Environmental fluctuations can generate asynchronous species’ fluctuations and community stability, due to compensatory dynamics of species with different environmental tolerances. We tested this hypothesis in intertidal hard-bottom communities of north-central Chile, where a persistent upwelling centre maintains a mosaic in sea surface temperatures (SST) over 10s of kilometres along the shore. Coastal upwelling implies colder and temporally more stable SST relative to downstream sites. Uni- and multivariate analyses of multiyear timeseries of SST and species abundances showed more asynchronous fluctuations and higher stability in sites characterised by warmer and more variable SST. Nevertheless, these effects were weakened after including data obtained in sites affected by less persistent upwelling centres. Further, dominant species were more stable in sites exposed to high SST variability. The strength of other processes that can influence community stability, chiefly statistical averaging and overyielding, did not vary significantly between SST regimes. Our results provide observational evidence supporting the idea that exogenously driven compensatory dynamics and the stabilising effects of dominant species can determine the stability of ecosystems facing environmental fluctuations. Forecasted scenarios of species loss, ecological phase shifts, and global environmental change have fuelled extensive research on community stability z, determines the contribution of statistical averaging to stability, with 1<z<2 meaning that increasing diversity increases and dampens population- and community-level variability, respectively z values <2 and disproportional increases of the temporal mean of an aggregate property can be caused by fundamental ecological mechanisms such as negative species interactions and resource complementarity, respectively The stabilising mechanisms listed above depend on the magnitude and type of temporal variation in species abundances. For example, asynchronous and compensatory dynamics of competing species with differential environmental tolerances can maintain stability, because the decrease of stress-intolerant species can be compensated by growth of others , where a persistent upwelling centre, extending ∼100 km, maintains significant mesoscale differences in climatic and oceanographic conditions This study was conducted as part of the activities carried out by the Changolab at the Centro de Estudios Avanzados en Zonas Áridas (CEAZA) and approved by its bioethics committee. In addition, the study conforms to the Art. §7 Law 19.300 “Bases Generales del Medio Ambiente”, which supports research on ecology and environmental science. No specific permits were required for the described field studies, because they were based on non-destructive estimations of species abundances and only few specimens  were collected when in situ identification was unfeasible. These species were not among endangered or protected species and locations were not privately-owned or protected by law.Observations were conducted on the north-central rocky shores of Chile, around a persistent upwelling centre . Punta LWe selected four sites that differed in their position relative to the PLV upwelling centre . The souMazzaella laminarioides and the large bull-kelp Durvillaea antarctica find their northern geographic range limit around this zone Perumytilus purpuratus, characteristic on the mid intertidal zone of south-central shores, become scarce and are replaced by increased empty space and patches of chthamalid barnacles Ulva compressa, U. rigida, and Pyropia sp. are persistent across the entire study region Scurria spp.), chitons, and keyhole limpets (e.g. Fissurella crassa). In the low intertidal zone, the kelp Lessonia nigrescens and the bull kelp Durvillaea antarctica form dense canopies and support a diverse assemblage of mobile invertebrates, including chitons like Enoplochiton niger and Chiton granosus, keyhole limpets like Fissurella limbata, and turban snails like Tegula spp. and Prisogaster nigerThe study region is characterised by spatial variation in terms of composition and abundance of species. For example, the dominant corticated red alga 2 plots located along ca. 20 m alongshore transects. The position of each plot was permanently marked with stainless-steel bolts and rings. Plots were haphazardly positioned, but positions were restricted to flat and gently sloped surfaces lacking crevices and tide pools. This protocol has been used in several studies on benthic diversity along Chilean coasts and elsewhere  species was estimated on each plot every three months between May 2009 and February 2012. Estimations were conducted during daytime low-tide intervals and to the nearest 1% by means of 0.25 mOur study represented a conservative estimate of the biodiversity at each site, because observations were focused on macro-epibenthic organisms for which appropriate taxonomic keys were available and therefore were possible to accurately be identified in the field in situ measurements of sea surface temperature (SST) to characterise the environmental variability. Between August 2009 and August 2011, we deployed at each site temperature data-loggers that recorded SST every 10 minutes. Each data-logger was enclosed into a PVC pipe, which was inserted into a concrete block moored at 1-m depth We used The magnitude of environmental fluctuations in each site was estimated using temporal variances calculated from the two-year daily SST measurements where the few missing observations in the time series were interpolated using a cubic spline. Temporal variances were calculated separately from daily to trimonthly temporal scales  using a running-mean filter.In order to illustrate multivariate patterns in community structure across sites, we used canonical analysis on principal coordinates CAP . CAP is S = μ/σ) of total percentage cover; i.e., the sum of the abundances of all species in the assemblage μ and σ were formally analysed to address whether mesoscale variation of stability was related to variations of mean abundances or temporal variability.Stability has several meanings in ecology, which include concepts like the magnitude of disturbances a system can tolerate (domain of attraction), the magnitude of changes driven by a disturbance (resistance), how long a the system requires to return to a specified fraction of its initial state (resilience) and how much a measure varies over time (variability) xφ statistic xφ describes the variance in the sum of all species abundances  relative to the squared sum of the standard deviations of all N individual species (ix). xφ is standardised between 0 (perfect asynchrony) and 1 ; and is independent of the magnitude and distribution of species abundances and variances, allowing quantitative comparisons of communities with different species richness. The xφ statistic uses the fact that the variance of an aggregate property can be partitioned into the sum of all species variances plus the sum of all pair-wise species covariances In order to measure community-wide synchrony in species abundances, we used the D is the Simpson dominance index, x is the relative abundance of the sth species, and S is the number of species in the sample. Statistical averaging effects depend on how the temporal variance in the abundance of a species changes with its temporal mean σ2 with the mean μ is described by Taylor’s power function, σ2 = z,c µ where c is a constant and z is the scaling coefficient z affects the strength of the statistical averaging, with 1<z<2 meaning that diversity increases and dampens the population- and community-level variability, respectively σ2 = zc µ results in a linear equation in the form of log (σ2) = c+z log (μ). We fitted this linear relationship to the entire dataset, combining all species. The z-values obtained from each regression were used as dependent variables. In addition, regression residuals for each species were used to estimate species-level stability, and thus infer whether species with larger percentage covers (i.e. dominant species) were more stable; larger negative residuals indicated higher species-level stability r) between residuals and the long-term means of species’ percentage covers. Significance was tested for each site by testing observed correlation coefficients against a null distribution of r coefficients generated from 10000 randomisations P≥0.25) error terms were removed and F-ratio statistics were then recalculated with a pooled denominator S and xφ were log10 transformed, and those of σ were square root transformed.We used separate 2-way nested analyses of variance (ANOVAs) with ‘region’ (2 levels: either north or south PLV) as a fixed factor and ‘site’ (2 levels) as a random factor and nested in ‘region’ to test the prediction that sites with different degrees of temporal variation in SST show significant differences in stability and the stabilising mechanisms. After running the full models, highly conservative (2 plots on a regular basis. Most sites were sampled every six months from 1998 to 2000 and then from 2003 to 2005 (see 2σ) by means of restricted maximum likelihood (REML). The regional trend in variance components was removed after calculating the residuals from locally weighted regression scatterplot smoothing (LOWESS) against latitude. The hypothesis that stability  differs between sites exposed to warmer and colder SST was tested with a 1-way ANOVA with ‘SST’  as fixed factor and the residuals of variance components as dependent variable. SST analyses were conducted in Matlab . All other estimations and statistical analyses were conducted in R 2.15.0 In addition to the datasets described above, we analysed a multiyear (1998–2005) dataset of species percentage covers estimated in 17 sites and spanning ca. 733 km of the coast (29.47°S–36.07°S). In each of these sites, species percentage covers were estimated on randomly located 0.25 m2005 see . Each siOur observations showed that SST was higher at Panul  and Guanaqueros , both sites located north of Punta Lengua de Vaca (PLV); than at Limarí  and Punta Talca , both located south of PLV. Temporal variances of SST were, on average, 50% larger at Panul and Guanaqueros that at Limarí and Punta Talca . The dif3, 396 = 60.059, P = 0.001). Pairwise comparisons and CAP ordinations , was significantly higher at northern sites .Community stability, measured as the ratio between the long-term mean and standard deviation in total community cover  was significantly larger north than south of PLV (σ) was significantly smaller at northern than southern sites  was related to higher stability and lower species synchrony (thus strengthened compensatory dynamics), but it was unrelated to other stabilising mechanisms such as statistical averaging and overyielding. Dominant species were significantly more stable than subordinate ones only in sites exposed to high SST variability, suggesting that particularly stable dominants may also have influenced community stability. Our findings agree with theoretical and empirical evidence for a positive effect of environmental fluctuations on compensatory dynamics. We suggest that ecological mechanisms evoking differential species’ responses to environmental fluctuations  and decreased standard deviation (σ) of total community cover. The marginal difference in μ between upwelling regimes suggests that decreased temporal variability at sites affected by low upwelling intensity was more important. Species dominance, statistical averaging, and overyielding did not vary between environmental regimes, indicating that reduced synchronisation in species abundances can be associated to lower temporal variability Following the stability metric we chose S = μ/σ,, higher The differences in terms of stability and compensatory dynamics between environmental regimes can be explained in the light of differential amounts of temporal variability, assuming that climate and broad-scale processes strongly influence population synchrony Heliaster helianthus; The effects of dominant species did vary between warm and cold sites. Only in the northern sites, residuals from Taylor’s power law estimated from dominant species were significantly lower than those estimated from subordinate species. This indicates that dominant species were less variable over time in sites exposed to warm and variable SST, and that such dominant species may have influenced the community-wide stability north Punta Lengua de Vaca. These communities were dominated by chthamalid barnacles, which have a complex life cycles with a planktonic stage that might last for several weeks, allowing barnacles to rapidly colonise disturbance-generated patches of empty substratum Mazzaella laminarioides’ leathery morphology can generate understorey environmental conditions different from the overall environment, and thus positive covariance among obligate understorey species as seen in other temperate shores Lithothamnion spp. are positively correlated with that of corticated algae M. laminarioides. These positive relationships might have contributed with synchronous species dynamics and thus with lower stability in both sites south PLV. Upwelling intensity, however, can dampen positive interactions, probably due to enhanced nutrient concentrations. Manipulative evidence shows that the turf-forming alga Gelidium chilense has stronger facilitative effects on recruitment of P. purpuratus on areas downstream of upwelling sites Following the negative relation between recruitment of chthamalid barnacles and upwelling intensity Here, we have presented multiyear observational evidence for a relationship between environmental fluctuations and community stability. We have observed contrasting structural configurations in low- vs. high-upwelling sites, defined by life histories that respond to different scales of environmental variation: life-histories that respond to broad-scale processes  dominated low-upwelling sites, while life-histories that respond to fine-scale environmental variation dominated high-upwelling sites. Our results indicate that higher environmental variability might lead to stronger compensatory dynamics and thus to higher community-wide stability. Such a relationship has been largely proposed by theoretical models and laboratory-based experiments, but rarely by field observations that integrate natural filters and broader environmental conditions. Still is necessary to test for differences in environmental tolerances between sites experiencing different levels of environmental fluctuations; manipulative experiments seem ideal to test if this assumption holds at the mesoscale. Our results support the idea that exogenously driven compensatory dynamics, in addition to stabilising effects of dominant species, can determine the stability of ecosystems facing environmental fluctuations.Table S1Summary of sampling sites used to estimate variance components (REML) of total community cover. Species percentage covers were estimated seasonally in each site from 1998 to 2005 (Sampling year). *Sites were categorised as experiencing warm or cold sea surface temperatures (SST), according to satellite imaging and in situ records (DOC)Click here for additional data file.


Variability in patients' postoperative pain experience and response to treatment challenges effective pain management. Variability in pain reflects individual differences in inhibitory pain modulation and psychological sensitivity, which in turn may be clinically relevant for the disposition to acquire pain. The aim of this study was to investigate the effects of conditioned pain modulation and situational pain catastrophizing on postoperative pain and pain persistency.Preoperatively, 42 healthy males undergoing funnel chest surgery completed the Spielberger's State-Trait Anxiety Inventory and Beck's Depression Inventory before undergoing a sequential conditioned pain modulation paradigm. Subsequently, the Pain Catastrophizing Scale was introduced and patients were instructed to reference the conditioning pain while answering. Ratings of movement-evoked pain and consumption of morphine equivalents were obtained during postoperative days 2–5. Pain was reevaluated at six months postoperatively.Z = 1.0, P = 0.3) or level of catastrophizing . In the acute postoperative phase, situational pain catastrophizing predicted movement-evoked pain, independently of anxiety and depression  whereas conditioned pain modulation predicted morphine consumption .Patients reporting persistent pain at six months follow-up (n = 15) were not significantly different from pain-free patients (n = 16) concerning preoperative conditioned pain modulation response (Preoperative conditioned pain modulation and situational pain catastrophizing were not associated with the development of persistent postoperative pain following funnel chest repair. Secondary outcome analyses indicated that conditioned pain modulation predicted morphine consumption and situational pain catastrophizing predicted movement-evoked pain intensity in the acute postoperative phase. These findings may have important implications for developing strategies to treat or prevent acute postoperative pain in selected patients. Pain may be predicted and the malfunctioning pain inhibition mechanism as tested with CPM may be treated with suitable drugs augmenting descending inhibition. Pain is an expected part of surgical recovery but effective pain management remains challenging dispositional catastrophizing , which probably measures different pain experiences than situational catastrophizing  Endogenous pain modulation has been experimentally investigated in humans via conditioned pain modulation (CPM) paradigms, during which central inhibition of a painful stimulus is induced by applying a second painful conditioning stimulus to a remote body region  To the best of our knowledge, no studies have yet combined preoperative assessment of CPM with measurements of cognitive and emotional processes in the prediction of postoperative pain. Such a study would add to our understanding of the causes underlying individual differences in pain and potentially enable identification of patients at high risk of developing persistent postoperative pain. In turn this may have important implications for developing multimodal pharmacological or psychological intervention strategies to treat or prevent postoperative pain in the future. We hypothesized that patients with a preoperative deficient endogenous pain inhibition capacity, expressed by less efficient CPM and/or high levels of situational pain-related catastrophic thinking, were more susceptible to postoperative pain and pain persistency.The current study was a prospective observational cohort study designed to investigate whether preoperative assessment of endogenous pain inhibition capacity can predict the course of postoperative pain in a consecutive cohort of patients undergoing surgical correction of funnel chest (pectus excavatum). Funnel chest is the most common congenital deformity of the anterior chest wall and the male/female ratio is approximately 6/1 for patients undergoing surgical correction of funnel chest at our institution. The deformity is primarily treated to achieve anatomical correction and thus avoid cosmetic and psychological inconveniences for the patient. Patients undergoing surgical funnel chest repair are generally healthy pain-free opioid naïve adolescents. However, they are at high risk of experiencing moderate to severe acute postoperative pain.A schematic illustration of the study protocol is depicted in The Regional Committee on Biomedical Research Ethics (M-20110064) and the Danish Data Protection Agency (J. no.: 2011–41–6061) approved the study. The study was registered in the clinicaltrials.gov database  and was conducted in accordance with the Declaration of Helsinki. Patients admitted to surgery received written information explaining the purpose of the study along with the appointment for surgery. Upon arrival at the department, the principal investigator (K.G.) restated the study purpose verbally to eligible patients. All participating patients provided written informed consent. Of note, the 15–17-year-old patients received special information adapted to their age and abilities, both in language and in content. To further aid decision-making, parents, next of kin, caretakers, or guardians who had parental rights and responsibilities of underage patients, received separate information in writing and also participated in the conversation about the study purpose and possible participation of the young. The Ethics Committee granted special dispensation for the underage patients to provide written informed consent autonomously.Patients were recruited consecutively from the Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Denmark, during a pre-determined one-year period (May 2011 to May 2012). Patients were prospectively enrolled according to the following inclusion criteria: (1) elective minimally invasive surgical correction of funnel chest (pectus excavatum); and (2) age ≥15 years. Exclusion criteria were: (1) previous thoracic surgical interventions; (2) presence of diseases affecting the central and/or peripheral nervous system; (3) presence of chronic pain conditions; (4) inability to speak and/or understand Danish; (5) inability to understand and participate in the experimental pain session; (6) presence of psychiatric disorders; (7) history of frostbite in the non-dominant upper limb; (8) presence of sores or cuts on non-dominant upper limb; (9) presence of cardiovascular disease; (10) history of fainting and/or seizures; (11) presence of fractures of the non-dominant upper limb; or (12) presence of Reynaud's phenomenon. Secondary exclusions included: (1) insensitivity to experimental cold pressor pain; (2) lack of epidural catheter placement; or (3) re-operation.et al. was used to explain the reporting of pain intensity and unpleasantness using an 11-point numerical rating scale (NRS), where 0 =  no pain/unpleasantness and 10 =  worst pain/unpleasantness imaginable TM Brush-05, Somedic AB, Hörby, Sweden) and mechanical dynamic hyperalgesia using a single von Frey monofilament  as previously described by Stubhaug et al.All preoperative assessments were conducted by the principle investigator (K.G.), the day before scheduled surgery in a quiet room with a constant temperature of approximately 20–22°Celsius. Initially, patients were informed briefly about the purpose of the study, excluding any information about examination of CPM and the focus on pain catastrophizing, and asked to read and sign a consent form. Patients were then asked to fill in the Spielberger's State-Trait Anxiety Inventory (STAI) and Beck's Depression Inventory (BDI). Subsequently, a standardized written statement based on instructions published by Price Mechanical Pressure Pain Threshold – the Test Stimulus. Pressure algometry was performed at the patients' quadriceps muscle (rectus femoris) 10 cm above the patella on the same side as the dominant hand. A handheld digital pressure algometer with a probe size of 1 cm2 was used . The pressure was gradually increased with a rate of 30 kPa per second. Patients were instructed to press a button when the feeling of pressure changed into a sensation of pain. This in turn froze the assessment parameter, i.e., the pressure measured in kPa at the pressure pain threshold on the algometer's display and the value was instantaneously stored it in the internal memory of the apparatus. The algometer was calibrated between patients using a manufacturer-supplied weight equal to 100 kPa.Cold Pressor Test – the Conditioning Stimulus. Patients were asked to place their non-dominant hand in a stirred ice water bath (1±1°Celsius) in a still position with their fingers spread for as long as possible. A cut-off time of 120 seconds was set for safety reasons. The CPM effect was evaluated by comparing the baseline pressure pain threshold with the pressure pain threshold following the cold pressor test. In order to control for individual variation in baseline measures, the proportion of difference in pressure pain thresholds from baseline was used rather than the raw difference, and expressed in percentages. This approach results in negative CPM scores for pain facilitation and positive CPM scores for pain inhibition. In other words a positive CPM score was considered indicative of effective endogenous pain modulation The tendency to engage in pain catastrophizing was assessed by means of the Danish version of the Pain Catastrophizing Scale (PCS) Pain catastrophizing shares variance with negative affect constructs such as depression and anxiety. It is thus important to statistically control for depressive symptoms and level of anxiety when investigating relations between pain catastrophizing and pain-related outcomes  Anxiety was measured with the Danish version of the state part of the Spielberger's State-Trait Anxiety Inventory (STAI) for adults Depression was assessed with the Danish version of Beck's Depression Inventory (second edition) (BDI) consisting of 21 items assessing psychological and physiological aspects of depression over the preceding two weeks The primary outcome measure was the presence of persistent postoperative pain at six months, and the potentially predictive role of CPM on such pain. Persistent pain was assessed according to responses to the Brief Pain Inventory – Short Form (BPI) Postoperative movement-evoked pain was self-reported by patients in a study-specific pain diary based on NRSs. It is recommended to use NRSs in adult clinical trials −1 day−1Data on postoperative opioid use following epidural analgesia , independent of route or mode of administration was extracted from the electronic medical records or pain diaries if the patient was discharged before postoperative day 5. Missing data on morphine consumption on postoperative days 4 and 5 were acceptably imputed using the fixed daily morphine dose prescribed at discharge. Doses of opioid other than morphine were converted to morphine equivalents using standard equianalgesic dose conversion ratios  and calculated as mg kget al. It has been suggested that using pain scores and analgesic requirements as isolated variables may prevent identification of the total benefit provided by the analgesic regimen. Hence, an integrated analgesic assessment score was calculated as described by Silverman −1) and cisatracurium (0.1–0.15 mg kg−1).All patients followed a standardized anesthesia regimen. Prior to the induction of general anesthesia, an epidural catheter placed at the thoracic levels 4–6. With the patient sitting in an upright position, using local anesthesia and a paramedian approach, the catheter was placed using a hanging drop technique. The epidural block was activated and tested with an injection of 0.5% bupivacaine with simultaneous intravenous infusion of a 6% hydroxyethyl starch. General anesthesia was initiated with intravenous injection of fentanyl (0.05–0.1 mg), propofol . The standardized analgesic regimen combined epidural analgesia, and non-opioid and opioid analgesics as described below. This regimen has previously been linked to enhanced recovery in fast-track surgery Epidural Analgesia−1 for the first 24 hours and with 0.25% bupivacaine for the next 24 hours. Maximum epidural infusion rate was set at 10 ml h−1. Epidural analgesia was discontinued and the epidural catheter was removed at 9:00 on postoperative day 2 in all patients.The preoperative epidural blockade was reactivated during surgery or upon arrival in the Post Anesthesia Care Unit at the discretion of the attending anesthetist and subsequently provided continuously for an additional 48 hours. Epidural analgesia was provided with continuous infusion of 0.25% bupivacaine + morphine, 50 µg mlNon-opioid Analgesia−1) and ibuprofen (1200 mg day−1) from the day of surgery.Non-opioid analgesic treatment was initiated in all patients with acetaminophen .Rescue AnalgesiaIntermittent epidural bolus injections were given if the patient was uncomfortable due to pain . If the patient required additional boluses within the hour, the continuous basal infusion was either increased or the epidural catheter was retracted to optimize the spread of injected solutions and avoid fluctuations in the level of analgesia. Additionally, bolus intravenous injections of 2 mg of morphine followed by an upward titration in 1–2 mg increments were available as rescue analgesia throughout hospitalization.The size of the study population was based on a conservative estimate for a predetermined 1-year study period. We wanted to avoid overlooking an actual difference in CPM effect between pain-free patients and patients developing persistent postoperative pain at six months. Power calculation was based on results from previous studies showing that 14% of patients undergoing similar chest wall surgery developed persistent postoperative pain at six months P values less than 0.05 were considered statistically significant unless stated otherwise. Categorical variables are presented as numbers (percentages); continuous variables with a normal distribution are described with mean ± standard deviation; whereas non-normally distributed data is described with median [interquartile range], unless stated otherwise. Non-normally distributed data was attempted log-transformed to normality.Stata/IC 12.1 for Mac (64-bit Intel)  was used for all statistical analyses. Two-tailed t test was used to test the null hypothesis that conditioning cold pressor pain had no effect on the response to pressure algometry. Spearman's rank correlation coefficients (ρ) were calculated as measures of dependence between preoperative, perioperative, and postoperative study variables including predictors, potential confounders and outcomes. Considering the small sample sizes and obviously skewed data, the Wilcoxon-Mann-Whitney rank sum test was used for primary analyses to compare the distributions of CPM responses, S-PCS, BDI, and STAI scores in pain-free patients and patients reporting persistent postoperative pain at six months.A paired S-PCS]). Hence, for each of the three mentioned multiple regressions (i-iii), log[S-PCS] was entered in Model 1; CPM response in Model 2; and log[S-PCS], STAI score, and BDI score in Model 3.For analyses of secondary outcomes, we performed three separate ordinary least squares (OLS) linear regressions of (i) movement-evoked pain; (ii) morphine consumption; and (iii) the integrated analgesic assessment score on each of the continuous covariates: conditioned pain modulation (CPM) and situational pain catastrophizing (S-PCS). State anxiety and depression were solely chosen to control the effect of pain catastrophizing on postoperative pain-related outcome measures. Following initial residual analyses it was found appropriate to log-transform the S-PCS score , and leverage versus squared residuals. If the leverage versus squared residuals plot revealed an observation with high leverage and large residuals, we performed regressions with and without this outlier to assess its potential impact. Additionally, we conducted robust regressions in which influential observations were automatically dropped, , and those with large absolute residuals were down-weighted. Model validation did not suggest severe assumption violation and the conclusions were broadly similar regardless of using model-based or robust standard errors.In order to interpret and visualize the regression models, we calculated adjusted means (predictive margins) with 95% confidence intervals (95% CI) of the patient's postoperative movement-evoked pain intensity given different log-transformed values of preoperative situational pain catastrophizing and of postoperative morphine consumption given different values of preoperative CPM.We included 51 patients  in the experimental protocol . Preopert = −4.0, P = 0.0003) (P = 0.04) and the integrated analgesic assessment score . We found no evidence of associations between measures of postoperative pain intensity and analgesic consumption. In other words, patients reporting postoperative pain of high intensity do not necessarily consume more morphine, and vice versa.There was a 17% increase in the mean pressure pain thresholds before and after cold pressor test  . Change Thirty-one patients (74%) responded to our questionnaire assessment at six months after surgery . Of thesZ = 0.99, P = 0.3).The median CPM response in patients reporting pain at six months after surgery was 1.2% ; in pain-free patients it was 9.0% . Accordingly, there was no significant difference between the two groups .The distributions of S-PCS scores were not significantly different for patients reporting pain at six months after surgery and pain-free patients, with median S-PCS scores of 11  and 15 , respectively  would yield predicted means at 4.8  and 6.0 , respectively. Similarly, a CPM response of e.g. 25% would yield a predicted mean postoperative morphine consumption of 1.0  mg kg−1 day−1 using the simple regression model in which patients' morphine consumption was uniquely predicted from their preoperative CPM response  consumed more morphine than patients with efficient CPM (positive CPM scores), and vice versa.Contrary to some previous studies et al. showing that pain catastrophizing in response to experimental heat pain accounted significantly for the variance in cesarean section pain Our findings extend previous observations To date, very few studies have assessed the CPM effect as predictor of analgesic efficacy and results diverge Preoperative CPM and situational pain catastrophizing were not predictive of the proposed integrated analgesic assessment score. One possible explanation for this may be attributed to our co-administration of several analgesics. Despite the use of a standardized analgesic regimen, we cannot rule out that the administration of several analgesics may have resulted in differential synergistic effects across patients. Another explanation for this observation may be based on evidence that a combined endpoint may cause problems if the predictor effect is in opposite directions for different outcomes included in the composite endpoint Some of the limitations of the present study have implications for the generalizability of findings. Although we used a highly standardized surgical procedure and protocols for anesthesia and analgesia in a homogenous study population, inevitable differences in anesthesia, analgesia and surgically induced tissue injury and inflammation may have affected postoperative pain perception differently across patients. However, we found no evidence of significant associations between measures related to the anesthesia, analgesia, and complexity of surgery and study outcomes. Furthermore, patients in this study were primarily pain-free male adolescents who were not suffering from chronic pain before undergoing surgery. The applicability of the present findings to patients suffering from pain-related conditions thus remains uncertain. We acknowledge that our results are based on a small sample and that results need to be confirmed and replicated in larger studies. However, the homogeneous patient sample free from the usual bias related to pain, social and economical parameters, the uniform surgical procedure and the fact that all patients were operated on by the same surgeon and investigated by the same investigator strengthen our findings.In conclusion, preoperative CPM scores and levels of pain catastrophizing in response to experimental pain were not predictive of persistent postoperative pain development. Analyses of secondary pain-related outcomes, however, indicated that reduced pain-inhibition may contribute to enhanced clinical postoperative pain and morphine consumption. If replicated and confirmed in larger samples, this may potentially enable clinicians to tailor individualized analgesic drug administration and thus hypothetically improve postoperative pain management in future patients.


Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions. DNA methylation occurs at most CpG dinucleotides in the mammalian genome in vivoSomatic Cell Nuclear Transfer (SCNT) is a promising tool for animal breeding. It has been successfully used to generate cloned animals for both agricultural applications and development of animal models for human diseases in vivo fetuses develop to birth with average litter than in litters of cloned pigs, despite the fact that 80–100 blastocysts are transferred in the cloning procedure. The early cloned embryos with aberrant reprogramming might be selected out and thereby fail to establish pregnancy. Thus, it's of great interest to see whether cloned animals with normal phenotype possess abnormal DNA methylation.The reprogramming by DNA methylation during normal development takes place in a much longer time frame than in SCNT. In SCNT incomplete or aberrant reprogramming may happen and this is postulated to attribute to the low efficiency of cloning and the improper development of cloned animals sometimes observed To date, only a limited number of studies have addressed gene expression patterns and DNA methylation status for cloned pigs produced by SCNT, and no studies have addressed genome-wide gene expression and DNA methylation patterns in parallel. We have developed a high-throughput method, Modified Methylation Specific Digital Karyotyping (MMSDK), to detect genome-wide DNA methylation patterns by combining methylation-sensitive enzyme enrichment of unmethylated DNA tags and massively parallel tag-sequencing technology All procedures involving animals described in present study were reviewed and approved by the Danish Experimental Animal Inspectorate (“Rådet for Dyreforsøg”), Danish Ministry of Justice. Procedures for transfer to recipient pigs of cloned embryos, and to have such piglets born and raised were conducted under approval ID no. 2004/561-925. All other procedures were also reviewed and approved by the Danish Experimental Animal Inspectorate, Danish Ministry of Justice; however, no specific approval ID is issued for other aspects of experimental animal use, such as animal care and killing of the animals.in vivo bred control pigs were obtained from the Faculty of Agricultural Sciences, Aarhus University. Liver and muscle tissue samples were collected from one litter of two male cloned pigs and one litter of two male control pigs both at six weeks after birth. The cloned pigs were from the same fetal cell line which originated from the ear cell of an offspring of a sow which is a hybrid of Landrace and Yorkshire and a boar of Duroc and were age, breed, and sex matched to control pigs from their conventionally bred counterparts. They were produced by HMC with pretreatment of HHP of the oocyte, as described previously, and were confirmed to be genetically identical using microsatellite DNA analysis. All pigs were placed in adjacent identical pens and given continuous access to a standard commercial feed ration and water Cloned pigs and Genomic DNA and total RNA were isolated from frozen tissue samples using DNeasy® Blood & Tissue Kit (QIAGEN) and TRIzol reagent (Invitrogen), respectively, following the manufacturer's protocols. RNA integrity was assessed by 1% agarose gel electrophoresis and by Agilent Bioanalyzer QC.High quality total RNA from each sample was used for microarray experiments. Microarrays were hybridized and scanned in Molecular Diagnostic Laboratory, Clinical Biochemiccal Department, Aarhus University Hospital/Skejby Sygehus. Briefly, cRNA probes were synthesized from 5 µg total RNA, labeled by biotin and hybridized to Affymetrix GeneChip® Porcine Genome Array , which contains a total of 24,123 probe sets, of which 23935 probe sets interrogate 23,256 transcripts in pig, which represents 20,201 genes, and 11,265 genes among them were annotated.pvclust” was applied to group samples based on similarity of gene expression data P value<0.05. Finally, DEGs were analyzed by Ingenuity Pathway Analysis  for their functional involvement in biological processes, pathways and molecular networks.After scanning, the raw expression data was generated by GeneChip Operating Software (GCOS) and assessed with affyQCReport to ascertain individual array quality, homogeneity between arrays, variance mean dependency, RNA degradation and data distribution. The raw expression data was processed with RMA (Robust Multi-Array) normalization, and absent/present (A/P) calls Synthesis of cDNA was performed with the iScript cDNA Synthesis Kit  from one µg of total RNA of the samples used for microarray. One µl (10 times diluted) cDNA product was used as template for quantitative real-time PCR analysis. Quantitative RT–PCR was carried out using LightCycler 480 SYBR Green I Master (Cat. 04887352001) on LightCycler 480 (Roche). Beta-Actin was chosen as reference gene for Q-PCR. Comparison of relative gene expression level among the samples was calculated as fold changes of the sample, in which the gene expression level is the lowest. Primers are listed in MluI (New England Biolabs), ligated to a biotinylated linker, and fragmented by NlaIII (New England BioLabs) cleavage following the protocol. As MluI only cuts unmethylated regions, the DNA fragments ligated with biotinylated linkers were captured by streptavidin-conjugated beads to separate unmethylated and methylated fragments. Controls were set up to monitor unspecific binding of fragments without biotinylated linker and were called bead-controls. Next the isolated DNA fragments were ligated with linkers (N) containing a MmeI recognition site, and then digested with the Type IIS restriction enzyme (tagging enzyme) MmeI (New England Biolabs) to generate short sequence tags (17-bp). Finally, the tags were ligated with P7 linker and amplified by PCR using Phusion polymerase (Finnzymes) and primers N and P7. PCR products were purified using a QIAquick MiniElute kit (Qiagen) and stored as library for each sample. Before Solexa sequencing, conventional clone sequencing was carried out to verify the library quality as described previously Eight MMSDK tag libraries, one library for each tissue and individual pig, were constructed followed the protocol described previously ftp://ftp.sanger.ac.uk/pub/S_scrofa/assemblies/PreEnsembl_Sscrofa) and annotated by blasting to the human genome in the RefSeq database (http://www.ncbi.nlm.nih.gov/refseq).The final build pig genome (Sscrofa10) was downloaded from Ensembl database , and the high-confidence type with a mapping quality score more than twenty (MQ20). As described in the MAQ manual, if a read can be mapped to several equally best positions, MAQ will randomly choose one position and give the alignment a zero mapping quality. In principle, a tag with MQ20 should have 1% error rate. SNPs and sequencing errors are considered in calculating this score. We used MQ0 as a low-confidence criterion for addressing whether a tag can be mapped back to the genome, and MQ20 as a criterion for collecting high-confidence tags used for data analysis.We extracted 17-bp tags from all reads of 1G Genome Analyzer for each sample with the Solexa pipeline, and applied MAQ  algorithm MluI site in a library with the total tag number in the library and clustered by hierarchical clustering using the “pvclust” package www.r-project.org, Vienna, Austria): This groups samples by measuring the similarity between two DNA methylation patterns represented by tags' positions and counts based on Pearson's correlation coefficient values. Concerning the uncertainty of clustering, pvclust calculates p-values for hierarchical clustering via multiscale bootstrap resampling. P value of a cluster is a value between 0 and 1, and indicates how strong the cluster is supported by data. Pvclust provides two types of P values (%) on the edge of the cluster: AU (Approximately Unbiased) P value and BP (Bootstrap Probability) value. AU P values are computed by multiscale bootstrap re-sampling, and BP values are computed by normal bootstrap re-sampling. Clusters with AU larger than 95% are strongly supported by data.For the analysis of tag information, we divided the tags into two categories considering whether they are located in repeat sequences according to RepeatMasker P values were calculated from raw tag counts and adjusted by False Discovery Rate (FDR) We used a Poisson-based Significance Analysis algorithm (SA) www.urogene.org/methprimer/index.html). These primers were designed to recognize regions without CpG sites to avoid amplification bias of methylated versus unmethylated sequences. BSP validation experiments were conducted as follows: 500 ng of genomic DNA was converted using ZYMO EZ DNA Methylation-Gold Kit™ according to the manufacturer's instructions. After purification the converted DNA, PCR amplification was carried out in a final reaction volume of 50 µl consisting of 3 µl purified conversion fractions, 4 µl 2.5 mM dNTP, 5 µl 10×buffer, 1 µl BSP primers, 0.5 µl JumpStart™ Taq DNA Polymerase and 36.5 µl water and the following thermal cycling program was 94°C 1 min, 30cycles of 94°C 10 s, 58°C 30 s, 72°C 30 then prolong with 5 min at 72°C and products could be hold at 12°C. Primers are listed in Specific validation regions were selected and bisulfite sequencing PCR primers were designed by online MethPrimer software . After RMA normalization (Robust Multi-Array) and absent/present (A/P) calls pvclust method. As shown in We performed hierarchical clustering on the gene expression profiles of eight samples using the A few transcripts seemed differentially expressing ≥2) in cloned pigs versus controls. A total of 116 transcripts, which accounted for 0.63% of total transcripts, seemed differentially expressed in muscle samples. Of the 116 transcripts, 67 transcripts were expressed at higher level in muscle samples of cloned pigs, whereas 49 transcripts were more abundantly expressed in control samples . In liveP value)). Among these networks, one molecular network with a function related to cellular growth and proliferation, cell death, and cancer was over-represented both in muscle    and livere = 19) . HoweverMluI/NlaIII tag library for methylation-tag mapping in the pig genome. In silico analyses showed that there are 23759 recognition sites for the MluI restriction enzyme in pig genome, and we obtained 47132 virtual fragments by simulating the digestion of MluI and NlaIII on pig genome under the hypothesis that all cytosines were unmethylated. 193 fragments between adjacent MluI recognition sites lacked NlaIII recognition site inside. The length distribution of the virtual MluI/NlaIII fragments is presented in MluI recognition sites  are located in promoter-associated CGIs in the pig genome using CpGProD (CpG Island Promoter Detection) software MluI recognition sites  are located within repeat sequences.We constructed a virtual MluI recognition sites in the pig genome. Among the 23759 MluI recognition sites, there were 358 “empty” sites to which no tags could be mapped back, giving 98.5% mappable sites. Among the 4699 sites located in repeat sequences and the 7028 sites located in CGIs, there were 64 and 63 “empty” sites, respectively giving 98.6% and 99.1% mappable sites. All information of the libraries for the samples is available in P values .For the analysis of the repeat families, we isolated the tags in different categories of repeat sequences and analyzed them according to MQ0 tag counts. The results of the tags collected in muscle and liver genomes from cloned and control pigs are summarized in P values. The result indicates a similar level of differently methylated cytosines for liver and muscle in cloned and control pigs. We identified 2167 and 2157 significantly different tag sites out of 14094 ones in muscle and liver, respectively , with a percentage of 15.38% and 15.30%. 747 and 881 tag sites out of the 2167 and 2157 ones were located in promoters associated with CGIs. P value)) ) . Notably value)) . Randoml value)) , probablBesides, the “gene expression and cellular growth and proliferation” network was significantly enriched with differentially methylated genes in muscle and liver, although genes involved were mostly different. This suggested that DNA methylation status in the promoter regions of certain cellular growth-related genes was altered in cloned pigs, indicating a potential risk of abnormal gene expression.We tried to correlate the DNA methylation changes with the gene expression profile changes by performing a cross-comparison between the genes neighboring the significantly different methylation sites and the differentially expressed genes, especially for the genes in the molecular networks highlighted by differentially expressed genes and genes containing differentially methylated genomic loci. We were not able to identify genes that differed significantly both in DNA-methylation and gene expression status between cloned and control pigs.In order to identify the mechanisms related to the inefficiency of SCNT technology, we compared global gene expression profiles between cloned animals and conventionally bred controls. A limited number of studies have already addressed this problem and demonstrated that SCNT embryos were able to undergo significant nuclear remodeling and their gene expression patterns were similar to those of artificial insemination (AI) embryos with only a small set of differentially expressed genes If cloned animals with abnormal phenotypes can be subjected to conventional breeding, all their offspring tend to show normal phenotypes, showing that the defects coming from the cloning procedure are due to inefficient or aberrant nuclear remodeling after SCNT rather than to genetic mutations In the present study, we have identified a similar methylation status of repetitive sequences in both muscle and liver tissues of cloned and control pigs . HoweverDNA methylation plays an important role in tissue differentiation, and numerous tissue-specific differentially methylated regions (T-DMR) have been reported throughout the mammalian genome We further compared genes neighboring the significantly different methylation sites with the set of differentially expressed genes. No overlapping genes could be detected . One explanation could be that only one-third of the genes in mammalian genome are regulated by DNA methylation The global gene expression profiles for all samples presented much less differences when compared with the DNA methylation patterns. Also, the observation that some genomic loci existed with a higher degree of DNA methylation should be considered together in relation to the normal phenotype of these cloned pigs. This implies that the developing cloned pigs can tolerate more alterations of DNA methylation in those genes than expected. One possible explanation could be that the alteration of DNA methylation in certain regions will not change the expression level of the genes, as many methylation tags are located in the regions, for which the regulation mechanism by DNA methylation is not yet clarified. Another possible explanation might be that most of the influenced genes are functionally irrelevant, while the important genes are correctly reprogrammed and expressed in phenotypically normal cloned pigs.It remains unclear whether the higher methylation state is biologically important and whether it increases the risk of developmental or pathological disorders. However, it is reasonable to infer that DNA methylation reprogramming in SCNT to some extent is a stochastic process. If the DNA methylation reprogramming is not even fulfilled completely in phenotypically normal cloned pigs, there is clearly a risk in cloned pigs not selected for a phenotypically normal appearance. In aborted and/or abnormal pigs, aberration in DNA methylation profiles and gene expression are expected to be more severe. Further work is required to study the epigenetic reprogramming and tissue development in cloned pigs with abnormal phenotypes as well as in neonatally dead or aborted cloned pigs.Figure S1Tag distribution for DNA methylation sequencing. Figure A presents the length distribution of DNA fragments digested with MluI in a digital enzyme cutting simulation of the pig genome. Table B presents the obtained sequence reads from MMSDK and the mapping results for cloned piglets and control samples, respectively.(PDF)Click here for additional data file.Table S1List of significantly (P<0.05) and differentially (FC≥2) expressed genes in muscle of cloned pigs.(PDF)Click here for additional data file.Table S2List of significantly (P<0.05) and differentially (FC≥2) expressed genes in liver of cloned pigs.(PDF)Click here for additional data file.Table S3IPA molecular network analysis of significantly and differentially expressed genes in muscle or liver of cloned pigs. Network score≥2 was considered as over-represented.(PDF)Click here for additional data file.Table S4Summary of all raw data of MMSDK, including DNA fragment ID, counts of mapped tags by low-confidence MQ0 and high-confidence MQ20 standards for all samples. Promoters associated with CGIs, repeat sequence information and annotation information are also given.(XLSX)Click here for additional data file.Table S5Summary table of SA analysis of DNA methylation in unique sequences, including DNA fragment ID, counts of mapped tags by high-confidence MQ20 standards, CGIs location, annotation information and SA P values.(PDF)Click here for additional data file.Table S6Summary table of molecular network analysis by Ingenuity Pathways Analysis of genes neighboring significantly-differently methylated tags in muscle and liver.(PDF)Click here for additional data file.Table S7Primer sequences using for Q-PCR.(XLSX)Click here for additional data file.Table S8Bisulfite sequencing PCR validation of differential methylation. In the MMSDK result, the numbers in the column of control 1 indicates the tag counts of control sample 1. In the BSP result, two samples of control pigs were pooled and the numbers in the column of control-sup indicates the clone counts that support the methylated C of control sample 1, while the Control-nonsup means for the reads that doesn't support.(XLSX)Click here for additional data file.


The lysosomal storage pathology in Mucopolysaccharidosis (MPS) IIIB manifests in cells of virtually all organs. However, it is the profound role of the neurological pathology that leads to morbidity and mortality in this disease, and has been the major challenge to developing therapies. To date, MPS IIIB neuropathologic and therapeutic studies have focused predominantly on changes in the central nervous system (CNS), especially in the brain, and little is known about the disease pathology in the peripheral nervous system (PNS). This study demonstrates characteristic lysosomal storage pathology in dorsal root ganglia affecting neurons, satellite cells (glia) and Schwann cells. Lysosomal storage lesions were also observed in the myoenteric plexus and submucosal plexus, involving enteric neurons with enteric glial activation. Further, MPS IIIB mice developed progressive impairments in sensory functions, with significantly reduced response to pain stimulation that became detectable at 4–5 months of age as the disease progressed. These data demonstrate that MPS IIIB neuropathology manifests not only in the entire CNS but also the PNS, likely affecting both afferent and efferent neural signal transduction. This study also suggests that therapeutic development for MPS IIIB may benefit from targeting the entire nervous system. We demonstrate characteristic lysosomal storage in the dorsal root ganglion (DRG) and enteric nervous system (ENS) in an MPS IIIB mouse model. Our data also showed significant impairment in pain sensory capacity as the disease progresses. These data suggest the potential role of the PNS neuropathy in MPS IIIB.Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23. The genotypes of progeny mice were identified by PCR, using primers targeting undisrupted mouse NaGlu Exon 6: 5′-TGGACCTGTTTGCTGAAAGC (sense) and 5′-CAGGCCATCAAATCTGGTAC (anti-sense), and the transgene (neomycin): 5′-TGGGATCGGCCATTGAACAA (sense) and 5′-CCTTGAGCCTGGCGAACAGT (anti-sense). MPS IIIB mice and their wild type (wt) littermates were used in this study. The animal studies have been approved by the IACUC committee in the Research Institute at Nationwide Children’s Hospital (IACUC#: 04804AR).The mouse model of MPS IIIB, generated by homologous recombination For tissue sample collection, age-matched MPS IIIB and wt mice (n = 4 each) were anesthetized with an intraperitoneal injection of avertin , and were then perfused transcardially with cold PBS  followed by 4% paraformaldehyde in phosphate buffer . Spinal cord and small intestine samples were fixed in 4% paraformaldehyde and processed for paraffin sections (4 µm). The sections were then processed for histology staining using toluidine blue (1%), which does not stain GAGs.Spinal cord, spinal nerve and samples of cervical, thoracic and lumbar DRG (n = 4 each) were fixed in 2.5% glutaraldehyde overnight and processed for plastic sections (1–2 µm). The samples were collected and processed at the same time. The sections were then assayed for TEM and/or histopathology with toluidine blue staining.488 or AlexaFluo568 (Invitrogen). The sections were visualized under a fluorescent microscope. GFAP- or Iba1-positive cells in grey matter of spinal cord in an area of 300×430 µm were counted on cervical, thoracic and lumbar sections from 6 mice per group. Cell counting was performed on 3 sections/level/mouse.Indirect IF was performed on the spinal cord and DRG paraffin sections (4 µm)(n = 4/group) following the protocol provided by the manufacturers, using a primary antibody against glial fibrillary acidic protein (GFAP)(Millipore), Iba 1 (Abcam), or LAMP-1, and secondary antibodies conjugated with AlexaFluoTotal RNA samples from lumbar spinal cord were assayed for CD45 expression by Real-time qRT-PCR, using a pair of primers for murine CD45 gene, as previously described. Touching for changes in the response to tactile stimulation was performed using touch test sensory monofilaments  Algesic response to thermal stimuli in mice was measured with the tail flick test The thermal nociceptive threshold in mice was assessed using a hot-plate analgesia apparatus (Columbus Instruments) t-test were used to analyze quantitative data. Unquantified data from touch tests were analyzed by chi-square test and logistic regression with repeated measurements (GENMOD) using SAS9.2.Means, standard deviation and student ), which are known characteristic storage pathology in the CNS of animals and patients with MPS IIIB. Our data also showed increase in LAMP-1 staining in DRGs (data not shown), indicating a degenerative pathology in the DRG. TEM also showed the destruction of myelin sheath in the spinal nerves from MPS IIIB mice , suggesting possible demyelination in the PNS. In addition, the myelin sheath destruction is ubiquitous in the spinal nerves, suggesting the involvement of both DRG sensory neurons and spinal cord motor neurons. Our data demonstrate a PNS component of neuropathology.Histopathology and TEM on DRG sections were performed to determine whether the lysosomal storage pathology affects the PNS in MPS IIIB mice in comparison with wt mice . Histopathology staining with toluidine blue showed wide spread lysosomal storage lesions in virtually all neurons, satellite cells (PNS glia) and Schwann cells in DRG from MPS IIIB mice Fig. 1a.Histopathology using toluidine blue staining was also used to probe lysosomal storage lesions in the intestine. We observed vacuoles, characteristic lysosomal storage lesions, in the neurons in myenteric plexus and submucosal plexus Fig. 1b To assess whether MPS IIIB affects sensory function, mice were tested for response to nociceptive stimuli by microfilament touch test , tail flick and hot plate assays (n = 10–15/group).Tactile testing showed that 6 mo old MPS IIIB mice were significantly less responsive to hind paw stimulation with 4.13 g (P<0.001) and 4.65 g (P<0.001) microfilaments on their feet Fig. 2a,Tail flick and hot plate tests were performed in mice at 2, 3, 4 and 6 mo of age . MPS IIIB mice exhibited significant increase in the latency to respond to heat stimuli in the tail flick Fig. 2b To assess neuropathological manifestations in lower CNS, the spinal cord samples from 6 mo old MPS IIIB and wt mice (n = 6/group) were assayed by histopathology, IHC and transmission electron microscopy. c, e) and in lateral horn neurons (data not shown), but not sensory neurons in the dorsal horn, even though adjacent microglia exhibited profound lysosomal accumulation pathology  transduction, it is not surprising that the profound lysosomal storage pathology in MPS IIIB leads to the functional sensory impairments. This is supported by a recent study showing impaired vision and hearing in MPS IIIB mice Given the fact that the MPS IIIB mouse model strongly resembles the disease in humans, it is reasonable to predict that the neuropathy also manifests the PNS in patients MPS IIIB. It is worth noting that MPS IIIB mice develop characteristic late-stage clinical disorders, such as urine retention, rectal prolapse and/or protruding penis; symptoms which may reflect severe neurological damages Our data have demonstrated neuropathology in the spinal cord of MPS IIIB mice that is global in distribution with differential cell-type presentation, similar to that observed in previous studies in mice and dogs Figure S1Distruction of myelin sheath in spinal nerve in MPS IIIB mice. TEM images of spinal nerve from 1 wt and 2 MPS IIIB mice (6 mo old). Scale bar = 2 µm.(TIF)Click here for additional data file.


Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be “domesticated” for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7–15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning  Since the late nineties, metagenomic-based methodologies have been applied to decipher the composition and gene content of bacterial communities in the environment, as well as to detect novel biomolecules for subsequent functional screening Several strategies can be followed to discover new bacterial products in metagenomic samples In all the above-mentioned methods, an assay designed to discover a single novel biomolecule must be applied to the whole clone library (often containing thousands of clones), finally giving only a few positive clones (if any), which makes the screening process very inefficient. However, the clones that are negative for that specific assay could also contain sequences with an interesting biotechnological potential; however, unfortunately in this case they would remain unexplored.Prior knowledge of clone sequences can help researchers to design the appropriate screening assays and, thus, to increase the biotechnological application potential of the library (novel biomolecule per clone library rate). However, sequencing clones one-by-one using any type of sequencing platform would be very time-consuming and expensive.Here, we propose a hybrid sequencing approach based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. This technique allows genetic information to be gathered from individual clones in a short time and with reduced sequencing costs. Instead of whole individual clone sequencing, we sequenced the clones in a pool and the medium-large insert (7–15 kb) cloning strategy allowed us to assemble these 358 sequenced clones correctly and assign them the correct Sanger reads of clone ends. Thus, the clone-end sequences maintain the link between the living clones and the annotated contigs from 454 assemblies, and so serve to locate the clone of interest in the clone library. The retrieved data facilitate planning consecutive biochemical assays for a given clone of interest. Moreover, the choice of the easy-handling plasmid vector enables an appropriate sub-cloning strategy to be designed for gene expression in suitable vectors/hosts. This article reports the characterization of the first metagenomic clone library from the fecal sample of a healthy human volunteer.The study was approved by the Ethics and Research Committee of the Centre for Public Health Research (CSISP) of Valencia, Spain. The volunteer involved in this study provided a written informed consent. One ml of fresh feces from the healthy volunteer was resuspended in 3 ml of salt solution (0.9% NaCl) by vortexing and then centrifuged at 2000 rpm for 2 min. The supernatant was transferred to a 15 ml tube. Microbial cells were purified as previously described Seven µg of extracted bacterial DNA was digested with EcoRI enzyme  at 37°C for 2 hours and then resolved on 0.5% TAE agarose gel at 15 V for 16 hours. DNA fragments measuring between 7 and 15 Kb were cut out from the gel without exposure to UV light. DNA elution was performed in the Elutrap device  running at 150 V for 3 hours. Amicon 50 K columns  were used for sample concentration and for the exchange of the electrophoresis buffer into water. Fragments shorter than 1.5 Kb were completely removed to allow ligation of longer ones by adding 100 µl of Agencourt Ampure Xp magnetic beads  to the DNA sample, diluted in 200 µl 10 mM Tris-HCl. DNA was bound to the magnetic beads on a magnetic particle concentrator  and purified by 70% ethanol. Size selected DNA was finally resuspended in 20 µl 10 mM Tris-HCl.Two µl of sample DNA was ligated to EcoRI pBluescript  with Takara DNA ligation kit  at 16°C overnight. The ligation reaction was transformed into One Shot TOP10 electrocompetent cells (Invitrogen) at 1800 Volts . Transformed cells were incubated in 1 ml of SOC medium at 37°C for 1.5 hours and then spread on LB agar plates containing ampicillin (100 µg/ml), XGAL (50 µg/ml), and IPTG (1 mM) and incubated at 37°C overnight.White colonies were picked and placed separately in 1 ml of LB with 100 µg/ml ampicillin into 96 well plate and left to grow overnight at 37°C. Plasmid minipreps were performed using 100 µl of solution 1 , 200 µl of solution 2  and 150 µl of solution 3 . Plasmid DNA was resuspended in 30 µl of water. Twenty ng of each of the 358 clones were pooled together.The shotgun library was prepared from 1 µg of the pooled sample according to manufacturer instructions . The sample was then sequenced on 1/8 of PicoTiterPlate by GS FLX+ system. Sequencing depth has been calculated in order to reach coverage of about 10×, distributed among the 358 clones.Plasmid DNA obtained by miniprep from each clone (about 60 ng) was sequenced separately by the Sanger method on DNA ABI 3730 (Applied Biosystems) using M13 forward or M13 reverse primers.http://www.sanger.ac.uk/resources/software/smalt/) was used and plasmid sequences coordinates were employed in the following assembly step to avoid unnecessary vector assembly. Pyrosequencing reads were assembled by MIRA3 applying typical de-novo genome 454 assembly parameters In order to remove vector sequences (2961 bp), Smalt 0.5.8. tool  were identified by Glimmer 3 A figure with the flow chart of the proposed approach is shown in http://www.ebi.ac.uk/ena/data/view/ERP001596).Sequences were deposited in EMBL-EBI Sequence Read Archive (SRA) with study number ERP001596  were excluded from the analysis.The hybrid assembly revealed that 57 contigs correctly matched more than one Sanger sequence, showing a probable partial digestion or that inserts can proceed from different original microbial genetic rearrangement. Only six Sanger sequences could not be mapped to 454 contigs.The assembly results show that on using the strategy of cloning the medium-large inserts (7–15 kb) into plasmids, there is no need for additional paired-end 454 sequencing; moreover, the coverage for correct assembling was sufficient. The sequence length of the pBluescript plasmid (2961 bp) is lower than the length of commercially available fosmid of BAC vectors (8–17 kb), which reduces the number of reads containing vector sequences.Out of 473 contigs, 316 were larger than 1000 bp and used for the analysis. Glimmer3 identified 1790 ORFs. The average length of ORFs was 249 amino acids, while the shortest and the longest ones were 38 and 2381 amino acids, respectively. HMMPfam annotated 742 different proteins in our assembly and Seg scanning application identified 1,859 matches see . The comThe clone library derived from the fecal sample of a healthy volunteer provided genetic information of several clones containing enzymes with previously known applications. Pseudomonas, Mycoplasma, Halobacterium, Lactobacillus, Lactococcusin vitroClone 2H2 , panel aClone 2C1 , panel bAnother enzyme of interest is choloylglycine hydrolase  found in contig 2B3 , panel cin vitro biofilm system that co-administration of antibiotics with alginate lyase from Bacillus circulans might benefit cystic fibrosis patients by increasing the efficacy of antibiotic in the respiratory tract Pseudomonas aeruginosa have become established in the patient’s respiratory tracts, they can rarely be eliminated by antibiotic treatment alone. Alginate lyase was also found to have application in plant culture techniques in vitro. It has been applied successfully for the extraction of protoplasts for food research and regeneration of a variety of algal species, including brown algae, and serves as an alternative for various mechanical and chemical methods We found alginate lyase in contig HK3UA , Panel dSeveral studies indicate an association between common neuro-developmental disorders and gut microbiota. The microbial colonization process triggers signaling mechanisms that can influence central nervous system development and might be linked to autism It is known that bacteria can mediate gene transfer, which has led to the utilization of various bacterial strains in gene therapy Cystathionine β-synthase (CBS) is a vitamin B6-dependent trans-sulfuration enzyme needed to synthesize cysteine from methionine. A CBS deficiency causes homocystinuria, a rare autosomal recessive disease, characterized by mental retardation, psychiatric disturbances, skeletal abnormalities, and vascular disorders ad hoc biochemical screening. The approach used in this paper started by analyzing 358 clones and ended up with 316 assembled and annotated contigs. The proposed method is perfectly scalable enabling work to tackle larger clone libraries, proportionally increasing sequencing efforts. This strategy enables the link to be maintained between the information and the living clones, providing annotation of the whole library; thus, particular clones of interest can undergo further testing by the most appropriate biochemical assay and/or sub-cloning for appropriate selection of vectors and/or host, if required.Metagenomics of the human microbiome can provide genetic information about the DNA of bacteria inhabiting human-related ecological niches, adapted to physiological conditions such as temperature, pH, redox potential, etc. The assembly and annotation of clones produced from a library of this kind reveal the presence of several proteins for which industrial or medical applications have already been reported. They can also be used to study proteins of unknown function. The screening of a clone library with inserts of 7–15 Kb by 454 pyrosequencing enabled us to obtain the annotation information of the genes present in each clone without the need of prior and expensive Figure S1Protocol flow chart. The figure summarizes the protocols applied to construct the clone library, pyrosequencing of pooled clones, individual clone Sanger end sequencing, contigs/clone-ends matching and annotations.(TIFF)Click here for additional data file.Figure S2Valine biosynthesis pathway. Green frames indicate enzymes found in the library.(TIFF)Click here for additional data file.Table S1Whole library InterPro annotation table. Columns describe: ORF name bounded to the original clone name in the library; amino acid length; InterPro inquired database; database match; match description; ORF match start position; ORF match end position; match p-value; date of search; InterPro match code; protein name; protein description.(ZIP)Click here for additional data file.


Mitochondrial disorders are difficult to diagnose due to extreme genetic and phenotypic heterogeneities.We explored the utility of targeted next-generation sequencing for the diagnosis of mitochondrial disorders in 148 patients submitted for clinical testing. A panel of 447 nuclear genes encoding mitochondrial respiratory chain complexes, and other genes inducing secondary mitochondrial dysfunction or that cause diseases which mimic mitochondrial disorders were tested.We identified variants considered to be possibly disease-causing based on family segregation data and/or variants already known to cause disease in twelve genes in thirteen patients. Rare or novel variants of unknown significance were identified in 45 additional genes for various metabolic, genetic or neurogenetic disorders.Primary mitochondrial defects were confirmed only in four patients indicating that majority of patients with suspected mitochondrial disorders are presumably not the result of direct impairment of energy production. Our results support that clinical and routine laboratory ascertainment for mitochondrial disorders are challenging due to significant overlapping non-specific clinical symptoms and lack of specific biomarkers. While next-generation sequencing shows promise for diagnosing suspected mitochondrial disorders, the challenges remain as the underlying genetic heterogeneity may be greater than suspected and it is further confounded by the similarity of symptoms with other conditions as we report here. Patients with mitochondrial diseases present with widely variable phenotypes that can affect any organ, at any age. This clinical variability and a lack of reliable diagnostic tests can make diagnosing these disorders very challenging. Currently, the diagnosis relies upon the enzymatic analysis of respiratory chain complexes (RCC) in muscle biopsy tissues,2. UnforRecently, targeted next-generation sequencing (NGS) has been utilized to diagnose patients with several types of disorders-11. For 148 patients were referred by experts in mitochondrial medicine, and all patients had diagnosed or suspected mitochondrial disorders according to the physicians. Limited clinical information was provided for the patients with the test requisition, and the clinical presentations that were provided varied widely. The most common clinical findings included developmental delay and hypotonia, and other common findings are listed . The exons of the genes of interest , as well as mitochondrial respiratory chain complexes assembly factors (n = 29), mitochondrial carriers (n = 17), genes for mitochondrial DNA synthesis, transcription, translation, mitochondrial biogenesis and dynamic (n = 71), genes for mitochondrial enzymes (n = 114), and other genes that may affect mitochondrial function secondarily or that cause similar clinical phenotypes (n = 127) . Data weIn each patient’s report, parental testing was recommended and offered at no additional charge to further elucidate the significance of all confirmed, possibly disease causing variants. This parental testing was performed by Sanger sequencing as described above, and results were used to further interpret variants found in the proband.A variety of quality control metrics were used to ensure that patient runs were of the highest quality. These included an examination of the percentage of reads mapping to the human genome, the percentage of reads mapping to the targeted regions, the average read depth of targeted regions, and the percentage of targets with greater than an average of 20× coverage per base with a quality score of Q ≥ 30. An external control was included in the eighth lane of every sequencing run and consisted of a previously characterized HapMap sample .On average, 67.4% of reads aligned to the human genome, and 61.2% aligned to the targeted regions. Average coverage of the targeted regions was 147× per base, and on average, 95.6% of targets had greater than 20× coverage per base with a score of Q ≥ 30. Internal and external controls were used to verify the quality of each sequencing run. In the external HapMap control, we identified on average 164.7 variants per run (95.7%) of the 172 previously characterized SNPs in this HapMap sample within the targeted regions. We also used a panel of 200 SNPs with high minor allele frequencies in the first 20 patients as an internal sensitivity control for all samples, and we identified an average of 194.3 (94.7%) of the SNPs in each patient.On average, we found 553 variants per patient in the targeted regions. Synonymous single nucleotide substitutions were the most common, followed closely by intronic/UTR (limited to 20 nucleotides from the exon boundary), and missense single nucleotide substitutions [NDUFS2 [NDUFAF5 [ETFB (Glutaric acidemia IIB)[PANK2 (Neurodegeneration with brain iron accumulation)[PCK2 (Phosphoenolpyruvate carboxykinase deficiency)[SLC3A1 (Cystinuria)[TYMP [OTC (Ornithine transcarbamylase deficiency)[SPAST (Spastic paraplegia 4)[SLC12A3 (Gitelman syndrome)[CLN6 (Ceroid lipofuscinosis 6)[CLN6 and other non mitochondrial nuclear genes were included in our panel as the associated clinical presentation can be easily confused with mitochondrial disease. PolyPhen-2 predictions for these variants are listed in Table in silico prediction models are known to not always be accurate[The variants identified as possibly disease-causing were located in the genes yndrome),23, NDUFiciency), NDUFAF5iciency), ETFB (Gmia IIB), PANK2 (ulation), PCK2 (Piciency), SLC3A1 tinuria), TYMP (Mlopathy), OTC (Oriciency), SPAST (legia 4), SLC12A3yndrome) and CLN6nosis 6). CLN6 anaccurate-37.trans, therefore consistent with AR disease inheritance , PDHA1 (PDH deficiency), ABCD1 (X-linked adrenoleukodystrophy), MECP2, CDKL5 (Rett syndrome), and SLC6A8 (creatine transport defect)   and one gene for the assembly of the complex I (NDUFAF5). In a total of 148 patients we analyzed, there was only one additional gene SCO2 in the category of primary mitochondrial disease causative genes, with variants that we classified as of unknown significance as we did not receive parental samples, but most likely associated with disease.Our data highlights the great underlying genetic heterogeneity of suspected mitochondrial disease, but also the difficulty that even experienced physicians endure when attempting to define mitochondrial disorders. Of note, possible disease-causing variants, confirmed by parental samples analysis, were found in only one gene that encodes an oxidative phosphorylation subunit of complex I , have disease that is probably not the result of direct impairment of energy production as we observed in our previous study. InsteadFrom our observation, it appears that the clinical differential diagnosis for mitochondrial disease could be even more challenging, as the diagnostic spectrum is much wider than previously thought. Some of our patients could have been diagnosed by standard biochemical or genetic studies, without NGS sequencing. Unfortunately, the lack of clinical phenotypes that are specific to these diseases causes physicians to have a difficult time identifying which biochemical studies should be ordered for each case. A good example is represented by the patient affected by CLN6, a lysosomal storage disorder in which the clinical presentations can easily mislead to focus on mitochondria, leading to muscle biopsy and RCC assay.GAA (Pompe disease), HLCS (Holocarboxylase synthetase deficiency), ABCD1 (X-linked adrenoleukodystrophy), PC (Pyruvate carboxylase deficiency), UBE3A (Angelman syndrome), ASL (Argininosuccinic aciduria), or ZFYVE26 (Spastic paraplegia 15)  disease, spinocerebellar ataxia or spastic paraplegia related genes. While these variants still require parental samples analysis to determine the clinical significance, these conditions may be an emerging group of mitochondrial disorders as supported by recent publications with evidence of mitochondrial dysfunction,50.Our study revealed that a broader diagnostic spectrum including neurodegenerative disorders or myopathy need to be considered during the clinical work-up of suspected mitochondrial patients and in developing future diagnostic tests. It appears that the current standard diagnostic algorithm along with the definition of mitochondrial disorders may require substantial revision to ultimately improve patient quality care. The clinical sensitivity of NGS assays for mitochondrial disorders will be further improved by targeting more genes and ultimately by whole exome sequencing with adequate coverage in the near future.J. DaRe is an employee of, and holds stock options for Transgenomic, Inc. Seattle Children’s Research Institute receives a licensing fee for all NuclearMitome tests run through Transgenomic, Inc. Transgenomic, Inc is financing the article-processing charge for this manuscript.VV and JTD equally contributed to this manuscript. JTD drafted the manuscript and analyzed and interpreted the data. VV selected and designed the targets capture, performed experiments, drafted the manuscript, analyzed and interpreted the data. JP analyzed the data. TT performed experiments. SHH conceived of the study concept, drafted the manuscript, and analyzed and interpreted the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/118/prepubList of Genes for Nuclear Mitome Test (447 genes).Click here for fileAverage number of variants per patients in targeted regions.Click here for fileList of disease with variants of unknown significance (VUS) detected in patients for which parental testing results are not available.Click here for fileVariants of unknown significance identified in autosomal dominant and X-linked genes with decreased suspicion after parental testing.Click here for fileVariants of unknown significance identified in autosomal recessive genes with decreased suspicion after parental testing.Click here for file


To gain insight into potential roles of isomiR spectrum and isomiRs with 3′ additions in pre-eclampsia, we performed a comprehensive survey of miRNA repertoire and 3′ addition events from placental samples with different degrees of pre-eclampsia by applying SOLiD sequencing platform.Over 30% isomiRs were detected with 3′ non-template additional nucleotides, especially for additional nucleotide of adenosine. However, these modified isomiRs showed a lower percentage of total miRNA expression (<15%). Generally, 1-3 abundant isomiRs from a given miRNA locus were identified, but none of them was detected with 3′ additions. Different miRNAs indicated various isomiR spectrums and expression patterns. The most abundant isomiR spectrum, isomiR profile and expression pattern always were stability, but herein we found several exceptions across samples, especially between normal and diseased samples. At isomiR level, we detected a distinct subset of differentially expressed modified isomiRs between normal and diseased samples or between mild and severe samples. Gene Ontology analysis of their experimentally validated target genes revealed enrichment for specific biological process categories.The phenomenon of multiple isomiRs, especially for isomiRs with 3′ additions, is not a random event during pre-miRNA processing. Varieties of isomiRs and expression patterns reveal potential functional implication and should be taken into account. The study enriches association of miRNAs and human disease, including potential roles of various miRNA variants and 3′ addition events. MicroRNAs (miRNAs), a class of endogenous small non-coding RNAs (∼22 nt), play pivotal post-transcriptional regulatory roles in normal physiological functions by targeting messenger RNAs for cleavage or translational repression Drosophila melanogasterDrosophilaP. trichocarpaHigh-throughput sequencing technologies are offering a broad and deep survey of the interesting and pivotal small non-coding regulatory molecules. Discovering and profiling of miRNAs have been widely studied in animals and plants, especially for various human diseases. Based on high-sensitivity and high-throughput sequencing datasets, more small RNAs are detected, including multiple miRNA variants (isomiRs) Pre-eclampsia is a relatively common human disease of pregnancy characterized by hypertension and proteinuria. It originates in the placenta and causes some maternal and fetal problems, and even might threaten maternal and perinatal survival. Abundantly and differentially expressed miRNA species in placental samples were reported by applying microarray analysis and real-time quantitative reverse transcription-ploymerase chain reaction Using the next generation sequencing technology, SOLiD System, we sequenced the small RNAs of placental samples from pregnant women with mild and severe pre-eclampsia and normal pregnant woman . AccordiIsomiRs with 3′ non-template additional nucleotides may have shorter, longer or consensus lengths with their canonical miRNA sequences. For example, modified isomiRs of hsa-miR-24 showed various length distributions, and additional nucleotide could be added 3′ end of canonical miRNA sequence, shorter or longer isomiRs . AlthougAccording to the sequence count of the most abundant isomiR from a single miRNA locus, we assessed the top 10 abundant miRNA species . Hsa-miRThe top 10 most abundant isomiR species with 3′ additions showed that some of them were yielded from the same miRNA locus . For exa2 of hsa-miR-143 was −5.29 (based on the most abundant isomiR) and −5.69 , respectively.Different estimation schemes were used to assess differentially expressed miRNAs across different samples. According to sequence count of the most abundant isomiR, fold change values of 15 miRNAs were over 4 or less than -4 between at least one pair of samples . Almost Meanwhile, at isomiR level, we also estimated differentially expressed isomiRs with 3′ additions. A total of 40 abundant modified isomiRs were analyzed, and 15 of them were differentially expressed at least between one pair of samples . SimilarMultiple isomiRs with various 5′ and/or 3′ ends and expression levels, including isomiRs with 3′ additions, were detected from a given miRNA locus . DespiteInterestingly, some miRNAs showed inconsistent the most abundant isomiR spectrums  across the different samples, especially between normal and diseased samples . DespiteAlthough multiple isomiRs with various 5′ and/or 3′ ends from a given locus were identified, they showed various expression levels. Generally, 1–3 isomiRs were abundantly expressed, whereas others always had fewer sequence counts . StrikinDue to high-sensitivity of high-throughput sequencing technologies, multiple miRNA variants with heterogeneous ends, lengths and expression levels, termed as isomiRs, were widely detected in animals and plants More importantly, we found bias of the degree of heterogeneity between 5′ and 3′ ends based on a comprehensive analysis without involved isomiRs with 3′ additions . 3′ isomDifferent miRNAs showed different type of isomiRs with various expression levels . RelativOverall, multiple isomiRs should not be a random result due to imprecise and alternative cleavage of Drosha and Dicer during pre-miRNA processing. The variety of isomiRs may play a role in regulating biological processes and reveal potential biological implication. The major reasons are as follows. (1) Type of isomiRs may be different across different miRNAs, and no strict correlation was detected between type of isomiRs and expression level of miRNA; (2) Most isomiRs were 3′ isomiRs with 3′ variations due to bias of cleavage, and these 3′ isomiRs always ensured the same identities with their canonical miRNAs; (3) Generally, 1–3 abundant isomiRs were identified from a given locus, but number of abundant isomiRs and dominant cleavage sites showed diversity across different miRNAs. Abundant isomiRs always were 3′ isomiRs and had the same 5′ ends and “seed sequences”, but none of them was detected with 3′ non-template additional nucleotides; (4) 3′ addition event was a widespread phenomenon. Additional nucleotide showed a strong bias towards adenosine. Modified isomiR also could be abundantly expressed despite it was not characterized as abundant isomiR from a specific miRNA locus; (5) The most abundant spectrum may be different in the same tissues from normal and diseased samples, even from samples with different degrees of disease. Despite different sequences were detected, they were 3′ isomiRs and had the same identities; and (6) IsomiR spectrums and their expression distributions always were stability, and their alternation may influence specific biological processes, even may contribute partly to pathogenesis of disease.xmiRNAs always showed 1–3 abundant isomiRs and other rare isomiRs, and expression levels of miRNAs might show different distribution patterns based on estimation methods of the most abundant isomiR and sum of all isomiRs, respectively Based on the inconsistent distributions, we then asked whether there were inconsistent differential expression profiles based on different estimation methods across samples. Here, despite detailed fold change values were different, consistent differentially expressed miRNAs were collected  across samples according to the most abundant isomiR and sum of all isomiRs, respectively . The conIncreasing evidence has demonstrated that miRNAs are subject to 3′ nucleotide additions, especially post-transcriptional non-template 3′ additions of adenosines or uridines Generally, isomiRs with 3′ additions were not abundant isomiR species from a given locus, although in some cases they also showed higher sequence counts . The nonTaken together, isomiRs with 3′ non-template additional nucleotides may be involved in pathogenesis of human disease. The phenomenon of 3′ addition events may influence miRNA stability and play a role in interactions of miRNA:target Placental samples of pregnant women were obtained from Zhongda Hospital, Nanjing, China. The institutional review board at Zhongda Hospital approved the tissue acquisition protocol to conduct the study. Written informed consent was obtained from each participant before tissue acquisition. Of these samples, one of them was from normal control pregnant woman, and others were from pregnant women diagnosed as mild and severe pre-eclampsia, respectively. Total RNAs of these samples were extracted with TRIzol (Invitrogen). Small RNAs were isolated from their total RNAs using mirVana™ miRNA Isolation Kit (Ambion). According to the protocol of SOLiD™ Small RNA Expression Kit (Life Technologies), purified small RNAs were subjected to miRNA library construction. Sequencing was carried out using SOLiD™ sequencing platform  at the State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, China.http://SOLiDsoftwaretools.com/gf/project/srna/), human other non-coding RNAs  were firstly filtered. The remaining sequencing reads were then analyzed by aligning to known human miRNA precursor sequences from the miRBase database  Sequencing files in colorspace were collected from SOLiD System based on a two-base encoding technology. According to SOLiD miRNA analysis pipeline Click here for additional data file.Figure S2Frequencies of heterogeneity of 5′ and 3′ ends. The frequency is estimated based on type of isomiRs without involved isomiRs with 3′ additions. 3′ isomiRs are quite prevalent than 5′ isomiRs across the three samples.(TIF)Click here for additional data file.Figure S3Distribution patterns of miRNAs and type of isomiRs. Expression distributions of miRNAs are assessed from lower to higher expression levels based on sum of all isomiR sequence counts, while their corresponding types of isomiRs show chaos distributions. No strict correlation is found between expression level of miRNA and its type of isomiRs. All of these miRNAs are abundantly expressed in corresponding sample (sequence count of the most abundant isomiR is over 999). Type of isomiRs is assessed based on their sequence counts (>99). To avoid great expression difference among miRNAs, some miRNAs with quite high expression levels are removed.(TIF)Click here for additional data file.Figure S4Percentage distributions of 3′ additions across different samples. Here we only consider those isomiRs that sequence counts are over 99. Percentage of 3′ additions based on (A) all type of isomiRs; (B) sequence counts of all isomiRs; (C) type of all isomiRs; (D) sequence counts of all isomiRs; (E) type of isomiRs with 3′ additions; (F) sequence counts of isomiRs with 3′ additions.(TIF)Click here for additional data file.Figure S5The 3′ non-template double additional nucleotides and their percentage across different samples.(TIF)Click here for additional data file.Table S1The number of total sequencing reads and reads that match to known miRNAs.(DOC)Click here for additional data file.Table S2Differentially expressed miRNAs with 3′ additions and their experimental validated gene targets from the miRTarBase database.(DOC)Click here for additional data file.Table S3Sequence count of miRNA (based on the most abundant isomiR) and its type of isomiRs across different samples.(DOC)Click here for additional data file.Table S4The most abundant isomiR sequence varies among different samples.(DOC)Click here for additional data file.