Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type “Zaosu” revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from “Red Zaosu” compared with “Zaosu”. Moreover, the GA levels in “Red Zaosu” seedlings were lower than those in “Zaosu” seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in “Red Zaosu”. Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in “Red Zaosu”. Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in “Red Zaosu” induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport “Red Zaosu” ( Unlike the common green stripes in watermelon, red stripes sometimes occur in apple, pear, and peach. The red appearance of these fruits results from anthocyanin accumulation5. Nevertheless, the genetic basis of this anthocyanin-associated striping trait is poorly understood, as are the mechanisms underlying the formation and distribution of the stripes.Fruit with stripes, which generally run longitudinally, can occur naturally. Alternating dark and light-green stripes are common in watermelon, pumpkin, and squash. Although such stripes form along the main carpellary vascular bundles7. The signaling networks behind the developmental and environmental regulation of anthocyanin biosynthesis are complex. Light stimuli are necessary for the production of anthocyanins in most plants. The ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1, ELONGATED HYPOCOTYL5, and PHYTOCHROME-INTERACTING FACTOR 3 are key components involved in the regulation of light-dependent anthocyanin biosynthesis8. The activities of ELONGATED HYPOCOTYL5 and PHYTOCHROME-INTERACTING FACTOR 3 can be modulated by DELLA proteins involved in gibberellin (GA) signaling10. The regulation of anthocyanin accumulation induced by different kinds of internal or external stimuli, such as jasmonate, temperature, sucrose, and nutritional starvation12, is affected by GA–DELLA signaling. Moreover, calcium signals may participate in anthocyanin biosynthetic regulatory networks that are induced by abscisic acid, sucrose, or environmental stimuli, including drought or low temperature15.Anthocyanins not only accumulate in flowers to attract insects for pollen dispersal or in fruit to attract animals for seed dispersal but also are produced in plants as radical scavengers in response to various biotic and abiotic stresses16. Among the flavonoid biosynthetic enzymes, dihydroflavonol 4-reductase, UDP-glucose: flavonoid 3-glucosyltransferase (UFGT), and anthocyanidin synthase are directly involved in anthocyanin biosynthesis16. The transcript levels of the genes encoding these anthocyanin biosynthetic enzymes are directly regulated by the MYB–bHLH–WD40 transcription complex17. Several MYB transcription factors, including PbMYB10, PbMYB10b (PbMYB114), and PbMYB9, are involved in pear anthocyanin regulation19.Anthocyanins, such as cyanidin 3-galactoside in pear, are derived from phenylalanine, the common precursor of flavonoid metabolites, through a series of enzymatic actions20. Anthocyanins accumulated mostly in leaf-derived tissues and produced red leaves and red-striped fruit skins in “Red Zaosu”  was confirmed to contribute to this atypical anthocyanin accumulation pattern in “Red Zaosu”. We observed that calcium-enriched xylem cells were spatially coordinated with anthocyanin distribution near vascular bundles, and this coordination was necessary for anthocyanin production in the fruit skin of the sport. PbGA2ox8-related anthocyanin accumulation was demonstrated to be sensitive to these calcium-enriched areas and resulted in the formation of anthocyanin-associated stripes near vascular bundles in pear fruit.In this study, pear GA 2-beta-dioxygenase 8 (P. bretschneideri Rehd.), “Zaosu” (P. bretschneideri Rehd.), “Red Anjou” (Pyrus communis L.), “Hong sichou” (P. communis L.), “Early Red Comice” (P. communis L.), “Dangshansu” (P. bretschneideri Rehd.), “Bartlett” (P. communis L.), and “Suisho” (Pyrus pyrifolia Nakai.) pear were collected from the horticultural research base of Northwest A&F University in Yangling District, Shaanxi Province, China. F1 plants from a “Red Zaosu” × “Yuluxiang” (Pyrus sinkiangensis Yu) cross were collected from the horticultural research base of the Henan Academy of Agricultural Sciences in Xinxiang County, Henan Province, China. “Red Zaosu” and “Zaosu” explants were propagated at the Laboratory of Fruit Trees Stress Biology of the College of Horticulture, Northwest A&F University. The detailed culture conditions, tissue information, information on the application of GA, and the calcium channel blocker lanthanum chloride (LaCl3) and sampling dates for these materials are listed in Supplementary Table “Red Zaosu”, a spontaneous bud sport of “Zaosu”, was first found in a commercial orchard in Linwei District, Weinan City, Shaanxi Province, China, in 2004. In this study, “Red Zaosu” 23. The functional annotations and gene ontology (GO) terms enriched in most DEGs were characterized using the pear genome as a reference. The annotations and GO terms enriched in 26 DEGs common to young leaves, mature leaves, and receptacles were further adjusted using any previously published orthologs. Variations in the expression levels and GO terms enriched in the 26 DEGSs were visualized using the GOChord R package24. The DEGs between red/green-striped regions of “Red Zaosu” fruit were clustered by the KEGG pathway enrichment analysis using DAVID25.The total RNA (3 μg) extracted from the receptacles, young leaves, and mature leaves of “Red Zaosu” and “Zaosu” and red/green-striped regions of “Red Zaosu” fruit were used for sequencing, with three biological replicates for each material assessed. Briefly, the total RNA was randomly fragmented, reverse-transcribed, amplified, and purified to form a cDNA library. After the cDNA library was assessed using the Bioanalyzer 2100 system , the library preparations were paired-end sequenced (100 bp) on a HiSeq 2500 platform . Clean reads were enriched by removing reads containing adapters, reads with multiple unknown bases, and low-quality reads from the raw data. Paired-end clean reads were aligned to the pear genome using TopHatPbActin  are listed in Supplementary Table −ΔΔCt) method.Purified RNA (2 μg) was reverse-transcribed to cDNA using the PrimeScript RT reagent kit with gDNA Eraser . The primer pairs for selected genes and 27. Fresh “Red Zaosu” flowers were immediately fixed in a formaldehyde–acetic acid–alcohol solution. The receptacles were separated, dehydrated, embedded in paraffin, cut into 10-μm slices, and stained with Fast green and safranin. The safranin-labeled xylem was imaged using a 450-nm excitation filter in combination with a 520–550-nm emission filter. Histological analysis was carried out using a BX51 + PD72 + IX71 microscopic imaging system .Fresh latitudinal sections of the receptacles of “Red Zaosu” and “Zaosu” were manually cut and temporarily preserved in a 5% (w/v) ascorbic acid solution during microscopic observation. Paraffin sectioning of the “Red Zaosu” receptacle was performed28. Briefly, the extraction buffer was 70% methanol containing 2% formic acid. Extracted anthocyanin was filtered through a 0.45-μm syringe filter prior to HPLC analysis. The anthocyanin concentration was determined by the absorbance at 520 nm on an HP 1200 liquid chromatograph equipped with a diode array detector . The anthocyanin in three biological replicates was quantified based on the calibration curve for a cyanidin 3-galactoside standard .Anthocyanin extraction and quantification were performed as previously describedPbGA2ox8 was divided into four overlapping fragments and amplified using McrBc-PCR. The primer pairs used are listed in Supplementary Table The DNA methylation level was analyzed using McrBC-PCR. In total, 1 μg of genomic DNA was isolated from the young leaves of “Zaosu”, “Red Zaosu”, and the 15F1 hybrid population. The isolated DNA was then digested with 40 units of the methylation-sensitive restriction enzyme McrBC  for 2 h, and the digestion buffer without GTPase was used as the negative control. The methylation level in the digested DNA templates was measured by semiquantitative PCR. The 1300-bp region upstream of the ATG translation site of 18. The full-length PbGA2ox8 complete coding sequence was PCR-amplified from “Red Zaosu” cDNA and then cloned into the pCambia1301 binary vector (replacing the GFP-coding sequence) using a ClonExpress One Step Cloning Kit (Vazyme) with homologous recombination technology. The primer pairs for selected genes and PbActin  are listed in Supplementary Table Agrobacterium tumefaciens strain GV3101 and suspended at 28 °C in the LB medium containing 10 mM MES and 20 μM acetosyringone with the appropriate antibiotics. Agrobacterium cells were harvested, resuspended in infiltration buffer  to a final OD600 of 0.8 and cultured for 4 h at room temperature before infiltration. Plant infiltration was performed by vacuum instead of injection. Young expanded leaves from cultured “Zaosu” explants were completely immersed and vacuumed in 2-mL Agrobacterium suspensions (containing pCambia1301-GA2ox8 and pCambia1301-GFP) at 25 kPa for 5 min. The GFP signals in leaves infiltrated with pCambia1301-GFP were imaged using a BX51 + PD72 + IX71 microscopic imaging system (Olympus). Infiltrated leaves were then cultured on MS medium containing different combinations of 0.1 M sucrose and 1 mM LaCl3  for 5 days at 1200 lx.The transient assay was performed as previously described, with a modified infiltration method4 extracted from mature fruit skin and “Red Zaosu” and “Zaosu” explants was quantified as previously described30. Three biological replicates were assessed. Samples of 200 mg were ground in 1.5 mL of extraction buffer  at 4 °C. The supernatant was filtered through a 0.22-μm syringe filter prior to HPLC analysis. LC-MS2 analysis was performed using an HPLC system (Agilent 1290) coupled to a SCIEX 6500 Qtrap (AB Sciex). Samples were injected onto ZORBAX SB-C18 columns . GA4 was screened and quantified using the multiple reaction monitoring model with a transition of 331.0 > 213.0.GA2, and 50 mM sorbitol) at 4 °C for 5 min in the dark. The calcium signal was assessed using a 450-nm excitation filter in combination with a 520–550-nm emission filter on a BX51 + PD72 + IX71 microscopic imaging system (Olympus). The total calcium concentration was quantified by flame atomic absorption spectrophotometry.The artificially shaded young leaves of “Red Zaosu” explants, artificially bagged carpopodia of “Red Zaosu”, naturally growing young leaves of “Zaosu” explants, and naturally growing carpopodia of “Zaosu” were incubated with fluorescence dye  between “Red Zaosu” and “Zaosu” Fig. , S2. ThePRODUCTION OF ANTHOCYANIN PIGMENT (PAP) type MYBs , the flavonoid biosynthetic genes flavonol synthase (FLS) and UFGTs, and the anthocyanin transporters GLUTATHIONE S-TRANSFERASE F12 (GSTF12) and DETOXIFICATION 35 (DTX35)  Fig. .To determine whether the 26 DEGs were associated with the red trait in “Red Zaosu”, their expression patterns were further investigated in pooled red/green-leafed segregants of “Red Zaosu” × “Yuluxiangli” crosses performed in 2015 (15F1R and 15F1G) and 2016 (16F1R and 16F1G). In total, 13 of the 26 DEGs, including all of the anthocyanin genes, were coexpressed with the red trait in “Red Zaosu” Fig. .PbGA2ox8 was unique in “Red Zaosu”  were clustered into group II, in which the anthocyanin accumulation was high but PbGA2ox8 was expressed at a low level. No progeny from the 15F1 or 16F1 cross was found in group II. Group III members, which had low anthocyanin concentrations and expressed low levels of PbGA2ox8, consisted of 55 anthocyanin-deficient 15F1 lines, 51 anthocyanin-deficient 16F1 lines, and the green pear cultivars, including “Zaosu”  was further isolated from “Red Zaosu” and “Zaosu” , anthocyanin biosynthetic gene PbUFGT1, and anthocyanin transporter PbGSTF12 were upregulated by PbGA2ox8 overexpression  of sis Fig. , b. The ion Fig. .Fig. 5An4 was detected in the fruit skins and leaves of “Red Zaosu” than in those of “Zaosu”, but the GA4 levels in the receptacles of these two cultivars were similar . GA4 repressed anthocyanin accumulation in “Red Zaosu” subcultured explants within 10 days, and paclobutrazol (PAC), an inhibitor of bioactive GAs, induced anthocyanin accumulation in “Zaosu” within subcultured explants after 10 days  near the fruit skin by loading them with violet ink or their observation in paraffinized sections Fig. . In “RedMoreover, large amounts of anthocyanin accumulated in epidermal cells in the leaf, petal, sepal, stamen, style, carpel, hypanthium, fruit stalk, and fruit skin of “Red Zaosu”. In contrast, limited anthocyanin production was observed in most tissues of “Zaosu“ . The red stripes on “Red Zaosu” fruit were enriched in anthocyanins, and the stripes appeared on the “Red Zaosu” fruit surface immediately after receptacle formation were maintained during all of the fruit developmental stages, accompanied by red dots derived from the stomata .The red coloration of the leaf and petal of “Red Zaosu” was dendritic and occurred exactly along the veins derived from vascular bundles . Tube-shaped organs, including the stamen and style, were also completely colored in “Red Zaosu” . Major vascular bundles in the receptacles of “Zaosu” and “Red Zaosu” were classified as lateral carpellary, dorsal carpellary, and ventral carpellary bundles. The red regions in “Red Zaosu” were near dorsal carpellary bundles and ventral carpellary bundles .In summary, anthocyanins were enriched near vascular networks in the epidermal cells of fruit, leaves, and flower organs in “Red Zaosu”.The xylem cells, which are located mostly in the main vascular bundles among VBs and nearby lenticels (fruit dots) in fruitlets, were spatially coordinated with the red stripes and red fruit dots on “Red Zaosu” fruit surfaces Fig. . Based oFree calcium labeled with Fluo-3/AM ester was unevenly distributed and enriched in xylem cells in the fruit and leaves of “Zaosu” and “Red Zaosu” Fig. . SimilarConsistently, the total calcium concentrations quantified by atomic absorption spectrophotometry in “Red Zaosu” leaves were slightly lower than those in “Zaosu” Fig. . The cal3 was applied to “Red Zaosu” fruit during its coloration. Hardly any anthocyanin accumulated in LaCl3-treated “Red Zaosu” fruit. The expression of anthocyanin-related genes was also blocked by LaCl3  have been mapped and identified as responsible for fruit color32. Other PAP-type MYB transcription factors, including MYB10 in sweet cherry, MYB10b in pear, and MYB10 in peach, have also been associated with anthocyanin accumulation in their respective fruit34. However, unlike these typical anthocyanin regulatory genes, in this study, we identified a novel gene that encodes GA2ox8, which contributes to the red fruit trait in the spontaneous bud sport “Red Zaosu”.Anthocyanin accumulation is responsible for the red coloration of some fruits, including apple, peach, strawberry, and pear. The key genes responsible for this trait have been studied. In apple, PbGA2ox8 was expressed exclusively in the red organs of “Red Zaosu”, but not any other red pear cultivars tested in this study -treated “Zaosu” explants . The irregular red stripes observed on the hypanthial surface in “Red Zaosu” were further shown to coincide with the distribution of VBs in the hypanthium (Fig. Regular striping is easily observed on the fruit and flower stalks of some watermelon cultivars. This type of stripe formation may be associated with morphogen diffusion from vascular bundlesium Fig. . SimilarPbGA2ox8-induced anthocyanin biosynthesis was inhibited by LaCl3, indicating that calcium signaling participates in the anthocyanin biosynthesis process (Fig. 16.After we identified the correlation between anthocyanin distribution and the vascular system, we determined that calcium was enriched in xylem cells near vascular bundles and in the stomata of both pear cultivars and spatially coordinated with the anthocyanin distribution in “Red Zaosu” Fig. . MoreovePbGA2ox8 is expressed at a high level with a low level of promoter methylation exclusively in “Red Zaosu”, resulting in a low GA level. This activates the DELLA-mediated anthocyanin accumulation pathway. GA2ox8-induced anthocyanin accumulation occurs preferentially near longitudinal arrays of vascular bundles, resulting in permanent red stripes (Fig. We propose the following mechanism of anthocyanin stripe formation in this spontaneous pear sport. pes Fig. .Fig. 9Merevised Supplementary figures and tablesDataset 3Dataset 1Dataset 2


The distinctive experience of pain, beyond mere processing of nociceptive inputs, is much debated in psychology and neuroscience. One aspect of perceptual experience is captured by metacognition—the ability to monitor and evaluate one’s own mental processes. We investigated confidence in judgements about nociceptive pain (i.e. pain that arises from the activation of nociceptors by a noxious stimulus) to determine whether metacognitive processes contribute to the distinctiveness of the pain experience. Our participants made intensity judgements about noxious heat, innocuous warmth, and visual contrast  and rated their confidence in those judgements . First-order task performance between modalities was balanced using adaptive staircase procedures. For each modality, we quantified metacognitive efficiency (meta-d’/d’)—the degree to which participants’ confidence reports were informed by the same evidence that contributed to their perceptual judgements—and metacognitive bias (mean confidence)—the participant’s tendency to report higher or lower confidence overall. We found no overall differences in metacognitive efficiency or mean confidence between modalities. Mean confidence ratings were highly correlated between all three tasks, reflecting stable inter-individual variability in metacognitive bias. However, metacognitive efficiency for pain varied independently of metacognitive efficiency for warmth and visual perception. That is, those participants who had higher metacognitive efficiency in the visual task also tended to have higher metacognitive efficiency in the warmth task, but not necessarily in the pain task. We thus suggest that some distinctive and idiosyncratic aspects of the pain experience may stem from additional variability at a metacognitive level. We further speculate that this additional variability may arise from the affective or arousal aspects of pain. Independently of metacognitive sensitivity, a person might show a metacognitive bias, that is, a tendency to be over- or under-confident regardless of whether the judgement was correct. These measures jointly characterise how people evaluate their perceptual decisions. Applied to judgements about nociceptive pain—i.e. pain that arises from the activation of nociceptors by a noxious stimulus , innocuous warmth, and visual gratings (contrast). They also rated their confidence in those judgements. We quantified metacognitive access using the ratio meta-d’/d’. This represents the efficiency with which confidence ratings discriminate between ‘correct’ and ‘incorrect’ trials, while controlling for differences in perceptual sensitivity . To exam22.110) for analyses of variance (ANOVAs) across our three conditions were higher than 3.00  or lower than 0.33  indicating how much more (or less) likely the data would be under the alternative hypothesis compared to the null hypothesis  for MATLAB 8.5.0 . The visual stimuli consisted of a central white fixation cross 2° across (luminance: 13.64 cd/m2) and Gabor gratings at 3° of visual angle , presented at ±7.5° eccentricity from the fixation cross. The background was a uniform grey screen (luminance: 3.66 cd/m2). The stimuli were displayed on a 17″ LCD monitor . The display was gamma-calibrated using a CS-100A photometer .Visual stimuli and response prompts were generated in the Cogent 2000 toolbox . The probe was affixed to a computer-controlled haptic device  that was used to jitter stimulus position and to bring the probe into contact with the hand dorsum with a light force of 0.2 N. Skin temperature on the hand dorsum was monitored with a spot infrared thermometer .2.3All participants completed a perceptual intensity discrimination task in three different modalities: visual contrast, innocuous warmth, and nociceptive pain. Participants also completed a manipulation check in which they rated the painfulness of stimuli used in the nociceptive pain and innocuous warmth tasks, to confirm that the temperature ranges were perceived differently. These four tasks were completed in two experimental sessions on separate days. The second session was done within three days of the first session, and at the same time of day. Each session lasted about 1.5 h. The nociceptive pain and innocuous warmth discrimination tasks were always done in different sessions to minimise effects of habituation, sensitisation, or receptor fatigue from repeated thermal stimulation. The order of these tasks was counterbalanced across participants. The manipulation check was always done in the second session, after both the nociceptive pain and innocuous warmth discrimination tasks had been completed. The visual contrast discrimination task was done in the first session with either the nociceptive pain or the innocuous warmth discrimination task. Task order in the first session was counterbalanced across participants.reference stimulus, which was presented at the same stimulus intensity (i.e. the same contrast or temperature) on every trial, and a test stimulus, whose intensity was adapted throughout the task using a continuous 2-down/1-up staircase procedure, in order to keep discrimination accuracy at approximately 70.7%  judgement. Participants were given a short break after every 20 trials. The first 20 trials were considered a practice block, and were not included in any statistical analyses. Each trial consisted of a ly 70.7% . The ord2.3.1not confident) to 4 (confident). Participants were encouraged to use the entire confidence scale over the course of the task. They used a numerical keypad to respond to both prompts , followed by two Gabor patches presented sequentially (200 ms each) with a 300-ms inter-stimulus interval (ISI). The first Gabor patch was presented either 7.5° to the left or 7.5° to the right of the fixation cross , and the second Gabor patch was presented in the other location, in order to mirror the spatial jittering procedure used for the innocuous warmth and noxious heat tasks see , 2.3.3.  prompts a.Fig. 1EThe reference stimulus was always presented with 50% contrast. The test stimulus started at 70% and was adapted throughout the task based on performance. It was increased by 3% following an incorrect response and decreased by 3% following two consecutive correct responses.2.3.2Participants sat with their left hand placed palm down on the table in front of them. Prior to the task, the baseline skin temperature on their left hand dorsum was recorded . Each trial began with a central fixation cross which remained on the screen until response prompts were displayed. The haptic device sequentially delivered two contact thermal stimuli (2000 ms each) to distinct locations on the left hand dorsum with a 3000-ms ISI. Stimulus location was jittered between four different locations on the hand dorsum to avoid peripheral effects such as receptor fatigue or persistent changes in skin temperature. The distance between these locations was adjusted for each participant based on hand size and shape, but was always at least 15 mm. After the offset of the second stimulus, a prompt appeared on the screen asking participants to report which stimulus was warmer. Then participants rated their confidence in their perceptual decision, as described in The reference stimulus was always 38.0 °C. The target stimulus started at 40.0 °C and was adapted throughout the task based on performance. It was increased by 0.5 °C following an incorrect response and decreased by 0.5 °C following two consecutive correct responses. The test stimulus was never increased higher than 43.0 °C—even if a participant made an incorrect response when comparing a 43.0 °C test stimulus with the 38.0 °C reference stimulus—to avoid delivering stimuli in the noxious heat range.2.3.3The procedure of the nociceptive pain discrimination task was the same as the procedure for innocuous warmth discrimination see , except 2.3.4not painful) to 4 (painful). The brief task consisted of 20 trials—10 of each stimulus temperature—in a randomised order.In each trial, a single thermal stimulus (2000 ms) was delivered to the left hand dorsum. The temperature of the stimulus was set to either the lowest temperature delivered in the nociceptive pain discrimination task (i.e. 45.0 °C) or the highest temperature delivered on any trial to each individual participant in the innocuous warmth discrimination task . These temperatures were chosen to ensure that even the most similar stimuli delivered in the nociceptive pain and innocuous warmth discrimination tasks were perceived differently. After stimulus offset, a prompt appeared on the screen asking participants to report how painful the stimulus was on a scale of 1  to check whether our staircase procedures were successful. Then we used participants’ 2IFC intensity judgements and confidence ratings to calculate signal detection theoretic measures of first-order perceptual sensitivity (d’), second-order metacognitive sensitivity (meta-d’), and metacognitive efficiency (meta-d’/d’) for each participant in each sensory modality. To do this, we used a single-subject Bayesian estimation approach, which tends to perform better than the maximum likelihood estimation and sum-of-squared error approaches when there are relatively few trials per subject and condition  to investigate whether individual differences in these four dependent variables were positively correlated across all possible pairs of sensory modalities in our design. For each condition and dependent measure, we report the mean and the 95% credible interval (CI). We used frequentist Steiger’s ge cocor  to compage cocor .http://www.jasp-stats.org). BF10 values indicate how much more likely the alternative hypothesis is than the null hypothesis, given the prior and the evidence  is considered to show strong support for the alternative (or the null) hypothesis  and tests that lack the statistical power to infer support for either the alternative or the null hypothesis (0.33 < BF10 < 3.00). Thus, when reporting the results of these tests below, we distinguish between tests showing evidence for a difference (or correlation) between conditions (BF10 > 3.00), tests showing evidence for no difference (or correlation) between conditions (BF10 < 0.33), and tests that were inconclusive (0.33 < BF10 < 3.00).All Bayesian hypothesis tests were performed in JASP  than in the visual contrast discrimination task , BF10 = 328, and the nociceptive pain discrimination task , BF10 = 5.09 × 104. The comparison between percentages of correct responses in the visual contrast discrimination task and the nociceptive pain discrimination task was inconclusive, BF10 = 0.47. These results indicate that our attempt to hold task difficulty constant across the three sensory modalities was not entirely successful. We placed a strict upper limit of 43.0 °C on the test stimulus in the innocuous warmth intensity staircase so that it would not increase into the noxious heat range. However, some participants gave incorrect answers even at the maximum temperature of the warm test stimulus, so overall performance in this modality was slightly worse than in the other two modalities. Such small but reliable differences in performance reinforce the need to appropriately control for perceptual sensitivity when quantifying metacognition.A Bayesian repeated measures ANOVA showed strong evidence for differences in the percentage of correct responses between sensory modalities, BF3.1.210 = 331.75. Follow-up Bayesian paired samples t-tests showed that perceptual sensitivity was lower in the innocuous warmth discrimination task  than in the visual contrast discrimination task , BF10 = 8.98, and the nociceptive pain discrimination task , BF10 = 74.90. There was no difference between perceptual sensitivity in the pain discrimination task and the visual discrimination task, BF10 = 0.24  between sensory modalities, BF0 = 0.24 a. This pr = 0.05, BF+0 = 0.26. The correlations between the pain and visual discrimination tasks, r = 0.15, BF+0 = 0.48, and the pain and warmth discrimination tasks, r = 0.27, BF+0 = 1.35, were inconclusive  between sensory modalities, BF0 = 0.12 b. Mean mr = −0.01, BF+0 = 0.20. The correlations between the visual and warmth discrimination tasks, r = 0.13, BF+0 = 0.42, and the pain and warmth discrimination tasks, r = 0.28, BF+0 = 1.44, were inconclusive  between sensory modalities, BF0 = 0.32 c. As a gr = 0.42, BF+0 = 10.20.  Further correlation tests indicated no positive correlation between metacognitive efficiency scores in the visual discrimination task and the pain discrimination task, r = −0.04, BF+0 = 0.17. The correlation between the warmth and pain discrimination tasks was low, but inconclusive, r = 0.12, BF+0 = 0.40 . We found a significant difference between the vision-warmth and vision-pain correlations, Z = 2.13, p = 0.033. This further supports the finding of greater shared variance in metacognitive efficiency between the visual and warmth discrimination tasks than between the visual and pain discrimination tasks. Comparisons between vision-warmth and pain-warmth correlations, Z = 1.29, p = 0.198, and between vision-pain and pain-warmth correlations, Z = −0.89, p = 0.372, were not significant. Our Bayesian correlation tests showed strong evidence for a positive correlation between metacognitive efficiency scores in the visual and warmth discrimination tasks, and moderate evidence N = 36). Single-subject estimates of metacognitive efficiency can be noisy, so our estimates of the correlation coefficients may have also been imprecise. To overcome this potential issue, we used a hierarchical Bayesian model to estimate the covariance in metacognitive efficiency between visual, warmth, and pain discrimination tasks. A hierarchical Bayesian model ensures that uncertainty in subject-level parameter estimates appropriately propagates through to uncertainty around estimates of cross-task covariance  between sensory modalities, BF0 = 0.22 d. Mean cr = 0.55, BF+0 = 134; vision and pain: r = 0.60, BF+0 = 589; warmth and pain: r = 0.78, BF+0 = 1.24 × 106; Bayesian Pearson correlations showed strong evidence that individual differences in metacognitive bias were positively correlated across all three sensory modalities  than the highest temperature stimulus in the innocuous warmth range . There was, however, some variability in how the stimuli were perceived, both between and within individuals  between intensity judgements of visual contrast, innocuous warmth, and nociceptive pain c. Some aMoreover, we found that individual differences in metacognitive efficiency were positively correlated between the visual contrast and innocuous warmth discrimination tasks c. Importagainst the existence of a correlation between metacognitive efficiency for vision and nociception  values greater than 1 c. Such ais trend . One posis trend . Our useIn addition, we examined metacognitive bias across vision, innocuous warmth, and nociceptive pain perception. There were no overall differences in confidence between modalities d, and inAltogether, the results of our correlation tests suggest that metacognition consists of both a modality-independent component (i.e. metacognitive bias) and a modality-dependent component (i.e. metacognitive efficiency). The former was a consistent trait of individuals, while the latter differentiated judgements about nociceptive pain. Further, our findings suggest that metacognitive ability does not dissociate between senses serving primarily regulatory or discriminative functions, as has been previously suggested for interoceptive and exteroceptive somatosensory modalities . HoweverConfidence is often modelled as the strength or quality of the evidence that contributes to a first-order decision . HoweverDefinitions of pain routinely insist on its subjective nature, and some hold the view that pain can never have any ‘ground truth’ in the physical properties of the world. Chronic pain conditions, which sometimes lack any apparent neurophysiological aetiology, might encourage this view. In our study, however, participants made judgements about pain that directly resulted from noxious thermal stimulation of nociceptive sensory pathways. Moreover, the 2IFC intensity discrimination task we used was specifically designed to test a discriminative aspect of nociceptive pain, similarly to our tests of innocuous warmth and visual contrast discrimination. By applying signal detection theory, we could determine how much participants’ pain reports were informed by the properties of the evoking stimulus (i.e. the first-order judgement), as well as how people experienced the processes that contributed to the formation of their pain reports . This method allowed us to investigate the relation between judgements about experimentally evoked pain and underlying nociceptive processes, without insisting that pain is reducible to nociception. An alternative approach could have been to ask participants to report which noxious stimulus was hotter, rather than which was more painful. Such an instruction may have induced them to focus on the thermal quality of the noxious stimulation instead of its painfulness. The potential impact of this manipulation on our findings is an open question, and would depend upon whether the unshared variance in metacognitive efficiency for nociceptive pain came from the noxious nature of the stimulus, or from the task requirement to judge pain levels.quality (painful vs non-painful) rather than comparing stimuli of different intensity (more vs less painful). This may have introduced some variance in metacognitive efficiency that was not shared with the other tasks. Future studies could explore these issues by using innocuous and noxious thermal stimulation parameters that separate more clearly along the dimension of painfulness (e.g. innocuous cool temperatures vs noxious heat stimuli).One limitation of our study was an inability to adjust the temperature ranges of innocuous warmth and noxious heat stimulation so that, for every participant, the latter always felt painful and the former never felt painful at all. We were constrained by safety considerations, which placed an upper limit of 50.0 °C on contact thermal stimulation. Additionally, we were constrained by the need to adapt the intensity of the test stimulus throughout the task, so that we could control first-order task performance and specifically test differences between modalities at the metacognitive level. For the innocuous warmth discrimination task, in particular, this often required a large difference between stimulus temperatures. Thus, we could not further separate the innocuous and noxious temperature ranges without compromising these important considerations, even though it meant that participants would sometimes perceive the upper end of the innocuous warmth range as somewhat painful, or the lower end of the noxious heat range as not at all painful . If, as To conclude, we demonstrated that confidence tracks perceptual intensity judgements as precisely for nociceptive pain as for other modalities. However, we found no correlation between metacognitive efficiency for nociception and for vision, and minimal correlation between metacognitive efficiency for nociception and for thermoception. Thus, second-order judgements about nociceptive pain level appear to involve an additional factor, which may be the arousal and/or affective responses typical of noxious stimulation. Metacognitive appraisal is closely linked to higher-order accounts of conscious experience . Our fin


Microtus ochrogaster). We raised animals under two conditions: “socially enriched” , or “socially limited” . Males raised under each condition were either administered daily doses of IN-OT or a saline control for 21 days from postnatal day (PND) 21–42. During this time, we assessed the prosocial behavior subjects demonstrated by evaluating juvenile affiliation (as subadults), alloparental care (as adults no longer being exposed to IN-OT), and partner preference tests to assess tendencies to form adult monogamous pairbonds. We found that “socially limited” males, exhibited increased social contact in juvenile affiliation tests at PND 35 and 42. These males were also more likely to form a partner preference than “socially enriched” males and formed stronger partner preferences overall. IN-OT did not alter these behavioral effects. We also found that “socially limited” males exhibited a distinct response to chronic IN-OT treatment. When compared to all other treatment groups, “socially limited” males that received IN-OT exhibited a greater amount of huddling behavior in the alloparental care test. This effect was, in part, explained by an absence of attack behavior, found only in these males. This study contributes to understanding the complex interactions between the developmental social environment, oxytocin, and social behavior.Interactions between social experiences at different stages of development  can profoundly shape the expression of social behavior. Rarely are the influences of more than one stage of developmental sensitivity to social environment investigated simultaneously. Furthermore, oxytocin (OT) has an extraordinary effect on a breadth of social behaviors, activationally or organizationally. The use of intranasal OT (IN-OT) has become increasingly common therapeutically in humans and scientifically in non-human experiments, however very little attention has been paid to the potential developmental consequences on social behavior that might result. We investigated the effects of early-life social environments and the impact of chronic IN-OT on social behavior at different stages of development in male prairie voles ( Social environments can vary tremendously across stages of development and profoundly shape the social behavior of an individual  density. Indeed, rats that receive higher amounts of maternal care develop increased densities of OTR in important parts of the brain that regulate social behavior, and they exhibit higher maternal care as adults  provides a non-invasive exogenous means to alter central levels of extracellular oxytocin in rodents  raises fundamental questions about the developmental mechanisms that shape adaptive behavioral repertories in adulthood. Moreover, because the social environment during development is complex and varies over time, interactions at different stages of development might create combinatorial interactions on behavioral and brain outcomes. We explored the dynamic interaction between early post-natal social environment, subadult social environment, and chronic dosing of extracellular OT In the current study, we hypothesized that the interaction between perinatal social environments  and subadult social environments (isolated housing or group housing) would result in distinct expression of prosocial behavior during adolescence and adulthood. Furthermore, we predicted that IN-OT would increase the prosocial behavior of “socially limited” prairie voles  to a greater extent than voles experiencing standard (and relatively enriched) rearing. This prediction was based on a previous study that showed “socially limited” voles exhibited higher densities of OTR in regions of the brain implicated in prosocial behavior, including the LS, prefrontal cortex (PFC), and basolateral amygdala .All subjects came from the first litter of breeding pairs created specifically for this experiment. The breeders originated from our colony of prairie voles, which were originally trapped in Champaign County, IL, USA. Animals were housed in standard polycarbonate rodent cages (29 × 18 × 13 cm) lined with Sani-chip bedding and provided nesting material, and kept on a 14:10 light-dark cycle. Animals were provided rodent chow  and water All breeding units created for this experiment had litters culled to 3–5 pups. Male subjects were assigned to groups at birth that exposed them to one of two different social experiences across pre-weaning and post-weaning development. One group of “socially enriched” males was first reared by both a mother and father , and then group-housed with a male sibling at weaning PND 21; . The secBetween PND 21 and 42, subjects received daily intranasal treatments of either saline or oxytocin (0.8 IU/kg) between 08:00 h and 12:00 h . This doSubjects performed a series of behavioral tests during and after the period of intranasal treatment. These included four juvenile affiliation tests, two alloparental care tests, and a partner preference test (see below).N = 11; “socially enriched” + OT, N = 10; “socially limited” + Saline, N = 12; “socially limited” + OT, N = 12.Immediately after intranasal treatment on PND 22, 28, 35, and 42, subjects were placed in a standard-sized cage (29 × 18 × 13 cm) for 30 min of acclimation prior to testing. After acclimation, unrelated and novel juvenile voles (between PND 15 and 21) were placed on the opposite end of the cage from the subject. We assessed the social contact time  between subjects and the juvenile voles during the 10-min test. The timing of these tests correspond with expected release of oxytocin in the brain after intranasal treatment , subjects performed the first of two alloparental care tests. Subjects were placed in a novel standard-sized cage (29 × 18 × 13 cm) to acclimate for 30 min. After acclimation, two unrelated neonates (between PND 2 and 5) were placed at the opposite end of the cage from the subject. The amount of huddling behavior and aggression was quantified during the 10-min test. Huddling behavior was scored as the total time the subject was stationary and completely covering at least one of the two stimulus pups in the test. Aggression was scored as any lunges and biting behavior. An experimenter watched the social interaction from a close distance but out of sight of the voles to ensure that if the subject behaved aggressively to the stimulus pup, the trial could be terminated before the stimulus animal was harmed. A second alloparental care test was implemented on PND 58, as just described. This allowed us to determine behavioral effects both immediately after and weeks after the period of chronic intranasal treatment. This schedule also allowed us to examine the effects of treatment on behavior at an age associated with subadult peripubertal animals (~PND 21–45) and adulthood (~PND > 45). There were two instances where the subject did not move from the corner of the cage at the start of the video ; these recordings were excluded from analysis. The final sample sizes analyzed for the PND 43 alloparental care test were: “socially enriched” + Saline, N = 11; “socially enriched” + OT, N = 9; “socially limited” + Saline, N = 11; “socially limited” + OT, N = 12.Last, on PND 60 subjects performed a partner preference test after 24 h of cohabitation with a sexually receptive female primed with dirty male bedding  according to social manipulation and intranasal treatment, and Tukey HSD determined significant post hoc comparisons between groups. We performed a Pearson’s chi-square test for independence to compare the proportion of subjects displaying a partner preference in the partner preference test. Both chi-square tests  were limited to analysis of main effects  due to sample size constraints when comparing all four groups. We considered an alpha ≤0.05 to be statistically significant for all tests. We report all F and p-values rounded to the nearest one-hundredth decimal place, except where p < 0.01, in which case we report them as such.All behavioral data were collected using Noldus Observer XT 14.0 . For the juvenile affiliation tests, we performed a two-factor ANOVA to compare social contact time between groups according to social manipulation and intranasal treatment, and we used Tukey HSD tests to determine significant F = 1.00, p = 0.32), but “socially limited” males weighed less than “socially enriched” males at PND 28  = 4.21, p = 0.05), PND 35  = 4.82, p = 0.03), and PND 42  = 4.73, p = 0.04). Intranasal OT treatment did not affect body mass at any stage of development. Post hoc comparisons showed no significant difference in body mass between any combination of the four groups . Body mass did not correlate with any behavioral test at the comparable age  = 1.10, p = 0.30) and PND 28  = 1.77, p = 0.19; F = 8.12, p < 0.01) and PND 42  = 5.86, p = 0.02), with “socially limited” males having more social contact time than “socially enriched” males  < 0.01, p = 0.98; PND 28: F = 0.27, p = 0.60; PND 35: F = 0.51, p = 0.48; PND 42: F = 0.22, p = 0.64). Likewise, there were no significant interactions between social environment and intranasal treatment at any age  = 0.53, p = 0.47; PND 28: F = 0.20, p = 0.66; PND 35: F = 0.02, p = 0.90; PND 42: F = 0.01, p = 0.92). Post hoc comparisons showed no differences between any of the four groups .Social rearing environment did not impact the amount of social contact time with juveniles at PND 22  = 4.43, p < 0.01, p = 0.01, p = 0.04, p = 0.99, A mixed-effects ANOVA with a repeated measure for the age of testing revealed a significant interaction between developmental time and social rearing environment on social contact with juveniles (p = 0.03). Post hoc comparisons showed that “socially limited” + OT males spent more time huddling than “socially limited” + Saline males (Dunn’s test: p = 0.01), “socially enriched” + Saline males (p < 0.01), and “socially enriched” + OT males , with post hoc comparisons showing that “socially limited” + OT males spent more time huddling with pups than “socially enriched” + Saline (p < 0.01), and “socially enriched” + OT males , “socially enriched” + OT males  and “socially limited” + Saline males . Similar incidents of attack behavior were found at PND 58 . At both PND 43 and PND 58, no “socially limited” + OT males attacked the pups. When comparing groups according to intranasal treatment, subjects receiving IN-OT  were less likely to attack than subjects receiving saline  at PND 43  = 3.89, p < 0.01) and “socially limited” + Saline (t(10) = 2.53, p = 0.02) males demonstrated a significant preference for the female partner based on side-by-side contact time with each female in the partner preference test (t(10) = 0.48, p = 0.32) and “socially enriched” + OT males (t(8) = 1.02, p = 0.17). Comparison of a partner preference score  revealed a main effect of social environment, with “socially limited” males having larger preference scores than “socially enriched” males  = 4.19, p = 0.05; p = 0.05; Only “socially limited” + OT  with a socially enriched environment (access to two parents and then a sibling). We found that males reared in “socially limited” environments engaged in more social contact with juveniles when they were subadults (PND 35 and PND 42) when compared to males reared in “socially enriched” environments. Furthermore, whereas “socially limited” males increased social contact with juveniles as they became older, males reared in “socially enriched” environments reduced social contact with juveniles as subadults. “Socially limited” males were also more likely to form a partner preference in adulthood when compared to “socially enriched” males. Lastly, chronic doses of IN-OT during post-weaning development led to a high degree of alloparental care behavior in “socially limited” males. This result suggests an additive or synergistic effect wherein a two-hit social deprivation treatment during postnatal development and IN-OT treatment promotes pro-social behavior in male prairie voles  engaged in more social contact in peri-adolescence ; and ii. Our resWe suspect that these inconsistencies between our study and others could be attributable to elements of our experimental design. These differences could include the impact of social isolation at the subadult life stage (which was not investigated in these other studies), the unintended consequences of the necessary handling associated with the delivery of intranasal OT or Saline, or the combination of manipulating social experience at two important stages of development (see below).First, several studies have found that pre-adult social isolation leads to an increase in social interaction . Interestingly, Bales et al.  demonstrvia internalization  is particularly high in juvenile prairie voles and is correlated with alloparental care , and BLA when compared to control subjects .GP conceptualized the experiment, developed the experimental design, analyzed data, and wrote the manuscript. AO conceptualized the experiment, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TSC2. Fourteen adults (2.4%) were newly diagnosed with SEGA during follow-up, and majority had mutations in TSC2. Our findings suggest that surveillance for new or growing SEGA is warranted also in adulthood, particularly in patients with mutations in TSC2.The onset and growth of subependymal giant cell astrocytoma (SEGA) in tuberous sclerosis complex (TSC) typically occurs in childhood. There is minimal information on SEGA evolution in adults with TSC. Of 2,211 patients enrolled in TOSCA, 220 of the 803 adults (27.4%) ever had a SEGA. Of 186 patients with SEGA still ongoing in adulthood, 153 (82.3%) remained asymptomatic, and 33 (17.7%) were reported to ever have developed symptoms related to SEGA growth. SEGA growth since the previous scan was reported in 39 of the 186 adults (21%) with ongoing SEGA. All but one patient with growing SEGA had mutations in  Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by hamartomas in multiple organs, with the brain being the most commonly affected organ , 2. SubeDiagnosis of SEGA has changed from pathology-based to imaging-based , 7, but Onset and growth of SEGA has been reported most commonly in the first two decades of life . In two Present guidelines recommend that patients with asymptomatic SEGA diagnosed during childhood should continue to be imaged periodically as adults to ensure that there is no growth . PatientSurgical resection  is the recommended intervention for acutely symptomatic individuals, while either surgical resection or medical therapy with mammalian/mechanistic target of rapamycin (mTOR) inhibitors can be effective for individuals with growing asymptomatic SEGA . TreatmeThis is the first study evaluating prevalence, growth, symptoms, and treatment patterns in a large prospective cohort of adults with TSC-associated SEGA.TOSCA, a large-scale non-interventional study in patients with TSC, was conducted at 170 sites in 31 countries. The study design and methodology of TOSCA has been published previously . The stup-value < 0.05.In this study, designed prior to the 2012 imaging-based consensus, prevalence, and growth of SEGA were defined as per clinical practice of the participating centres. We evaluated SEGA manifestations among adult patients (>18 years) enrolled into the TOSCA study. SEGA-related questions included in the case report form (CRF) were presence of single or multiple SEGA, newly diagnosed SEGA, SEGA growth, clinical signs, and symptoms associated with SEGA and information regarding SEGA treatment. In addition, possible associations of SEGA prevalence with genotype were analysed using a Chi-square test. Statistical significance was set at Statistics were descriptive considering the exploratory nature of this study. Categorical data were reported as frequencies and percentages, and continuous variables were expressed as mean (± standard deviation) or as median (range), unless stated otherwise.The study was designed and conducted in accordance with the Good Clinical Practice principles, the Declaration of Helsinki and all local regulations. The institutional review board or ethics committee at each participating site approved required TOSCA-related documents. Written informed consent was obtained from all patients, parents or guardians before enrolment.A total of 2,214 patients with TSC were enrolled in TOSCA study, and data were analysed for 2,211 patients. In the final analysis performed on data collected until August 2017, a history of SEGA was reported in 30.3%  of patients. Other neuroimaging features reported included cerebral white matter radial migration lines in 25.5, cortical tubers in 87.2, and subependymal nodules 82.9%.Of the 803 adult patients included in the final analysis, a history of SEGA was reported in 220 patients (27.4%). The demographic of the adult patients with SEGA are shown in The median interval between consecutive scans was 1 year (range <1–34 years). During the study period (up to 5 years), 14 new diagnoses of SEGA were made . The oldest patient with a newly reported SEGA was 57 years. Of the 186 adults with ongoing SEGA, 153 (82.3%) remained asymptomatic, and 33 (17.7%) were reported to ever have developed symptoms related to SEGA growth in the past, including primarily increase in seizure frequency (15.6%), behavioural disturbance (13.4%), and headache (10.8%), either alone or in combination with other symptoms . Over tiTSC2 mutation compared to those with a TSC1 mutation . However, there was no significant difference in multiple (p = 0.1158), bilateral (p = 0.1062), or growing SEGA (p = 1.0000), and presence of SEGA-related symptoms (p = 0.2598) between those with TSC1 and TSC2 mutation. The median age at SEGA diagnosis was higher in patients with TSC1 mutations  compared to patients with TSC2 mutations , but this difference was non-significant , 220 of whom had SEGA (27.4%). During the 5 years follow-up period of the study, 23.2% of adults reported that the SEGA was still ongoing.TSC2 in the large majority of cases (85.7%). Other risk factors such as contrast enhancement of SEN in the caudo-thalamic groove were beyond the scope of this study.The occurrence of new SEGA after the age of 18 years was relatively low (2.4%) but more common than previously thought . In thisAnother key finding was that SEGA growth since previous scan (mean time of 1.5–2.3 years between previous scan and last assessment) was observed in 21% of our adult patients. Although not negligible, this is less frequent compared with children. In a cohort of 58 patients , Tsai et al. reported similar results, with SEGA growth in children being significantly higher than in adults (75.6 vs. 16.5%) .TSC2 since they seem to be at a higher risk for newly occurring SEGA and SEGA growth in adulthood as well as to individuals with intellectual disability who might not be able to verbally express SEGA-related symptoms. Importantly, certain SEGA-related symptoms  are not limited to signs of increased intracranial pressure, and therefore, parents and patients should be informed about all relevant symptoms which require referral for medical evaluation, particularly sudden behavioural changes such as acute-onset and unexplained aggression, academic difficulties or any other acute and unexplained manifestations of TSC-associated neuropsychiatric disorders (TAND)  –18.We acknowledge the limitations intrinsic to a large-scale, international, non-interventional/observational study. These included the fact that participants were recruited from expert TSC centres around the world and the fact that data on SEGA diagnosis, growth and SEGA-related symptoms were collected as reported per clinical practice. However, these limitations are, at least in part, offset by the large-scale and “real-world” nature of the cohort across multiple centres and countries. Being an observational study, detailed information on the treatment initiated for SEGA at adult age were not collected. The very low number of missing data for SEGA reflects good quality of data collection for this specific manifestation.TSC2.Findings from this large international study highlight the need for continued monitoring for SEGA growth in adults with ongoing SEGA. Clinicians and adults with TSC should be aware of the potential new onset SEGA in adults with SEGA-related symptoms, especially in the presence of mutations in http://www.encepp.eu/ (EU PAS Register Number EUPAS3247).Novartis supports publication of scientifically rigorous analysis which is relevant to patient care, regardless of a positive or negative outcome. Qualified external researchers can request access to anonymised patient-level data, respecting patient informed consent, by contacting study sponsor authors. The protocol can be accessed through the EnCePP portal ă de Bioetică a Medicamentului şi a Dispozitivelor Medicale; Comitato Etico Milano area 1 c/o ASST FBF Sacco-P.O. L. Sacco; Comité de Ética de la Investigación de Centro Hospital Universitario Virgen del Rocío; Comité Ético de Investigación Clínica Fundació Sant Joan de Déu Generalitat de Catalunya. Departament de Salut; Comité Ético de Investigación Clínica Hospital Infantil Universitario Niño Jesús; Consejería de Sanidad Dirección General de Salus Pública Junta de Castilla León; Dirección General de Asistencia Sanitaria, Consejería de Sanidad Gobierno del Principado de Asturias; Dirección General de Planificación, Ordenación Sanitaria y Farmacéutica e Investigación, Consejeria de Sanidad y Política Social Región de Murcia; Ethics Committee at Moscow Institute of Pediatrics and Pediatric Surgery; Paula Stradina kliniskās universitātes slimnicas, Attistibas biedribas Kliniskās izpētes Etikas komiteja, Ethics Committee for Clinical Research; The First Affiliated Hospital of The Fourth Military Medical University; Zhongshan hospital Fudan University.The study protocol and all amendments were reviewed and approved (if applicable) by independent ethics committee/institutional review board for each centre: National Hospital Organization Central Ethics Committee; Gazi University Clinical Research Ethics Committee; Independent Multidisciplinary Committee on Ethical Review of Clinical Trials; Peking Union Medical College Hospital; Commissie Medische Ethiek UZ Brussel; CNIL , CCTIRS (Comité Consultatif sur le traitement de l'information en matière de recherche dans le domaine de la santé); CEIC-E ; Ethikkommission der Medizinischen Universität Graz; North Wales REC–West; Regionala Etikprövningsnämnden i Göteborg; REK–Regionale komiteer for medisinsk og helsefaglig forskningsetikk; Komisja Bioetyczna przy Instytucie Pomnik Centrum Zdrowia Dziecka; Ethikkommission bei der Ludwig-Maximilians-Universitat München; Hokkaido University Hospital Independent clinical research Institutional Ethics Committee; Medical Juntendo University Institutional Ethics Committee; National Center for Chile Health and Development of IRB; Osaka University Hospital of IRB; Ethics Committee at Moscow Institute of Pediatrics and Pediatric Surgery; Peking University First Hospital; Sanbo Brain Hospital Capital Medical University; Tianjin Children's Hospital; Childrens Hospital Of Fudan University; Zhongshan Hospital Fudan University; Fudan University Shanghai Cancer Center; The Second Affiliated Hospital of Guangzhou Medical University; The First Affiliated Hospital, Sun Yan-Sen University; The First Affiliated Hospital of Guangzhou Medical University; Shenzhen Children's Hospital; West China Hospital, Sichuan University; Xijing Hospital; Children's Hospital of Chongqing Medical University; Wuhan Children's Hospital; The second affiliated hospital of Xi'an jiaotong university; Guangdong 999 brain hospital; Seoul National University Hospital Institutional Review Board; National Taiwan University Hospital (NTUH) Research Ethics Committee (REC); Institutional Review Board of the Taichung Veterans General Hospital; Institutional Review Board of Chung Shan Medical University Hospital; Institutional Review Board, Tungs' Taichung MetroHarbor Hospital; Institutional Review Board of National Cheng Kung University Hospital; Metro South Human Research Ethics Committee; Sydney Children's Hospital Network Human Research Ethics Committee; St Vincents Hospital Human Research Ethics Committee; Royal Melbourne Hospital Human Research Ethics Committee; Siriraj Institutional Review Board; The Institutional Review board, Faculty of Medicine, Chulalongkorn University, 3rd Floor, Ananthamahidol Building, King Chulalongkorn Memorial Hospital; The committee on Human Rights Related to Research Involving Human Subjects; Institutional Review board, Royal Thai Army Medical Department IRB RTA, 5th Floor, Phramongkutklaowejvitya Building, Phramongkutklao College of Medicine; Research Ethics Committee, Faculty of Medicine, Chiang Mai University; Research and Development, Queen Sirikit National Institute of Child Health; Human Research Ethics Committee, Faculty of Health Sciences, University of Cape Town; Shaare Zedek Medical center Helsinki committee; Sheba Medical center Helsinki committee; Tel Aviv Sourasly Medical center Helsinki committee; General University Hospital of Patras Ethics Committee; Pendeli Children's Hospital Ethics Committee; General University Hospital of Athens G. Gennimatas Ethics Committee; Evaggelismos General Hospital Ethics Committee; General University Hospital of Thessaloniki AHEPA Ethics Committee; General University Hospital of Ionnina Ethics Committee; METC UMC Utrecht; Direcció General de Regulació, Planificació i Recursos Sanitaris; Comité Ético de Investigación Clínica del Hospital Universitario Vall d'Hebron de Barcelona, Generalitat de Catalunya, Departament de Salut; Comité Ético de Investigación Clínica Hospital Universitario La Paz; Dirección General de Ordenación e Inspección, Consejería de Sanidad Comunidad de Madrid, Servicios de Control Farmacéutico y Productos Sanitarios; Comité Etico Investigación Clínica del Hospital Universitario y Politécnico de La Fe; Dirección General de Farmàcia i Productes Sanitaris, Generalitat de Valencia; Comité de Ética de la Investigación de Centro de Granada; IACS ; Comité Etico Investigación Clínica Regional del Principado de Asturias; Comité Etico Investigación Clínica Hospital 12 de Octubre; Comité Etico Investigación Clínica Hospital Universitario Virgen de la Arrixaca; Sección de Ordenación e Inspección Farmacéutica Departamento de Salud; Comité Ético de Investigación Clínica del Hospital Universitario del Río Hortega de Valladolid; CES (Comissão de Ética para a Saúde), Centro Hospitalar de Lisboa Ocidental, EPE; CES (Comissão de Ética para a Saúde), Centro Hospitalar do Porto, E.P.E; CES (Comissão de Ética para a Saúde), Centro Hospitalar Lisboa Central, EPE; CES (Comissão de Ética para a Saúde), Hospital Garcia de Orta, EPE; CES (Comissão de Ética para a Saúde), Centro Hospitalar de São João, EPE; CES (Comissão de Ética para a Saúde), Hospital Professor Doutor Fernando Fonseca, EPE; CES (Comissão de Ética para a Saúde), Centro Hospitalar do Algarve, EPE (Unidade de Faro); LUHS Kaunas Regional Biomedical Research Ethics Committee; Paula Stradina kliniskās universitātes slimnicas, Attistibas biedribas Kliniskās izpētes Etikas komiteja, Ethics Committee for Clinical Research; Komisija Republike Slovenije za medicinsko etiko; Comitato Etico Indipendente Presso La Fondazione Ptv Policlinico Tor Vergata Di Roma; Comitato Etico Regione Calabria Sezione Centro c/o A.O.U. Mater Domini Di Catanzaro; Comitato Etico Azienda Ospedaliera Universitaria Di Cagliari; Comitato Etico Cardarelli-Santobono c/o Ao Cardarelli; Comitato Etico Per La Sperimentazione Clinica Delle Province Di Verona E Rovigo, Presso Aoui Verona; Eticka Komise Fn Brno; Eticka Komisia Dfnsp Bratislava; Eticka Komisia Pri Dfn Kosice; Eticka Komisia Bratislavskeho Samospravneho Kraja; Comisia NaţionalAJ, EB, MB, PC, JF, MF, CH, SJ, JL, AM, RN, VS, MS, RT, BZ, and JK: designing the study, patient accrual, clinical care, data interpretation, drafting, revising, final review, and approval of the manuscript. TC, VC, LD'A, GBA, PV, CF, FO'C, JQ, YT, and SY: designing the study, trial management, data collection, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript. RM: designing the study, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript. SS: designing the study, trial statistician, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript.AJ, EB, TC, VC, PC, GBA, PV, JK, JF, MF, CF, CH, SJ, RN, FO'C, JQ, MS, RT, JL, AM, SY, MB, and BZ received honoraria and support for travel from Novartis. VC received personal fees for consulting, lecture fees, and travel from Actelion, Bayer, Biogen Idec, Boehringer Ingelheim, Gilead, GSK, MSD, Novartis, Pfizer, Roche, and Sanofi; grants from Actelion, Boehringer Ingelheim, GSK, Pfizer, and Roche; personal fees for developing educational material from Boehringer Ingelheim and Roche. PV has been on the study steering group of the EXIST-1, 2, and 3 studies sponsored by Novartis and co-PI on two investigator-initiated studies part-funded by Novartis. RN received grant support, paid to her institution, from Eisai and lectures fees from Nutricia, Eisai, Advicenne, and GW Pharma. YT received personal fee from Novartis for lecture and for copyright of referential figures from the journals and received grant from Japanese government for intractable epilepsy research. SJ was partly financed by the EC Seventh Framework Programme , the Polish Ministerial funds for science (years 2013–2018) for implementation of international co-financed project and the grant EPIMARKER of the Polish National Center for Research and Development No STRATEGMED3/306306/4/2016. JK, PC, CH, JL, and JQ received research grant from Novartis. RM and SS are employees of Novartis, while LD'A was a Novartis employee at the time of manuscript concept approval. This study was funded by Novartis Pharma AG. All authors approved the final version of the manuscript prior to submission.


Stress-related processes have been implicated in the associations between lower socioeconomic status (SES), central adiposity, and cardiovascular disease risk. This study analysed the impact of SES and central adiposity on cardiovascular, inflammatory and neuroendocrine stress responses, and associations with cytomegalovirus (CMV) infection in a sample of 537 men and women aged 53–76 years (mean 62.89 years). SES was defined by grade of employment , and central adiposity was indexed by waist-hip ratio (WHR). Cardiovascular, inflammatory and cortisol responses were monitored during administration of a standardized mental stress testing protocol and salivary cortisol was measured repeatedly over the day. Lower SES was associated with raised systolic and diastolic blood pressure (BP), plasma interleukin (IL-6), fibrinogen, C-reactive protein, and salivary cortisol, and a large WHR accentuated SES differences in fibrinogen, C-reactive protein, and likelihood of CMV seropositivity, independently of general adiposity indexed by body mass index. During mental stress testing, return to resting levels (recovery) following behavioural challenge in systolic and diastolic BP and heart rate was impaired among lower SES participants, particularly those with large WHR. Lower SES participants had greater cortisol concentrations across the day, but this pattern did not vary with WHR. These findings extend the evidence relating lower SES to stress-related biological risk factors for cardiovascular disease, and indicate that central adiposity may augment these effects. LifestyCentral or visceral adiposity is a growing health problem, and is a pivotal risk factor for cardiometabolic disease . Fat depSince central adiposity is more common among lower than higher SES groups, it is possible that the two share common pathways to cardiovascular risk through disturbed stress biology. However, the combined effect of lower SES and central adiposity has not previously been examined. We therefore investigated the impact of SES and central adiposity on inflammatory, hemodynamic and neuroendocrine responses to psychological stress in a sample of healthy middle-aged and older men and women drawn from the Whitehall II epidemiologic cohort. We hypothesized that lower SES and greater central adiposity would be associated with delayed cardiovascular (BP and heart rate) recovery following acute stress, with greater inflammatory responses to stress, greater cortisol output over the day, and with higher baseline levels. We further conjectured that these two factors might be synergistic, with the greatest responses emerging among lower SES individuals with greater central adiposity. Because the focus of this study was on central adiposity, body mass index (BMI) was included as a covariate in the analyses to take account of overall adiposity.We also evaluated associations between SES and central adiposity and cytomegalovirus (CMV) serostatus. Herpesviruses such as CMV are frequently acquired early in life, and are more common among lower SES individuals as defined by education, poverty or parental occupational status . Positiv22.1We recruited a sample of 543 healthy white European men (n = 294) and women (n = 249) aged 53–76 years (M = 62.89 ± 5.65) from the Whitehall II epidemiological cohort of British civil servants . To be i2.22). Waist circumference was assessed using a metal tape halfway between the bottom rib and iliac crest, and hip circumference was measured at the level of the great trochanters. The measurements were conducted twice to ensure accuracy, and WHR was calculated as waist circumference/hip circumference.Questionnaires were completed before the mental stress testing sessions that were conducted individually in a light and temperature controlled laboratory in either the morning or the afternoon between 2006 and 2008. At the start of the session, anthropometric measurements were taken. Height was measured to the nearest 0.1 cm using a stadiometer and weight was measured in kilos (kg) using Tanita Scales. Body Mass Index (BMI) was subsequently calculated using this information . There followed a 30 min resting period, the last 5 min of which was used to measure baseline systolic and diastolic BP and heart rate. At this time the first blood sample was drawn into EDTA and citrated tubes, and a saliva sample was taken using Salivettes .Two 5-minute behavioural tasks that have been used to induce stress in a number of previous studies were then administered in a counterbalanced order . These tA second blood sample and a saliva sample were taken immediately after the tasks, while systolic and diastolic BP and heart rate were measured continuously throughout the tasks and recovery period. Further blood samples were obtained 45 min and 75 min after task completion, with saliva samples at 20, 45 and 75 min during the recovery period.Following the mental stress testing session, participants were asked to take 5 saliva samples over the next day, following methods previously used in this laboratory . Samples2.3Blood samples were centrifuged immediately at 2500 rpm for 10 min at room temperature and the separated plasma was aliquoted into 0.5 ml portions stored at −80 °C until further analysis. Three inflammatory markers were analysed. Plasma IL-6 was assayed using a Quantikine high sensitivity two-site enzyme-linked immunosorbent assay (ELISA) from R&D Systems . The sensitivity of the assay ranged from 0.016 to 0.110 pg/ml and the intra and inter assay CVs were 7.3% and 7.7% respectively. Fibrinogen was measured from citrated blood with an automated Clauss assay in a MDA-180 coagulometer  using the manufacturer's reagents and the International Fibrinogen Standard, with inter- and intra-individual CVs of 8%. High sensitivity C-reactive protein was assayed in duplicate with a high sensitivity enzyme immunoassay kit . Glycated haemoglobin (HbA1c) was measured using a Tosoh G7 high performance liquid chromatography analyser  calibrated to Diabetes Control and Complications Trial standards. CMV IgG antibody titres were measured from thawed serum samples using a solid-phase enzyme immunoassay system as described previously . A sampl2.42 tests for categorical variables. Cardiovascular data were averaged over 4 periods: the last 5 min of the baseline period (baseline), the two behavioural task trials, and 40–45 min and 70–75 min following tasks (recovery phase). Heart rate variability was modelled as root mean square of successive differences (RMSSD) over the same time periods, and was log transformed before analysis because of negative skew. The total cortisol output over the stress session was computed using the area under the curve (AUC) method  method , and a sBaseline values of biomarkers were analysed by analysis of covariance with SES (grade of employment) and central adiposity (WHR) group as between-person factors, covarying for age, sex, and smoking status, since these potentially influence results. Analyses of salivary cortisol also included time of day of laboratory session (morning/afternoon) as a covariate. In order to identify effects related specifically to central adiposity, BMI was added as a further covariate to all analyses. Linear contrasts tested gradients across SES groups. The number of participants included in each analysis differed because of missing data, so the sample sizes are indicated in Stress responses were analysed using repeated measures analysis of variance with SES and WHR as between-person factors and trial as the within-person factor. There were 4 trials in the cardiovascular and IL-6 analyses: baseline, during task for cardiovascular variables and immediately after tasks for IL-6, and 45 and 75 min after tasks. Fibrinogen was sampled at 3 time points since levels return to baseline promptly after stress: baseline, immediately after tasks, and at 45 min . SalivarC-reactive protein and HbA1c were measured once, and were analysed in the same fashion as the baseline values of biomarkers. We analysed CMV serostatus using logistic regression, and present the odds of CMV positivity  adjusted for age, sex, smoking status and BMI, with the higher SES / small WHR group as the reference category.We also carried out two sensitivity analyses. First, we investigated the impact of WHR modelled as a continuously distributed as opposed to a categorical variable. Because of sex differences in WHR, normalised scores were used in these analyses. Second, we reanalysed all data with only SES and WHR in the models, without covarying for age, sex, smoking status, and BMI.3p = 0.015, since the lower SES participants were slightly older on average. Lower SES participants were more likely to be unmarried (p = 0.007), while educational attainment was positively associated with SES, with a substantially greater number of higher than lower SES volunteers being educated to University level (p < 0.001). There was no difference in the proportion of participants in the small and large WHR categories across SES groups, and no difference in gender distribution. As might be expected, BMI, average waist circumference and average hip circumference were all greater in the large than small WHR groups (p < 0.001). Additionally, there was a non-linear SES difference in waist circumference (p = 0.007), with slightly larger waists on average in intermediate than higher or lower SES respondents. There was a moderate positive correlation between WHR and BMI (r = 0.41), but tests for multicollinearity (variance inflation factors and tolerance) were all within acceptable ranges. Measures of task appraisal showed no differences related to SES or WHR in the extent to which tasks were perceived as difficult, involving, or controllable (results not shown).There were 207 higher, 209 intermediate and 121 lower SES participants as defined by occupational grade . There w3.1p < 0.001), with lower BP in higher status participants. There were no SES differences in heart rate or heart rate variability at baseline. Systolic BP was not related to WHR, but both diastolic BP and heart rate were higher in the large WHR category after adjusting for covariates  = 5.83, p = 0.016 and F  = 4.40, p = 0.037). Baseline plasma IL-6  = 3.03), fibrinogen  = 4.83) and C-reactive protein  = 2.29) all showed significant gradients across SES groups , with higher levels of inflammation in lower SES groups after adjustment for covariates. There was an SES gradient in baseline salivary cortisol as well  = 3.67, p = 0.009), with a higher concentration in lower SES participants. There were no significant differences in HbA1c levels. The interaction between SES and WHR was significant for plasma fibrinogen,  = 9.46, p < 0.001), and C-reactive protein,  = 4.20, p = 0.016). In both cases, the highest values were observed in the lower SES/large WHR group.Baseline levels of cardiovascular, inflammatory and other biological measures are summarized in 3.2p = 0.005), and a 3-way SES by WHR by trial cubic effect  = 3.24, p = 0.040). In the case of diastolic BP, there were main effects of WHR  = 5.73, p = 0.017) and SES  = 7.21, p < 0.001), plus significant SES by trial  = 4.50, p < 0.001), and SES by WHR by trial cubic effects  = 3.36, p = 0.036). These results are summarised in p < 0.001) and 75 min following stress  = 12.70, p < 0.001). SES effects remained significant after additional control for baseline and levels during tasks  = 3.57 and 3.93, p = 0.008 and 0.011 respectively), so were not secondary to any differences in baseline or stress reactivity. Additionally, SES and WHR interacted in recovery at 45 min  = 4.01, p = 0.019). These results are summarized in p = 0.003), with a SES and WHR interaction at 45 min showing that central adiposity moderated these SES differences  = 4.03, p = 0.018). As in the case of systolic BP, SES differences in recovery remained significant following additional adjustment for baseline and task levels at 45 min  = 4.57, p = 0.004) and 75 min  = 6.94, p = 0.002).Repeated measures analysis of variance of systolic BP identified a main effect of SES  = 8.17, p < 0.001), together with an SES by trial interaction  = 3.58, p = 0.034), but no 3-way interaction. In contrast with the BP results, there was a significant difference in heart rate reactions across SES groups  = 4.36, p = 0.004), with larger increases in the higher SES compared with lower SES participants (see p = 0.004), with more effective recovery among higher SES participants (p < 0.001), falling from baseline during tasks, and recovering above baseline after tasks. This pattern did not vary with SES, but there was a significant WHR by trial interaction  = 2.81, p = 0.048). Responses to tasks were comparable, but the large WHR group showed better post-stress recovery than the small WHR group at 45 min (p = 0.037).The analysis of heart rate revealed an SES by trial interaction  = 4.02, p < 0.001) and a main effect of WHR  = 4.34, ants see . Howevericipants . These r3.3p < 0.001). There was also a significant SES effect  = 2.30, p = 0.033), with higher IL-6 concentrations in lower SES participants after adjustment for age, sex, smoking status and BMI (p = 0.030). The large WHR group showed smaller increases in IL-6 concentration over the session after adjustment for covariates, but there was no interaction between SES and WHR. The pattern of plasma fibrinogen responses differed from that for IL-6, in that increases with tasks were immediate and transient  = 4.99, p = 0.007). There was a significant main effect for SES, together with an SES by WHR interaction  = 8.29, p < 0.001). As can be seen in Plasma IL-6 increased following stress, showing a delayed response with raised levels 45 and 75 min after tasks  = 125.5,  and BMI . The acup = 0.041, mirroring the socioeconomic gradient at baseline described in p = 0.017). The elevated cortisol in the lower SES group is apparent throughout the session, but there is no clear relationship with stress responsivity or recovery.The salivary cortisol AUC showed a main significant effect of SES  = 2.10, 3.4p = 0.009). The prevalence of positive CMV serostatus was 44.8% in the higher SES/small WHR group compared with 66.0% in the lower SES/large WHR group.A substantial proportion of respondents were positive for CMV, indicating that past infection was relatively common. 3.5p = 0.006), with greater cortisol output among lower SES individuals  = 4.93, ividuals . There w3.6The first sensitivity analysis investigated whether associations would be different if WHR modelled as a continuous variable rather than categorising individuals as having larger or smaller WHR. The results, summarised in The second sensitivity analysis assessed the associations of SES and WHR with biology in the absence of statistical adjustment for age, sex, smoking status and BMI. The results are summarised in 4This study investigated the separate and combined associations between lower SES and central adiposity and a range of stress-related biological processes. We hypothesised that lower SES and greater central adiposity (waist-hip ratio) would be associated with impaired post-stress recovery in cardiovascular parameters, with heightened inflammatory and neuroendocrine responses to stress, and with greater cortisol output over the day. We also conjectured that the combination of lower SES and greater central adiposity would be particularly toxic. The results confirmed and extended previous studies of SES and cardiovascular, inflammatory and neuroendocrine factors, and identified important interactions between lower SES and central adiposity in relation to inflammatory markers, CMV infection, and post-stress recovery of cardiovascular function.Associations between SES and acute cardiovascular stress responsivity have been investigated in a number of studies. A meta-analysis in 2018 showed no consistent relationship between SES and BP or heart rate reactivity, but differences emerged in post-stress recovery, albeit in a sample of only 6 studies . The preAlthough central adiposity did not affect cardiovascular reactivity or recovery directly, we observed interactions between SES and WHR in the post-stress recovery profile of systolic and diastolic BP. Blood pressure recovery was markedly impaired in the lower SES /large WHR group. For example, the decrease in systolic BP from task levels at 45 min averaged 26.6 mmHg in the higher SES/small WHR group, 22.15 mmHg in the lower SES/small WHR group, but only 16.22 mmHg in the lower SES/large WHR group . ImpaireResting values of all three inflammatory markers in this study  were inversely associated with SES in a graded fashion . This reThe cortisol responses to this protocol were small, although individual differences still predicted variations in cardiovascular risk and telomere attrition longitudinally . SalivarThe absence of differences in cortisol responses between WHR groups was unexpected, since several previous studies have shown associations . It is nThe interaction between lower SES and greater central adiposity in the proportion of respondents with CMV infection has not been documented before . A socioThis study was carried out with a well characterised population that was selectively sampled to encompass a broad range of SES. Existing cardiovascular disease was excluded, so that associations are not secondary to differences in health status. The size of the sample was large by comparison with many investigations of stress-related inflammatory responses. However, the selection process resulted in a sample of individuals with relatively normal body weights, and only 14% had a BMI ≥ 30. The impact of central adiposity may be greater among people who have more general obesity. The study was carried out with men and women of white European origin, and results may not generalise to other groups. SES was defined by occupational status, and other criteria such as educational attainment or income might generate different results. The cross-sectional design makes it impossible to draw causal conclusions about the directionality of effects. It is likely that there is a bidirectional relationship between central adiposity and stress biology, with greater abdominal fat contributing to the expression of adipokines and other markers, while stress stimulates more rapid accumulation of fat stores. Findings may also be limited by having only one session of mental stress testing and sampling cortisol over a single day. Nonetheless, the findings extend the evidence relating lower SES to stress-related biological risk factors for cardiovascular disease, while indicating that central adiposity may augment SES differences in inflammation and impaired recovery in cardiovascular function following acute mental stress.The authors have no conflicts of interest to declare.British Heart Foundation (RG/10/005/28296).This research was supported by the


Kümmell’s disease  cannot heal spontaneously. Bone-filling mesh container (BFMC) can significantly relieve pain, help the correction of kyphosis, and may prevent cement leakage. This pilot study may provide the basis for the design of future studies.To compare the effectiveness and safety of BFMC and percutaneous kyphoplasty (PKP) for treatment of Kümmell’s disease.From August 2016 to May 2018, 40 patients with Kümmell’s disease were admitted to Guizhou Provincial People’s Hospital. Among them, 20 patients  received PKP (PKP group) and the other 20 received BFMC (BFMC group). Operation time, Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), Cobb’s angle changes, and related complications were recorded.p < 0.05). Postoperative Cobb’s angle of both groups postoperatively was lower than preoperatively (p < 0.05). Cement leakage occurred in eight vertebrae (8/20) in the PKP group and in one vertebra (1/20) in the BFMC group. No complications such as pulmonary embolism, paraplegia, or perioperative death occurred during operation in both groups. Adjacent vertebral refractures occurred in five patients (5/20) in the PKP group and in four patients (4/20) in the BFMC group, with no significant difference in the incidence rate of refractures in both groups but the material is too small to verify statistically.All patients underwent operations successfully. VAS scores and ODI of both groups at each postoperative time point were lower than preoperatively, with statistically significant difference (Both PKP and BFMC technologies can significantly relieve pain and help the correction of kyphosis while treating Kümmell’s disease. Moreover, the BMFC may prevent cement leakage. Osteoporotic vertebral compressive fractures are common lesion in the elderly , 2. MostPatients with Kümmell’s disease are often multi-diseased and are in poor physical condition, and crevasse filling with bone cement may theoretically stabilize the spine and obviously relive pain. For such patients, the most commonly used methods at present are percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) . HoweverInclusion criteria are as follows: (1) single vertebral Kümmell’s disease without neurological symptoms; (2) patients aged above 60 years; (3) severe osteoporosis ; (4) radiological imaging examination showing vacuum sign, fissure sign, or sclerosis at the fractures site. Exclusion criteria are as follows: (1) need decompression to relieve nerve compression; (2) multiple-level compression fractures; (3) history of vertebroplasty or spinal surgery; (4) vertebral fractures or vertebral space occupying lesion due to tuberculosis, tumor, or otherwise; (5) coagulation dysfunction; (6) surgery not tolerable due to severe internal medicine diseases.Forty patients with Kümmell’s disease were included between August 2016 and May 2018, 22 patients with thoracic vertebrae, and 18 with lumbar vertebrae lesions. The patients were randomly divided into 2 groups, each with 20 patients. The vertebral body distribution and other general conditions are shown in Table All patients were taken in prone position. The skin of the surgical site was disinfected and draped routinely. One percent lidocaine was given as local anesthesia, and the puncture needle was inserted into the vertebral posterior margin of the vertebral pedicle. The balloon was embedded into the anterior 1/3 of the vertebral body by solid vertebral drill for dilatation. After the balloon was withdrawn, polymethyl methacrylate bone cement was injected with a bone cement injector guided by a C-arm  in the PKP group. However, in the BFMC group, after the balloon was withdrawn, BMFC  and special kyphoplasty equipment  were inserted. The material of the bag body is 75D/36F high-strength wire comprising 100% polyethylene terephthalate with a fineness of 166.5 dtex and a strength of 6.75 CN/dtex. The single-layer bag is made of a relatively thick high-strength wire that exhibits good biocompatibility . PolymetPatients returned to the wards without symptoms from lower limbs or respiratory discomfort after operation and were required to stay in bed for 6 h.Operation time, Visual Analogue Scale (VAS) , and OswCement leakage, incidence rate of cement leakage, and leakage locations could be observed through intraoperative C-arm and postoperative CT reexamination  , 15. ComF represented significant test statistics of results mean difference at different times, while p < 0.05 meant that differences were considered statistically significant.Measurement data were expressed as mean ± standard deviation, while non-normally distributed data were expressed as median and quartile. Preoperative and postoperative ODI, kyphotic angle, and operation-related complications were statistically analyzed and evaluated by using repeated measure analysis of variance. VAS was ranked data, which was verified by adopting rank sum. Value p < 0.05, Table The operation time of the PKP group was 33–55 min ([43.30 ± 7.20] min) and 32–55 min ([43.80 ± 7.61] min) in the BFMC group, with no statistical significance between the two groups. The VAS score of the PKP group at day one before and after operation as well as at 1 and 6 months after operation were 7 points (7–8 points), 2 points (2–3 points), 2 points (2–3 points), and 1 point (1–2 points), respectively, while those of the BFMC group were 7.5 points (7–8 points), 2 points (2–3 points), 1 point (1–2 points), and 1.5 points (1–2 points), respectively. VAS scores of both groups at each postoperative time point were lower than preoperatively, with statistically significant difference ° and (16.90 ± 1.37)°, respectively, while that of the BFMC group was (23.16 ± 0.83)° and (16.79 ± 1.59)°, respectively. Values of postoperative Cobb’s angle of both groups were lower than those of preoperative Cobb’s angle, with statistically significant difference  in the PKP group had cement leakage into the intervertebral disk and paravertebral tissues during operation, but no patient showed any clinical symptoms. Five patients had leakage into intervertebral space, two into paravertebral tissues or paravertebral veins, and one into the spinal canal. Only one vertebra (1/20) in the BFMC group had cement leakage into paravertebral tissues, with a cement leakage rate obviously lower than that of the PKP group, with a statistically significant difference p < 0.0, Table 5There were no perioperative complications. Adjacent vertebral fractures occurred in 5 (5/20) patients in the PKP group and in 4 (4/20) patients in the BFMC group, which showed no statistical difference between the two groups.Osteoporotic vertebral compression fractures are a common fractures in the elderly. Most patients can have symptoms relieved after several weeks of non-surgical treatment, but about 1/3 of the patients have continuous pain and discomfort and about 10% of the patients will develop vertebral collapse due to post-traumatic osteonecrosis . In 1895. However, in previous studies, it has been reported that PKP has advantages over PVP in reducing the cement leakage rate [At present, non-surgical and surgical treatments are used to treat Kümmell’s disease. Most of Kümmel’s diseases often develop into chronic back pain or even cripple . Surgicaage rate –27. In oage rate  found thp < 0.05). Oswestry Disability Indexes (ODI) at each postoperative time point are all lower than preoperative ones, and the differences are statistically significant (p < 0.05). Postoperative Cobb's angle value postoperative is lower than that preoperative,with statistical difference (p < 0.05), which indicates that both simple percutaneous kyphoplasty and percutaneous kyphoplasty with BMFC have good curative effects of significantly relieving pain, increasing vertebral strength, effectively preventing the aggravation of kyphosis, and obviously improving patients’ living quality.In our study, VAS scores of patients in both groups at each postoperative time point are all lower than preoperative ones (In the current study, cement leakage was found in only one case in the BFMC group, with significantly lower leakage rate than that in PKP group, but it should be regarded cautiously due to the small sample sizes. The BFMC used in the current study is a newly developed inflatable mesh-bag-shaped bone filler that is produced by Shandong Guanlong Medical Products Co. Ltd. Shandong, China. During the cement injection of PVP augmentation, bone cement can fully diffuse through specially designed meshes, and intravertebral cement distribution can be controlled by mesh bag to reduce the risk of cement leakage.Follow-up reveals that some patients have adjacent vertebral fractures that often occur in the inferior adjacent vertebral body of the diseased vertebra. Five patients in the PKP group have postoperative adjacent vertebral refractures, compared with 4 patients in the BFMC group, but with no statistical difference. The causes for adjacent vertebral refractures may be the high strength of polymethyl methacrylate bone cement, which generates “pillar effect” after being injected into the vertebra , which tIn our material of mainly Kümmell’s disease in phases I and II, a few have only spinal canal compression without neurological symptoms in phase III Fig. . The recIn conclusion, while both BMFC and PKP can relieve pain, restore vertebral height, and correct kyphosis, BFMC may also reduce the incidence rate of cement leakage during operation for patients with Kümmell’s disease in phase III with posterior wall breakage and spinal canal compression but without neurological symptoms. Therefore, considering the effectiveness and safety of the treatment of Kümmell’s disease in phase III with bone cortex breakage, BMFC should be further tested.As BMFC technology has been applied for treatment of Kümmell’s disease for a short time, this pilot study may provide the basis for the design of future studies. There are few reports about mesh bag and long-term follow-up data, and large-sampled, multi-center and long-term follow-up studies need to be conducted.