survival were observed between patients with available donor DNA (74%) and without donor DNA .This discovery study was set up to explore the potential associations between promoter polymorphisms in the genes encoding for complement regulatory proteins and kidney transplant outcomes. A limitation of this study is the relatively small sample size for a SNP association study and lack of replication cohort. The sample size has likely also attributed to differences in patient characteristics  when stratifying for CD46 SNP A donor genotypes, since we would not expect an association between these factors and a donor polymorphism. Retransplantation and PRA are general markers of immunization and can include both cellular and humoral immunization . We triein vitro  and performed in accordance with the Declaration of Helsinki.TK-H and LM performed the research; AZ, LM, HO, and MV participated in data analysis; AZ, LM, and HO participated in research design; AZ, LM, HO, and MV wrote the paper. All authors provided final approval of the version to be published.AZ has received a travel grant and speakers fee from Astellas Pharma and is on the Dutch Novartis Transplant Advisory Board. LM has received a travel grant from Astellas Pharma. All other authors have no conflict of interest to disclose.


Purpose: To investigate the differences in neural control of back muscles activated during the eccentric vs. the concentric portions of a cyclic, submaximal, fatiguing trunk extension exercise via the analysis of amplitude and time-frequency parameters derived from surface electromyographic (SEMG) data.Methods: Using back dynamometers, 87 healthy volunteers performed three maximum voluntary isometric trunk extensions (MVC's), an isometric trunk extension at 80% MVC, and 25 cyclic, dynamic trunk extensions at 50% MVC. Dynamic testing was performed with the trunk angular displacement ranging from 0° to 40° and the trunk angular velocity set at 20°/s. SEMG data was recorded bilaterally from the iliocostalis lumborum at L1, the longissimus dorsi at L2, and the multifidus muscles at L5. The initial value and slope of the root mean square (RMS-SEMG) and the instantaneous median frequency (IMDF-SEMG) estimates derived from the SEMG recorded during each exercise cycle were used to investigate the differences in MU control marking the eccentric vs. the concentric portions of the exercise.Results: During the concentric portions of the exercise, the initial RMS-SEMG values were almost twice those observed during the eccentric portions of the exercise. The RMS-SEMG values generally increased during the concentric portions of the exercise while they mostly remained unchanged during the eccentric portions of the exercise with significant differences between contraction types. Neither the initial IMDF-SEMG values nor the time-course of the IMDF-SEMG values significantly differed between the eccentric and the concentric portions of the exercise.Conclusions: The comparison of the investigated SEMG parameters revealed distinct neural control strategies during the eccentric vs. the concentric portions of the cyclic exercise. We explain these differences by relying upon the principles of orderly recruitment and common drive governing motor unit behavior. Progressive trunk muscle resistance training is widely recommended to increase back muscle strength, endurance and power, which in turn allows one to maintain pain-free functional performance of trunk movements and to build injury resilience , the geometrical distribution of active muscle fibers, the muscle length and the change in muscle length during muscle contractions  data could shed light on the neuromuscular control mechanisms underlying concentric and eccentric extensions of the trunk. SEMG data recorded during voluntary muscle contractions is regarded as the spatio-temporal superposition of the motor unit action potential (MUAP) trains of active MUs. The physiologic correlates contributing to the SEMG data amplitude are mainly the number of active MUs and their firing rates, whereas changes in the frequency content of the SEMG data correlate with changes in the conduction velocity of the active muscle fibers De Luca, .in vitro studies have demonstrated a close relationship between muscle fiber size, type and the median frequency of the SEMG suggesting that relative fatigue-related changes in the SEMG spectrum could indicate neural control properties of muscle fibers of the activated muscle  to derive the frequency content of SEMG data are only suitable for the analysis of stationary time series. SEMG data collected during dynamic contractions is non-stationary. Hence, techniques suitable for the analysis of non-stationary time series need to be applied to analyze SEMG data collected during dynamic contractions  were enrolled in the study. Volunteers were recruited via personal contacts, presentations at community centers for older adults, presentations at companies in the area close to the Karl-Landsteiner institute of outpatient rehabilitation research, and personal contacts with staff at an outpatient rehabilitation institute.2.Physical Medicine and Rehabilitation physicians screened all participants. Volunteers were considered eligible to participate in the study if they were healthy, performed normal physical activity (but did not participate in competitive sports more than 2 times per week) and were not affected by any conditions that could preclude their participation in vigorous exercise. Volunteers were not enrolled in the study if (1) they were unable to follow verbal instructions, (2) experienced more than 5 mild back pain episodes or episodes leading to referral for physical treatment (VAS > 30) lasting more than 2 days within the past year, (3) had a history of spine surgery, (4) were pregnant, (5) were affected by any medical condition that could interfere with maximum strength or sub-maximum endurance testing, or (6) had a BMI exceeding 35 kg/mThe study protocol was approved by the Ethics' Committee of the city of Vienna. Before inclusion, all participants provided written informed consent. The data collection was carried out in accordance with the Declaration of Helsinki. Participants received a financial compensation for the time spent participating in the study.Each study participant completed the following procedures. (1) Collection of basic anthropometric measures and administration of questionnaires to assess subjects' motivation and physical activity level. (2) Performance of warm-up and maximum isometric back extension tests using a dynamometer . (3) Rest for approximately 20 min, during which the SEMG electrodes were positioned. (4) Performance of one sustained isometric back extension for 30 s at 80% of the maximum voluntary contraction (MVC) level performed with the Technogym dynamometer “Total Trunk” . (5) Rest for approximately 5 min and performance of a task consisting of cyclic extensions of the trunk at 50% MVC on the Technogym dynamometer “Total Trunk” . All tests were supervised by 3 experienced examiners  and a certified clinical psychologist (BP). If the SEMG data recorded during the experiment was affected by significant artifacts, a second experimental session was conducted 1 or 2 days after the first one and all experimental procedures were repeated a second time.The maximum isometric back extension torque was required to set submaximum loads for the electromyographic fatigue tests. Each study volunteer was measured using a dynamometer . The device has been described in a previous publication  device. This device is more suitable to collect reliable SEMG recordings than the device that we used to collect the maximum isometric back extension torque data. It is equipped with a dorsal sacral pad instead of a back pad thus providing better access to the back extensor muscles than the DAVID® device. Otherwise the Total Trunk machine is built in a way that is similar to the DAVID® device. It is equipped with footplates adjustable according to the length of the lower leg, knee pads adjustable according to the thigh length, a pelvic belt, and an adjustable seat to accommodate for subjects of different height. The static study consisted of a sustained isometric extension of the trunk at 80% MVC. The dynamic study consisted of cyclic trunk extensions at 50% MVC. A 50% MVC was chosen because a relatively large proportion of the motor units would be recruited in the back extensors, blood supply to the back muscle fibers likely be partially suppressed only and at this load was demonstrated to be the minimum threshold to induce spectral SEMG muscle fatigue in the back extensors . Before positioning the electrodes, the skin was cleaned with alcohol wipes and, if needed, the area of interest was shaved. Electrodes were positioned bilaterally on the multifidus muscle at L5, the longissimus dorsi muscle at L2, and the iliocostalis lumborum muscle at L1. The electrodes were positioned according to the SENIAM project recommendations  was attached to the lever arm of the dynamometer in a standardized position and used to monitor the range of movement and movement velocity during the exercise of cyclic extensions of the trunk. The triaxial accelerometer acquired preamplified signals with a dynamic range of ±1.5 g and a bandwidth from DC to 50 Hz. The accelerometer signals were sampled at 160 Hz with a resolution of 8 bits using the EMG Works® acquisition software.Study participants completed the International Physical Activity Questionnaire (IPAQ) , members of the research team instructed them to perform a warm-up exercise consisting of extensions of the trunk at very low force level. These trunk extension exercises allowed subjects to familiarize themselves with the equipment and the experimental procedures. Thereafter, subjects performed 2 consecutive isometric maximum voluntary contractions (MVCs) under the supervision of research personnel. If the 2 tests varied by more than 10%, or if the peak moment was achieved later than 3 s after the onset of the contraction, additional trials were performed until a consistent  maximum force output was achieved. The best value observed during these tests was recorded and stored. Verbal instructions and encouragement were standardized.After placing the SEMG electrodes, study participants were instructed to position themselves for testing with the “Total Trunk” device  using the same settings that were used for the DAVID® device. Subjects maintained a 30° anteflexed trunk position for at least 30 s while performing a contraction at 80% MVC level. The MVC force output was derived from the best maximum trunk extension moment obtained with the DAVID® device. The torque generated by the subject was calculated as the mathematical product of the force recorded by the load cell of the dynamometer and the distance of the back restraint from the load cell.The cyclic exercise of trunk extensions was performed from a seated position with arms and hands positioned in a standardized way Figure  using thImmediately after the cyclic exercise of trunk extensions, study participants were asked to rate the perceived exertion of the back muscles using the Borg VAS scale.MATLAB routines  were used to process the SEMG data collected during the experiments. The SEMG data was filtered using a 20 Hz high-pass Butterworth filter and a 500 Hz low-pass Butterworth filter. RMS-SEMG estimates were derived from the 2nd to the 5ths of the sustained 80% MVC contractions, as described in a previous manuscript  were estimated for each data segment and each electrode recording site. IMDF-SEMG estimates were derived using a Cohen class time-frequency transformation Cohen, . SoftwarThe linear regression of the RMS-SEMG estimates derived from the SEMG data collected during the cyclic exercises was used to estimate the rate of change in RMS-SEMG values, which in turn was considered a measure of localized muscle fatigue. Consistently with the work by Larivière et al. , the RMSasin denotes the arcsine (inverse sine function), acc is the acceleration measured along the z-axis of the accelerometer positioned on the Total Trunk device, and g is the gravitational acceleration. This is an approximation of the actual orientation of the trunk angular displacement that we deemed suitable for the purpose of segmenting the SEMG data and comparing different cycles of the exercise. An approximation of the trunk angular velocity was derived from the estimated trunk angular displacement by low-pass filtering the angular displacement time series with an 8th order low-pass Butterworth filter with a cut-off frequency of 1 Hz and by estimating its first derivative. Figure The orientation of the lever arm of the Total Trunk device was considered as a proxy of the trunk angular displacement. Hence, the trunk angular displacement was derived from the acceleration measured along the vertical axis  of the accelerometer positioned on the Total Trunk device. The orientation α was estimated using the formula α = asin(acc/g), where The following dependent variables were used in the analysis: MVC as a measure of trunk extensor strength; mean normalized (80% isometric back extension) RMS-EMG and absolute IMDF-SEMG values derived from the initial values of linear regression models; mean RMS-SEMG and IMDF-SEMG slopes normalized by the RMS-SEMG and IMDF-SEMG initial values.Secondary analyses were carried out on the phase duration and the acceleration of the concentric and eccentric portions of the cyclic exercise, and the fatigue scores reported by study participants immediately after completion of the cyclic exercise of trunk extensions.p < 0.05.All statistical analyses were carried out using the R [2016] environment for statistical computing. Inspection of the analyzed variables showed clear deviations from normality, especially in terms of skewness. Thus, we used tests based on comparisons of median values Wilcox,  which arTable 2 for the concentric portion of the exercise and 0.37 ± 0.01°/s2 for the eccentric portion of the exercise.The analysis of the trunk angular velocity and acceleration measured using the accelerometer positioned on the lever arm of the Total Trunk device showed minor differences between the concentric and eccentric portions of the exercise. The peak trunk angular velocity was found to be 55.0 ± 0.6°/s for the concentric portion of the exercise and 56.7 ± 0.6°/s for the eccentric portion of the exercise. The peak trunk acceleration was found to be 0.46 ± 0.01°/sThe RMS-SEMG initial values for the concentric portions of the cyclic exercise were found to be different from the initial RMS-SEMG values for the eccentric portions of the exercise. Table During the cyclic exercise, the RMS-SEMG values generally showed significant increases for the concentric portions of the exercise but no such changes for the eccentric portions of the exercise. Table Table Table In this study, we relied upon amplitude and time-frequency parameters derived from the SEMG data to explore differences in the neural control of the back extensor muscles during the concentric vs. the eccentric portions of a cyclic exercise consisting of repetitive trunk extensions. Our findings revealed that: (1) the RMS-SEMG initial values for the eccentric portions of the exercise were considerably lower than the values for the concentric portions of the exercise; (2) the RMS-SEMG slopes generally showed a more pronounced increase in SEMG data amplitude during the concentric vs. the eccentric portions of the exercise; (3) the IMDF-SEMG initial values for the eccentric portions of the exercise were similar to the values observed for the concentric portions of the exercise; (4) the IMDF-SEMG slopes showed a similar compression toward the lower frequencies of the SEMG data for the concentric and the eccentric portions of the exercise; and sex and age did not affect the observed relationship.It is worth noticing that the RMS-SEMG initial values for the concentric portions of the cyclic exercise exceeded the RMS-SEMG values observed for the 80% MVC tests. It must be noted that the torque generated by the dynamometer during the cyclic exercise was equal to 50% of the force generated by the subjects during the MVC tests. In contrast, the RMS-SEMG initial values for the eccentric portions of the cyclic exercise were approximately 50% of those for the concentric portions of the exercise. Given the quasi-linear relationship between the RMS-SEMG value and the extension force generated by back muscles during isometric contractions    during cyclic dynamic contractions of muscles of the upper or lower limbs (De Luca et al., One might raise concerns that the level of performance of the cyclic exercise investigated in the study might have biased the results herein presented. It is worth noticing that a significant effort was devoted to instruct and supervise subjects during the performance of the cyclic exercise. Analyses of the data collected using the movement sensors attached to the device utilized in the study to perform the cyclic exercise assured us that the range of motion and the movement velocity of the trunk were virtually the same during the eccentric vs. the concentric portions of the cyclic exercise.In conclusion the comparison of amplitude and time-frequency parameters of the SEMG data revealed differences in neural control strategies between the concentric and the eccentric portions of a cyclic exercise of repetitive trunk extensions. We propose that such differences be primarily explained by relying upon the concepts of orderly recruitment of MUs and common drive. The observed contraction-specific differences in MU behavior are likely mediated by muscle specific differences in muscle spindle density and related differences in excitatory feedback that might be related to the higher intrinsic twitch force properties of muscles fibers during eccentric contractions compared to concentric contractions.Ethics Committee of the City of Vienna—Number: “EK 11-064-VK-NZ" Full address: Ethikkommission der Stadt Wien Magistratsabteilung 15, Gesundheitsdienst der Stadt Wien 3., Thomas-Klestil-Platz 8 Town Town Tel.: 01/4000-87523.GE conceived of the study together with TK and its design, he supervised the study, interpreted the results and provided the first draft the manuscript. TK participated in the study design, carried out the screening of patients, and contributed to drafting the manuscript. LU and SR collected and processed the data and contributed to the discussion of the findings. JK was responsible for technical equipment, preliminary testing and supervision of data quality control and part of the data processing. RH collected data and performed the statistical analyses under the supervision of PM. PB supervised the EMG data processing and PM supervised the statistical analyses. All authors critically revised the manuscript for important intellectual content and gave final approval. PB critically revised the language content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stringent donor-screening criteria and legislation prohibiting payment for donor gametes have contributed to the radical decline of donor insemination (DI) using sperm provided by Canadian men. Thus, many individuals rely on imported sperm. This paper examines the feasibility of an altruistic sperm donation (ASD) program to meet the needs of Canadians.Using Canadian census data, published literature and expert opinions, two population-based, top-down mathematical models were developed to estimate the supply and demand for donor sperm and the feasibility of an ASD program.It was estimated that 63 donors would pass Canadian screening criteria, which would provide 1,575 donations. The demand for DI by women was 7,866 samples .Considerable effort would be necessary to create the required increase in awareness of the program and change in societal behaviour towards sperm donation for an ASD program to be feasible in Canada. Since 2000, there has been a marked decline in the supply of donor sperm provided by Canadian men. This decline coincides with the introduction of new, more stringent regulations for donor screening and sample testing in 2000, and the proclamation of a criminal law prohibiting payment for donor gametes in 2004. Despite this law, and with limited altruistic alternatives, the importation of sperm from paid donors in the US and Europe has been sanctioned by Health Canada. Since an altruistic donation program is required under the new legislation, the current study was designed to model the number of altruistic donors would be required to satisfy the demand for donor sperm in Canada.Two population-based, top-down mathematical models were developed to estimate both the supply and demand for donor sperm. “Supply” factors included: population (age and location), ethnic origin, health policy criteria, donor behaviour, and medical eligibility. “Demand” factors included: estimates of utilization by single women, lesbian couples and heterosexual couples.The evidence-based analysis suggested that only 63 donors would pass Canadian screening criteria, which would provide 1,575 donations. However, the demand for donor insemination was estimated to be 7,866 samples. This results in a shortage of 6,291 donations or 252 donors. These analyses suggested that sperm supplied by donors from other countries is still necessary. The assumptions used in these models suggested that an altruistic program was unlikely to be sustainable unless significant changes occur in the awareness and societal behavior towards sperm donation.Using sperm from a third party to overcome infertility is an important option for many women. For some heterosexual couples and most single and lesbian women seeking to conceive, sperm from a fertile, healthy and carefully screened “donor” male remains the most appropriate choice. In particular, for women without a male partner, donor insemination (DI) provided through medical means is the safest and most effective option available. As a reflection of this, single and lesbian women now generate the largest demand for DI .Assisted Human Reproduction Act in 2004 which prohibited “payment for gametes” , 2313, 2n = 4,307,710). It was assumed that 25% of these men would be aware of the need for altruistic sperm donors. Of those, 15% of men would be willing to donate their sperm and 3% would present themselves for all necessary screenings and sample collections. The model assumed that 1.3% percent of men would pass the medical eligibility criteria and 25 allowable live births  .The base-case model predicted that a total of 63 sperm donors  providing 1,575 donations would be available Table  resultinThe best-case analysis predicts a total of 3,797 Canadian sperm donors representing 0.0881% of the eligible male population. In this analysis, the demand for donor insemination remained the same as the base-case analysis Table . Using tThe worst-case scenario, where the availability of sperm donors remains the same as currently available and the donor insemination demand maximized, the total demand by women seeking donor insemination would come from 32,412 women or 0.58% of the eligible population  can be modified through changes in health policy or recruitment strategies. The current guidelines regarding health policy put very few social restrictions on the type of men who are eligible to donate sperm, with the only major exceptions being age and being from a high-risk of human immunodeficiency virus (HIV) population . If Canada were to adopt a policy similar to that of France and require donors to be married and have at least one offspring, it would significantly exacerbate the shortage of potential sperm donors. Male behaviour also appears to be a key factor that affects supply: even with the best-case estimates, the potential pool of sperm donors was 0.0881% of the eligible population, with one of the largest barriers to sperm donation being the inconvenience of donating . Del ValThere have been a variety of methods used in other jurisdictions in order to increase the number of sperm donors, including: 1) asking couples seeking donor insemination to recommend to family and friends the need for sperm donors; 2) asking patients that are about to undergo a vasectomy if they would consider donating sperm; 3) mirror gamete donation, where the partner with usable gametes donates in exchange for the gametes needed ; and, 4) media campaigns to increase awareness of altruistic sperm donation –30. WhetSeveral limitations of this study exist. Firstly, the model assumes that estimates obtained from the literature are reflective of Canadian values and attitudes. Some of the parameters in the analyses were based on studies conducted in other countries. In addition, several parameters, such as Canadian male behaviour towards altruistic sperm donation, require further study. Due to the limited amount of primary research on this topic, all studies that helped identify appropriate variable estimates were used. Furthermore, it was assumed that estimates used for behaviour variables were independent of ethnicity and geography. This may be inaccurate since there is evidence in the literature indicating that certain ethnic groups are more willing to donate than others and there may be differences between people’s propensity to donate from rural rather than urban regions –33. SomeAnother factor that was not accounted for in the model was whether having the option of being an anonymous or non-anonymous donor affects the availability of donor sperm. Surveys of sperm donors’ attitudes and motivations toward donor anonymity conducted in Denmark  found thThe analysis presented here did not consider the effect of payment on male behaviour towards sperm donation given that payment for sperm donation is illegal in Canada. Evidence suggests that payment does change male willingness to donate, especially among younger donors –39. A stThe model created estimated that the potential Canadian donor availability within an altruistic donor sperm program was only 63 donors. Assuming a maximum of 25 live births per donor, only under the best-case scenario with optimized awareness could a Canadian altruistic sperm donation program meet the expected demand. It is expected that the demand will increase due to the increase in funding that provincial governments are infusing into in vitro fertilization.


Pythium insidiosum, causes the life-threatening infectious condition, pythiosis, in humans and animals worldwide. Affected individuals typically endure surgical removal of the infected organ(s). Detection of P. insidiosum by the established microbiological, immunological, or molecular methods is not feasible in non-reference laboratories, resulting in delayed diagnosis. Biochemical assays have been used to characterize P. insidiosum, some of which could aid in the clinical identification of this organism. Although hydrolysis of maltose and sucrose has been proposed as the key biochemical feature useful in discriminating P. insidiosum from other oomycetes and fungi, this technique requires a more rigorous evaluation involving a wider selection of P. insidiosum strains. Here, we evaluated 10 routinely available biochemical assays for characterization of 26 P. insidiosum strains, isolated from different hosts and geographic origins. Initial assessment revealed diverse biochemical characteristics across the P. insidiosum strains tested. Failure to hydrolyze sugars is observed, especially in slow-growing strains. Because hydrolysis of maltose and sucrose varied among different strains, use of the biochemical assays for identification of P. insidiosum should be cautioned. The ability of P. insidiosum to hydrolyze urea is our focus, because this metabolic process relies on the enzyme urease, an important virulence factor of other pathogens. The ability to hydrolyze urea varied among P. insidiosum strains and was not associated with growth rates. Genome analyses demonstrated that urease- and urease accessory protein-encoding genes are present in both urea-hydrolyzing and non-urea-hydrolyzing strains of P. insidiosum. Urease genes are phylogenetically conserved in P. insidiosum and related oomycetes, while the presence of urease accessory protein-encoding genes is markedly diverse in these organisms. In summary, we dissected biochemical characteristics and drew new insights into clinical identification and urease-related evolution of P. insidiosum.The oomycete microorganism,  Pythium insidiosum), which has been increasingly reported in tropical and subtropical countries  and morphologically similar fungi. Although the use of these biochemical assays in the clinical identification of P. insidiosum is promising, it requires further evaluation with a more extensive selection of P. insidiosum strains.Biochemical assays may be used to characterize sidiosum . Recentlsidiosum . They prP. insidiosum. The strains tested had different geographic origins  and were isolated from different hosts   . Initialoformans . Since tvailable , we wereThis study was approved by the Committee on Human Rights Related to Research Involving Human Subjects, at the Faculty of Medicine, Ramathibodi Hospital, Mahidol University .P. insidiosum isolated from humans (n = 14) or equines (n = 10) with pythiosis and from the environment (n = 2), were available for this study , Biosafety Level 2 precautions were followed throughout this study (https://www.cdc.gov/biosafety). All of the organisms were retrieved from stock cultures, and maintained on Sabouraud dextrose (SD) agar at 37 °C for at least three passages. SD agar plugs (5 mm in diameter) from one-week-old, actively growing cultures of P. insidiosum were then prepared  from yellow to pink (urease assay); (ii) from brown to black (esculin hydrolysis assay); (iii) from green to blue (citrate hydrolysis assay); (iv) from dark blue to yellow ; and (v) from blue to colorless (DNase assay). All biochemical assays were performed in duplicate.To set up biochemical assays, 10 different routinely available agars were each prepared in test tubes , using ingredients purchased from BD Difco and BBL (if not stated otherwise), and the recommended protocols of the manufacturers. These agars included: urea agar (urease assay), Simmons’citrate agar (citrate hydrolysis assay), bile esculin agar (esculin hydrolysis assay), DNA agar (DNase assay), and purple agar base (sugar hydrolysis assay) with 2% (wt/v) dextrose, lactose, maltose, sucrose , trehalose , or xylose. A 5 mm diameter agar plug of an actively growing colony of each P. insidiosum, 19 related oomycetes, and two diatoms (https://blast.ncbi.nlm.nih.gov/).The Oomycete Gene Table is an online comparative genomic analysis tool, derived from sequence similarity based gene grouping of the genome sequences of  diatoms  ; NP_850239 ; NP_173602 ; and NP_180994 ) , and two Illumina-derived genomes of the P. insidiosum strains Pi07  and Pi45 (http://www.ncbi.nlm.nih.gov/) and the cut-off E-value ≤10−6.The urease and urease accessory protein sequences of the plant , UREG))  were retand Pi45 , using tP. insidiosum , related oomycetes, and diatoms (outgroup)   was execogeny.fr . In brieogeny.fr . Poorly ogeny.fr . Phylogeogeny.fr . The phyogeny.fr .P. insidiosum identified in the genomes of P. insidiosum strains Pi35 , Pi07 (accession number LC325168 for Ure1), and Pi45  have been submitted to the DDBJ database.Sequences of the putative urease genes of P. insidiosum included in the current study were derived from different sources  and geographic origins . Based on the growth rates, P. insidiosum can be divided into two groups: (i) fast-growing strains , and (ii) slow-growing strains  (Twenty-six strains of strains) . Each grP. insidiosum hydrolyzed esculin in the presence of bile but failed to breakdown citrate and two sugars . The majority of the strains can hydrolyze dextrose , maltose , sucrose , trehalose , and DNA , while those that cannot utilize these substrates were almost all slow-growing. Unlike the other fast-growing strains, Pi03 did not hydrolyze sucrose. With regard to the urease assay, 71% (n = 12) of the fast-growing and 78% (n = 7) of the slow-growing strains could catabolize urea. Biochemical characteristics of some representative strains at day 0  and day 2 post-inoculation  were displayed in As summarized in A. invadans, which contained three copies of this gene  in the genomes of three representative P. insidiosum strains  the clade of Aphanomyces and Saprolegnia species; and (iii) the clade of Albugo species. Most of the organisms contain one copy of the urease-encoding gene, except A. invadans (three copies) and P. insidiosum strain Pi45 (two copies). Four urease-encoding sequences from the P. insidiosum strains Pi07, Pi35, and Pi45 were grouped together, and placed more proximally to non-insidiosum Pythium, Phytophthora, Phytopythium, and Hyaloperonospora species than to other oomycete species.A set of 24 urease-encoding sequences identified in the genomes of  diatoms , were su diatoms : (i) theP. insidiosum (P. insidiosum strains tested (including the strains CBS 574.85 and ATCC 28251 of the current study) could utilize urea and these sugars. This is especially important considering maltose and sucrose are two key sugars that were thought to differentiate P. insidosum from other pathogenic oomycetes and fungi .A capacity to hydrolyze esculin, but not citrate, lactose and xylose, was the shared biochemical characteristic found in all 26 strains of sidiosum , consistsidiosum . This find fungi . Failuresidiosum . BecauseP. insidiosum. Efficient breakdown of urea can be observed in many slow-growing strains , and not in all fast-growing strains   the urea-hydrolyzing, slow-growing, Clade-I strain Pi07; (ii) the urea-hydrolyzing, fast-growing, Clade-II strain Pi35; and (iii) the non-urea-hydrolyzing, fast-growing, Clade-III strain Pi45. All three strains contain Ure1 orthologous sequence, which significantly matched the plant Arabidopsis urease (URE) , suggesting that the presence of Ure1 genes in the genome is not necessarily associated with the ability to hydrolyze urea in P. insidiosum.Unlike the hydrolysis of sugars and DNA, the ability to utilize urea was not associated with growth rate in C 28251) . The inaArabidopsis urease ) are necessary for maturation and activation of the nickel-containing metalloenzyme urease . The ures urease . In addi strains  may be dP. insidiosum from all three phylogenetically distinct clades, although gene duplication could occur in some strains   containing just a few genes in the genera Hyaloperonospora, Albugo, Aphanomyces, and Saprolegnia.Genome analyses demonstrated that urease- and accessory protein-encoding genes are conserved in  strains . We usedutgroup) . The ureutgroup) , and theutgroup) . Howeverutgroup) , rangingP. insidiosum, cautioning the use of related biochemical assays for pathogen identification. Unlike the hydrolysis of sugars, the ability to hydrolyze urea was not associated with P. insidiosum growth, as many slow-growing strains, and not all fast-growing strains, can utilize urea, even though the urease- and accessory protein-encoding genes are present and highly conserved in both urea-hydrolyzing and non-hydrolyzing strains of P. insidiosum. Future investigations on expression and regulation of the urease and accessory protein-encoding genes could elaborate the urea metabolism and its potential role in pathogenicity in P. insidiosum. Gain and loss of urease and accessory protein-encoding genes occurred in the genomes of oomycetes and diatoms as their evolutions diverged. In the current study, we dissected several biochemical characteristics, and provided new insights into urease-based evolution of P. insidiosum.No unique biochemical characteristic is observed among different strains of 10.7717/peerj.4821/supp-1Supplemental Information 1E-values, identity, and similarity in reference to the urease of P. insidiosum) of each organism is summarized in the table.Information on species, strain, source of genome data, sequence identification number (ID) and gene ID (or position) for retrieving the urease sequence, protein length, and pairwise sequence alignment output , Pi07 (accession number LC325168 for Ure1), and Pi45  have been submitted to the DDBJ database.Sequences of the putative urease genes of Click here for additional data file.


Objective: Postoperative delirium (POD) is a frequent complication after surgery and its occurrence is associated with poor outcomes. The pathophysiology of this complication is not clear, but identification of risk factors is important for positive postoperative outcomes. The purpose of this study was to investigate the associations between the preoperative expression levels of microRNA (miR)-146a, miR-125b, and miR-181c in cerebrospinal fluid (CSF) and serum and the development and severity of POD.Methods: Forty elderly patients aged 65 years old and older admitted for elective total hip/knee replacement under spinal anesthesia. Preoperatively, baseline cognitive function was assessed using the Mini-Mental State Examination. Each patient was interviewed daily on the first and second postoperative days. Delirium was diagnosed using the Confusion Assessment Method, and delirium severity was measured using the Memorial Delirium Assessment Scale (MDAS). Preoperative serum and CSF miR levels were determined by quantitative real-time PCR (qRT-PCR).Results: POD was detected in 27.5% (11/40) of patients. Up-regulation of miR-146a and miR-181c in CSF and down-regulation of miR-146a in serum were observed preoperatively in patients who developed POD, while patients with and without POD did not differ in serum or CSF levels of miR-125b. Delirious patients had higher CSF/serum ratios of miR-146a and miR-181c levels than non-delirious patients. The lower CSF miR-146a and CSF/serum miR-146a ratios were significantly associated with milder POD severity, represented by a lower MDAS score.Conclusion: The dysregulation of preoperative miR-146a and miR-181c in CSF and serum was associated with the development and severity of POD. These NeurimmiRs might participate in the neuropathogenesis of POD, pending further investigations.Clinical trial registration: this study was registered at ClinicalTrials.gov (NCT02817386). Postoperative delirium (POD), an acute, transient, fluctuating disturbance in attention, cognition, and level of consciousness, is a common (15–53%) postoperative complication , and it Neuroinflammation plays a crucial role in POD . SurgicaMicroRNAs (miRs) are endogenous, short, non-coding RNAs. Mature miRs are single-stranded RNA molecules of ∼20-25 nucleotides that act as important post-transcriptional regulators of gene expression by binding with their target mRNAs . SeveralTherefore, the objective of this pilot study was to assess whether the preoperative expression levels of miR-146a, miR-125b, and miR-181c in cerebrospinal fluid (CSF) and serum were associated with POD. We hypothesized that preoperative expression levels of these NeurimmiRs in CSF and serum would be associated with the development and severity of POD. The findings of this investigation may be helpful to create a correlation between the NeurimmiRs expression levels and development of POD, which would promote more studies to investigate the role of NeurimmiRs in the neuropathogenesis of POD and facilitate more miRs biomarker studies of POD.This study was performed in accordance with the Declaration of Helsinki and was approved by the research ethics committee of Zhongnan Hospital of Wuhan University. With written informed consent, we performed a prospective, observational study, which was registered at ClinicalTrials.gov (NCT02817386).Figure 1, flow diagram). After reviewing patient medical records, patients were excluded if they had (1) a past medical history of neurological or clinically evident neurovascular disease ; (2) inability to read or severe visual or auditory deficits; (3) Mini-Mental State Examination (MMSE) scores of 26 or less; (4) American Society of Anesthesiologists (ASA) score  greaterEach patient was interviewed preoperatively on the first and second postoperative days. The MMSE was administered one day before the scheduled surgery. The assessment of delirium was performed on the first and second days after surgery between 8:00 am and 10:00 am. A visual analog scale (VAS) score of 0–10  unless clinically indicated. All the details of clinical care were documented in case report forms.g for 10 min in 4°C for the collection of supernatants, followed by centrifuging again at 12,000 g for 10 min in 4°C for the collection of pure serum  were collected in additive-free vacuum blood tubes from the enrolled patients before anesthesia. Then, serum specimens were isolated by two steps of centrifugation. The blood samples were centrifuged at 3000 re serum , which wThe CSF (4 ml) was collected in an RNase-free Eppendorf tube during spinal anesthesia prior to administration of the local anesthetic. The samples were centrifuged immediately at 3000 rpm at 4°C for 10 min to remove cells , and onlFigure 2A), was used as an endogenous control for the validation of all the selected candidate miRs. The primer sequences were obtained from Invitrogen Biotechnology Co., Ltd  and are listed in Table 1. In the preliminary experiment, the single peak was observed in each melting curve for candidate miRs and U6 snRNA, which indicate good primer specificity. Quantitative real-time PCR (qRT-PCR) was performed using a StepOne real-time PCR system  with the SYBR Premix Ex Taq  according to the manufacturers’ instructions. Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to the internal control U6. miR expression levels were obtained by relative quantification using the 2-ΔΔCt method. Findings greater or less than 1 were considered to indicate overexpression or underexpression, respectively. All the values were standardized relative to the non-POD values, which were represented as a value of 1.We extracted total RNA from 2 ml of CSF and 300 μl of serum using TRIzol reagent  in accordance with the manufacturer’s protocol. The RNA concentration and purity were detected using a NanoDrop ND-1000 spectrophotometer   quartile of NeurimmiRs levels and the combination of the participants in the second, third, and fourth (highest) quartiles of miR levels. Finally, we applied simple linear regression to determine the associations between the NeurimmiRs levels in CSF and/or serum and MDAS scores, and we used multiple linear regression to determine the associations after adjustment for age, sex and total RNA concentration. The regression coefficient ± standard error (SE) was used to illustrate the association between NeurimmiRs levels and MDAS scores.The data are expressed as the mean ± standard deviation (SD), the median and interquartile range  or a number (%). POD incidence is presented as a percentage. The Kolmogorov–Smirnov method was used first to test the normality of all the variables. The Mann–Whitney P < 0.05. SPSS statistical software, version 21.0 , and GraphPad Prism software, version 6.01 , were used for data analysis.Because this was a pilot study, no power calculations were performed; instead we aimed to recruit the maximum number of patients possible with the resources available, which we estimated to be 40. Statistical significance was set at Figure 1. Therefore, 40 patients (n = 40) remained for analysis. The demographic and clinical data of the participants are summarized in Table 2, and the characteristics of the excluded participants are shown in Supplementary Table Eighty-six eligible participants were screened, 52 of whom provided informed consent for the study. Twelve participants were excluded from the study. The reasons for dropouts are shown in n = 11 of the 40 patients). Five of 11 patients who developed POD were male. The median of the highest MDAS score of delirious patients was 13 (11–18) (median and 25–75 percentile), which was higher than that of the non-delirious patients . The median of the highest MDAS score of all the participants over the first two postoperative days was 7 (5–11). Because The incidence of POD observed at either of the two postoperative assessments was 27.5% ] was not significantly different from the score those who did not develop delirium .Postoperatively, the highest VAS scores did not differ between patients with delirium 2 (1–3) and without delirium [3 (2–4), P = 0.432).Thirty-seven patients received propofol during the operation for sedation. The mean dose of propofol for patients who developed POD (268.6 ± 33.4 mg) was not significantly different from those who did not develop POD . Therefore, it was used for normalization purposes in the current study. We compared the preoperative levels of three candidate NeurimmiRs  in CSF and/or serum in the participants with POD and those without it. The Mann–Whitney test showed that patients with delirium had a higher CSF miR-146a level, a lower serum miR-146a level, and a higher CSF/serum miR-146a ratio compared to non-delirious patients . However, no significant difference was observed between patients with or without delirium with regard to miR-125b levels in CSF and serum . Additionally, for patients who ultimately developed POD, the CSF level of miR-181c, as well as the ratio of CSF/serum miR-181c, was significantly increased compared to patients who did not develop delirium . However, patients with and without POD did not differ statistically with regard to the serum levels of miR-181c .U6 has been used before as an internal control in CSF  and seruFigure 3A) than those of the patients in the combination of the second, third, and fourth quartiles of CSF miR-146a. Similarly, the highest MDAS score of the patients in the first quartile of CSF/serum miR-146a ratio was lower than that of the patients in the combination of the second, third, and fourth quartiles of CSF/serum miR-146a ratio . Conversely, the highest MDAS score of the patients in the first quartile of serum miR-146a was higher than that of the patients in the combination of the second, third, and fourth quartiles of serum miR-146a . However, no significant difference in the highest MDAS score was observed between the patients in the first quartile and the patients in the combination of the second, third, and fourth quartiles of CSF miR-181c or CSF/serum miR-181c ratio .Next, we investigated whether the levels of miR-146a and miR-181c in CSF and serum, as well as the ratios of CSF/serum miR-146a and miR-181c, were associated with POD severity. MDAS has been used to assess the severity of delirium symptoms based on 10 features . In the Supplementary Figure S1) and using unadjusted simple linear regression, we found that the preoperative CSF level of miR-146a, as well as the ratios of CSF/serum miR-146a and miR-181c, were significantly correlated (positively) with the highest MDAS score. However, the preoperative serum level of miR-146a was significantly correlated (negatively) with the highest MDAS score (Table 3). Multiple linear regression, after adjusting for age, sex, and total RNA concentration, showed that the preoperative CSF miR-146a level, as well as the ratios of CSF/serum miR-146a and miR-181c, remained significantly correlated (positively) with the highest MDAS score (Table 3).Then, we assessed the relationship between the NeurimmiRs levels in CSF and/or serum and MDAS scores. The data were fit by linear regression analysis  in CSF and serum and POD in 52 older adults who underwent total hip and knee replacement under spinal anesthesia. We found up-regulation of miR-146a and miR-181c in CSF and down-regulation of miR-146a in the serum of patients who developed POD. Additionally, the delirious patients had higher CSF/serum ratios of miR-146a and miR-181c levels than the non-delirious patients. We also found that lower CSF miR-146a and CSF/serum miR-146a ratios were significantly associated with milder POD severity, as represented by a lower MDAS score. Taken together, these findings suggested that miR-146a and miR-181c might participate in the neuropathogenesis of POD, pending further investigation.Previous studies have demonstrated that miRs are stably expressed in various body fluids , and theThe incidence of POD following total joint replacement in our study (27.5%) was within the reported range of 3.6–41% . For exaDespite an increase of studies focused on the identifications of POD risk factors, the POD molecular mechanisms are still largely unknown  and notaIncreasing evidence has demonstrated the involvement of miR-146a in the regulation of inflammation in human neurological disorders. In physiological conditions, transcription of miR-146a occurs at baseline levels; however, initiation of proinflammatory Toll-like receptor (TLR) signaling immediately results in strong co-induction of its expression through a mechanism that is largely NF-κB-dependent . In addiIn our study, the up-regulation of miR-146a and miR-181c in CSF and the down-regulation of miR-146a in serum were observed in patients who developed POD, while no difference was observed between patients with or without POD with regard to miR-125b levels in CSF and serum. Up-regulation of miR-146a has been reported in the brains of human beings and in mouse models of prion disease , consistIn comparison with sevoflurane anesthesia, propofol anesthesia has been shown to be associated with a lower incidence (6.9%) of POD in elderly patients . In the In the current study, we not only applied the MMSE to exclude participants with dementia, but we also compared differences in the MMSE at baseline between the participants with or without POD. We found that, for patients who subsequently developed POD, the preoperative MMSE score was not significantly different from that of subjects who did not develop delirium. However, it is known that prior cognitive impairment is a major risk factor for delirium . MoreoveThere were some potential limitations of our study. First, the current pilot study was not sufficiently powered to provide strong evidence for the role of NeurimmiRs in POD due to the small sample size and high dropout rate. A larger study with adequate power is indicated to validate our results. Second, we removed the cells from the CSF according to the methods as described by In summary, dysregulation of preoperative miR-146a and miR-181c in CSF and serum was associated with the development and severity of POD. To date, it remains unknown whether there is a causal relationship or an association between these NeurimmiRs expression levels and the development of POD. Hence, further studies are required to clarify this question. These studies will also hopefully facilitate miRs biomarker studies of POD.RD contributed to study design, data collection, statistical analysis, and manuscript preparation. LS performed qRT-PCR. YL involved in data collection. XY performed neuropsychological testing. MP contributed to study concept and design, manuscript preparation and review. ZZ performed statistical analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.