N = 90), we demonstrate that intensity in fact has a paradoxical role. Participants were asked to rate and classify the authenticity, intensity and emotion, as well as valence and arousal of the wide range of vocalizations. Listeners are clearly able to infer expressed intensity and arousal; in contrast, and surprisingly, emotion category and valence have a perceptual sweet spot: moderate and strong emotions are clearly categorized, but peak emotions are maximally ambiguous. This finding, which converges with related observations from visual experiments, raises interesting theoretical challenges for the emotion communication literature.Vocalizations including laughter, cries, moans, or screams constitute a potent source of information about the affective states of others. It is typically conjectured that the higher the intensity of the expressed emotion, the better the classification of affective information. However, attempts to map the relation between affective intensity and inferred meaning are controversial. Based on a newly developed stimulus database of carefully validated non-speech expressions ranging across the entire intensity spectrum from low to peak, we show that the intuition is false. Based on three experiments ( Importantly, both discrete-emotion and dimensional theories predict this pattern of results, although by different mechanisms: either maximized distance to other emotions by the increasing recruitment of diagnostic content  or through maximized distance in the affective space encompassed by the dimensions valence and arousal8 . In other words, the prevailing approaches conjecture less confusion or ambiguity for the classification of highly intense expressions than for intermediate ones, as the distinctiveness of emotion expressions is predicted to increase with increasing intensity.Whether conveyed by the face, body, or voice, expressions of emotion are ubiquitous. The inferred meaning of the expressions is, generally speaking, substantially aligned with the affective content expressed, and it is intuitive to suggest that the stronger the expressed affective state the more clear-cut the inferred emotional meaning. Indeed, a body of research suggests that high-intensity emotion expressions are better ‘recognized’11 and vocal12 expressions of peak emotional intensity. In the latter study, vocalizations of extreme positive valence could not be disambiguated from extreme negative valence. Moreover, these authors demonstrated a trend opposite the predicted relation for peak intense positive situations: the reactions of real-life lottery winners were rated more negatively as hedonic intensity (in this case cued by the prize sum) increased. They argue that peak emotion expression is inherently ambiguous and reliant on contextual information14.This generalization has been challenged by the discovery of perceptual ambiguity for facialone aspect of affective experience  is hard to differentiate. Valence along with arousal are thought to constitute essential building blocks of core affect16. Hence, its compromised perceptual representation invites the speculation that peak intense vocalizations do not convey any affective meaning. But it is not known whether arousal, an equally fundamental property of affect, is similarly indistinctive. Moreover, the data raise the question whether individual emotions of the same or opposing valence can be differentiated, or if only peak positive affect is unidentifiable.The research on the ambiguity of intense expressions is intriguing, but key issues lack sufficient evidence to refine our theoretical understanding. The studies on peak emotion elegantly contrast positive and negative affect. As such, peak emotion, or if affective expressions are generally more ambiguous than previously thought13. The one group of studies largely base their interpretation on results obtained with moderately intense emotion expressions; peak intensity emotional states were not examined. On the other hand, the data challenging this interpretation exclusively address peak emotional states. In summary, the data motivating these ideas are too sparse to adjudicate between the theoretical alternatives.These considerations are important to understand the complex role of emotion intensity. From an analytic perspective, the two types of studies yielding the contradictory evidence are difficult to compare. The contrast differs between the groups of studies . Additionally, it is unclear whether ambiguity is specific to Various questions arise. First, what underlies the perceptual ambiguity, that is, which aspects of emotion lack a differentiable perceptual representation? Are valence, arousal, and emotion category equally affected—and is ambiguity a general property of emotion communication? Second, how does affective information vary as a function of emotion intensity, if not linearly, as previously assumed—and what are the resulting theoretical implications? We illuminate the seemingly contradictory findings and provide insight into the processes of nonverbal emotion expression.18. They are largely shaped by physiological effects on voice. Such effects, associated with sympathetic activation or arousal, can be perceived through characteristic changes in vocal cues20 and play a role especially in the communication of strong emotion22. Specifically, for nonverbal expressions arising from extreme situations, little voluntary regulation and socio-cultural dependency are expected24. Emotionally intense vocalizations—in negative as well as positive contexts—oftentimes encompass harsh sounding call types such as screams, roars, and cries27. On a functional account, the characteristic acoustic structure  seem ideal to capture listener attention27. Importantly, acoustic signatures are linked to high attention and salience as well as the perception of arousal across species and signal modalities33. Their biological relevance thus seems irrefutable.Nonverbal vocalizations reflect variable degrees of spontaneity, cognitive control, social learning, and cultureThough valence and arousal are equally fundamental in emotion theoretical frameworks, it is implausible to assume that the human voice does not signal physical activation or arousal in the most extreme instances of emotion. In fact, from an ethological perspective, a perceptual representation of arousal as well as the specific intensity of the emotional state seem essential, even when overall valence and the specific type of emotion cannot be identified.Variably Intense Vocalizations of Affect and Emotion Corpus (VIVAE). The corpus, openly available (http://doi.org/10.5281/zenodo.4066235), encompasses a range of vocalizations and was carefully curated to comprise expressions of three positive  and three negative affective states , ranging from low to peak emotion intensity. Perceptual evaluations were performed by N = 90 participants, who in three separate experiments classified , rated the affective dimensions valence and arousal (Experiment 3), and rated perceived authenticity (Experiments 1 and 3). We hypothesized that listeners would be able to classify emotional categories significantly above chance (Experiments 1 and 2) and to rate the affective properties of the stimuli congruently with the expressed affective states (Experiment 3). The critical hypothesis was as follows: All judgments were examined as a function of emotion intensity, which we expected to have a systematic effect on stimulus classification (Experiment 1) and on perceptual ratings (Experiments 2 and 3). Following the theoretical frameworks, we predicted that intensity and arousal would be classified clearly over the range of expressed intensities, while, in line with recent empirical data, the amplifying role of emotional intensity on the classification of valence and emotion category would plateau at strong emotion. Peak emotion should be maximal in received intensity and arousal—however, valence and emotion category would be more ambiguous. Together, we conjectured a paradoxical effect of the intensity of expressed emotion on perception, a finding not easy to accommodate by current versions of categorical and dimensional theories of emotion.To address specifically the influence of emotional intensity on emotion perception, we use nonverbal vocalizations from a newly developed database, the emotion categorization task  = 12.91 for achievement, 21.57 for anger, 13.85 for fear, 19.54 for pain, 18.02 for pleasure, 13.54 for surprise, Bonferroni-corrected ps < 0.001, ds > 2.36). Of the expressions with incongruent emotion classification, positive expressions were more likely to be misclassified as negative (t(29) = − 5.36, p < 0.001, d = − 0.98), whereas negative expressions were equally likely to be confused within as across valences (t(29) = 0.95, p = 0.35, d = 0.17).In the emotion rating task  = 215.55 for achievement, 178.15 for anger, 135.53 for fear, 173.63 for pain, 171.93 for pleasure and 124.06 for surprise, ps < 0.001; all but one pairwise comparisons contrasting the matching scale ratings with the other scale ratings were significant at p < 0.001; p = 0.04 for achievement-surprise). Above chance classification for each emotion is reported in Supplementary Table Comparing participants’ ratings on each of the six emotion scales in the Congruence between expressed and perceived intensity is reflected in the monotonic increases depicted in Fig. F = 15.10, p < 0.001, ηp2 = 0.08) and intensity  = 266.07, p < 0.001, ηp2 = 0.68), and a significant interaction  = 9.91, p < 0.001, ηp2 = 0.03). Planned comparisons confirmed systematic differences in participants’ ratings, with low  < moderate  < strong  < peak emotion intensity ratings . Post hoc comparisons of the interaction are reported in Supplementary Fig. For the Experiment 1, the Emotion × Intensity rmANOVA revealed significant main effects of emotion  = 17.05, p < 0.001, ηp2 = 0.05), intensity  = 204.00, p < 0.001, ηp2 = 0.57), and a significant interaction  = 10.85, p < 0.001, ηp2 = 0.04). In line with the results from Experiment 1, planned comparisons confirmed an increase in participants’ intensity ratings from low to peak emotion intensity .Results were replicated in the ask Fig. b: We fouM = 4.30, ) and positive expressions   = 0.42, p = 0.52). In Experiment 2, intensity ratings were higher for negative  compared to positive expressions   = 16.43, p < 0.001, ηp2 = 0.01). As expected, the main effect of intensity was significant for both groups . The interaction of valence and intensity was significant in both groups  = 4.48, p = 0.008, ηp2 = 0.001; Expt2, p = 0.007, ηp2 = 0.002). Post-hoc comparisons revealed that for Experiment 2, differences between positive and negative valences were significant at higher intensities  for positive and negative strong intensity; Ms = 5.43,  and 5.64,  (p = 0.02) for positive and negative peak intensity), but not at weaker intensities . For Experiment 1, the same trend was shown but did not reach significance.The effect of valence on intensity ratings was assessed in Valence × Intensity rmANOVAs. Here, results differed between the two experimental groups. In Experiment 1, intensity ratings did not differ significantly between negative  = 16.33, p < 0.001; ηp2 = 0.02), perceived intensity increased significantly in line with intended intensity  in trials of incongruent emotion classification, and did so also in the case of incongruent valence classification . Cumulatively, the data on intensity ratings show the coherence between expressed and perceived intensity across all tested contrasts.The detected effect of expressed on perceived intensity persisted for trials in which emotion was not classified concordantly. Despite differences between congruently and incongruently classified trials  = 20.41, p < 0.001, ηp2 = 0.26 and intensity levels, F = 81.66, p < 0.001, ηp2 = 0.13 The interaction Emotion × Intensity was significant, F = 39.70, p < 0.001, ηp2 = 0.31. Four  out of six emotions featured lower classification accuracy for peak compared to strong, moderate, and low intensity . The opposite pattern was shown for achievement (ps < 0.001), whereas accuracy for fear was uniform across intensity levels.Separate ANOVAs were computed to assess the effect of valence, emotion, and emotion intensity on classification accuracy in Experiment 1 Fig. c. ClassiF = 2.95, p = 0.10. Again, the main effect of intensity was significant, p < 0.001, ηp2 = 0.28. Planned comparisons confirmed the pattern shown in Fig. M = 57.78%, 95% CI , which were not significantly different from moderate intensity expressions, M = 54.17%,  (p = 0.053). The decrease in accuracy from moderate to low intensity  was significant (p = 0.004). Classification accuracy for peak intensity was lower than for strong, moderate, and low intensity M = 43.11%, , ps < 0.001. The interaction between valence and intensity  = 5.21, p = 0.002, ηp2 = 0.02) corresponded to a significant difference in accuracy between low and moderate intensity only for positive but not negative expressions, along with a significant drop in accuracy for peak compared to all other intensity levels for expressions of either valence  = 21.29, p < 0.001, ηp2 = 0.23, intensities, F = 75.28, p < 0.001, ηp2 = 0.13, and an interaction, F = 32.83, p < 0.001, ηp2 = 0.33. Post hoc comparisons of the interaction replicated the pattern obtained in Experiment 1.In parallel to Experiment 1, separate ANOVAs were computed to assess the effect of valence, emotion, and emotion intensity on classification accuracy Fig. d. The EmF = 1.59, p = 0.22. The main effect of intensity was significant, p < 0.001, ηp2 = 0.32, as well as the interaction of valence and intensity, F = 3.45, p = 0.044, ηp2 = 0.03. Accuracy was lower for low  compared to moderate intensity , and lower for peak , whereas no difference was found between moderate and strong intensity (p = 0.095). The significant interaction stems from higher derived accuracy for expressions of negative valence at the outer intensity levels , but no significant differences at the centered intensity levels.For the Valence × Intensity rmANOVA, no main effect of valence on classification accuracy was found, M = 68.47%) compared to strong  and moderate intensity , yet peak and low (M = 72.61%) did not differ significantly (p = 0.11). The highest valence confusion occurred at peak intensity expressions of positive valence, where the classification accuracy of 53.67% was only marginally above chance (50%), t(29) = 1.86, p = 0.04, d = 0.34. In Experiment 2, correct valence classification dropped significantly for peak (M = 75.56%) compared to low , moderate , and strong intensity . Again, congruency of expressed and perceived valence was lowest for positive peak emotional states (63.11%).A second comparison across the two tasks examined how well listeners could distinguish positive from negative expressions. Valence classification accuracy (derived from forced choice judgements in Experiment 1), like emotion categorization, followed a paradoxical pattern. Accuracy was lower at peak  = 72.60, p < 0.001. This relationship is characterized by higher ratings in arousal for sounds which are rated as either highly pleasant or highly unpleasant. In addition, the relationship in our sample is asymmetrical: Negatively rated stimuli show higher arousal ratings (M = 4.82) than positively rated stimuli (M = 4.56), confirmed by a significant Wilcoxon test .Figure ask Fig. . The U-sMs = 3.63 95%, CI  > 4.33, ) > 5.13,  > 5.79, , ps < 0.001), the pattern of valence ratings showed interesting confusion and variation in participants’ agreement  = 183.91, p < 0.001). A factorial logistic regression quantified the effect of expressed valence and intensity on congruent or incongruent valence rating. Positive expressions, especially high and peak intensity expressions, were more likely to be rated of negative valence , accounting for the higher number of stimuli perceived as negative.While arousal ratings increased from low to peak intensity  cannot be retrieved easily from peak emotion expressions. However, arousal and emotion intensity 34. A direct comparison of accuracy scores across studies should be treated with caution, as, for example, the number of emotion categories varies across studies, and so does their intensity—here shown systematically to affect emotion classification. Furthermore, substantial differences exist in the tested stimulus sets themselves, that is stimulus production and selection procedures as well as stimulus sources . One speculative, but interesting possibility is that the lower convergence observed in our data reflects the heterogeneity allowed for in the stimulus material.In addition to the reported parabolic relationship of emotional intensity to classification accuracy, overall accuracy scores of individual emotions, although above chance, were far from perfect and in fact relatively low compared to previous research37. Likewise, the data challenge the conception that valence and arousal are equivalent elements in the composition of core affect16. Future work will need to investigate whether valence and arousal really share the same level of representation. Information on arousal is already available at early processing stages41 and may serve as an attention-grabbing filter, ensuring the detection of biological relevance in the most extreme cases. Valuation likely constitutes a more complex process, perhaps secondary in peak emotion.The data are incompatible with the view of diagnostic emotion expression suggested by basic emotion theorieshttp://doi.org/10.5281/zenodo.4066235), systematically manipulating emotion intensity. In line with previous research4, the data underscore that emotion intensity constitutes a prominent property of vocal emotion communication. In our population of listeners , we report compelling specific effects of intensity. Forced choice judgements and emotion ratings both revealed an inverted U pattern: The expressed emotion was most accurately classified for moderate and strong intensity expressions; low intensity expressions were frequently confused, and the least accurately classified were peak intensity expressions. The higher ambiguity of peak states was reflected in both lower valence and lower emotion classification accuracy. At the most extreme instances of emotion, the evaluation of ‘affective semantics’, i.e., valence and emotion type, is constrained by an ambiguous perceptual representation.We exploited a new database of human vocal emotion expressions , though their indication of ‘relevance’ remains unaltered. We speculate that this central representation of ‘alarmingness’, i.e., biological relevance of highly intense expressions, comes at the cost of other affective semantics, including valence and type of affective state. The latter might rely on contextual cues, underlining the role of top-down modulations and higher-order representations of emotional states43.In other words, for a given expression, despite the unreliable identification of the 44. Screams, for example, have spectro-temporal features irrelevant for linguistic, prosodic, or speaker identity information, but dedicated to alarm signals. The corresponding unpleasant acoustic percept, roughness, correlates with how alarming the scream is perceived and how efficiently it is appraised26. One hypothesis that arises is that information is prioritized differently as a function of emotion intensity. At peak intensity, the most vital job is to detect ‘big’ events. A salient, high arousal signal may serve as an attention-grabbing filter in a first step, and affective semantic evaluation may follow. In contrast, intermediate intensity signals do not necessarily elicit or require immediate action and can afford a more fine-grained analysis of affective meaning. A possible neurobiological implementation is that information is carried at different timescales, and ultimately integrated in a neural network underlying affective sound processing45. Concurrent functional pathways allow a rapid evaluation of relevance for vocal emotions of any valence, occurring at early processing stages and via fast processing routes47. Though perceptually unavailable, it might well be that information is objectively present in the signal, as has been shown for facial and body cues of extreme emotion11. The conjecture that a similar pattern might also exist in vocal peak emotion would resonate with the interpretation of the findings as temporally masked affective value and emotion information through the central representation of salience—via arousal and emotion intensity.In nonverbal vocalizations, the effects of increased emotional intensity and arousal have been linked to acoustic characteristics that attract attention48. The database comprises six affective states (three positive and three negative) at four different intensity levels . The six affective states—achievement/triumph, anger, fear, pain, positive surprise, sexual pleasure-represent a suitable, well-studied sample of affective states for which variations in emotion intensity have previously been described10.The stimuli are 480 nonverbal vocalizations, representing the Core Set of a validated corpusVocalizations were recorded at the Berklee College of Music . Ten female speakers, all non-professional actors, were instructed to produce emotion expressions as spontaneously and genuinely as possible. No restrictions were imposed on the specific sounds speakers should produce, only that vocalizations should have no verbal content as in words  or interjections . Following a technical validation, the Core Set was developed as fully crossed stimulus sample based on authenticity ratings. Stimuli were recorded with a sampling rate of 44.1-kHz (16-bit resolution). Sound duration ranges from 400 to 2000 ms.emotion categorization task ; thirty  to the emotion rating task; and thirty participants  to the dimensional rating task  would allow us to detect an effect as small as ηp2 = 0.005 (Cohen’s f = 0.06) with a power of 0.80. The experimental procedures were approved by the Ethics Council of the Max Planck Society. Experiments were performed in accordance with relevant named guidelines and regulations. Participants provided informed consent before participating and received financial compensation. All participants were native speakers of German, reported normal hearing, and no history of psychiatric or neurological illnesses.A total of ninety participants were recruited through the Max-Planck-Institute for Empirical Aesthetics (MPIEA), Frankfurt. Thirty participants were assigned to the www.neurobs.com), through DT 770 Pro Beyerdynamic headphones. Sound amplitude was calibrated to a maximum of 90.50 dB(A), resulting in 43 dB(A) for the peak amplitude in the quietest sound file. Each stimulus was presented once in pseudorandomized order. No feedback regarding response accuracy was provided.The studies took place at MPIEA. The 480 stimuli were presented using Presentation (Version 20.0) software , fear (Angst), pain (Schmerz), achievement (Triumph), positive surprise (Positive Überraschung), and sexual pleasure (Sexuelle Lust), plus a ‘none of the specified emotions’ option (Keines der Genannten). Next, participants were asked to indicate how intensively they believed the speaker had experienced the emotional state, reaching from minimally intense  to maximally intense . Finally, participants indicated how authentic they perceived the expression, from not at all (“gar nicht”) authentic to fully (“vollkommen”) authentic. The order of the 7AFC and intensity rating tasks was counterbalanced across participants. After the authenticity rating, the next stimulus was played automatically.each vocalization. As in the categorization task, emotion intensity was rated. The order of emotion ratings and emotion intensity ratings was counterbalanced across participants.Participants completed ratings for each emotion. They were instructed to indicate how clearly they perceived the specified emotion in the expression. A judgement from not at all (“gar nicht”) to completely (“völlig”) was performed on each of the simultaneously presented scales. Thereby, from none to all emotions could be identified to various extents in Participants were asked to judge each stimulus on the dimensions valence and arousal. Valence was rated from negative to positive and arousal from minimal to maximal. The scales were presented subsequently on individual screens, in counterbalanced order across participants. Authenticity judgements were performed in the same format as described for the categorization task.All statistical analyses and data visualizations were performed using R Studio.We refer to “classification accuracy” as the consistency of speaker intention and listener perceptual judgements. In Experiment 1, this corresponds to the percentage of correct classification of emotions. A measure of accuracy was also obtained from the emotion ratings performed in Experiment 2 by defining the response as a match whenever the highest of the ratings was provided on the intended emotion scale, and a miss whenever rated lower on the intended than on any other scale. As additional indices that take into account response biases, we report unbiased hit rates, differential accuracy, false alarm rates, and detailed confusion matrices of the response data in the Supplemental Information.p < 0.001), Greenhouse–Geisser corrections were applied. For readability, we report uncorrected degrees of freedom and adjusted p values. Pairwise comparisons were adjusted using the Tukey’s HSD correction in the emmeans package50.Intensity ratings and classification accuracy were tested with repeated measures analyses of variance (rmANOVA) to assess the effects of affective stimulus properties  and their interactions. Normality was screened; sphericity was assessed using Mauchly’s sphericity tests. When sphericity could not be assumed (https://osf.io/jmh5t/.Authenticity ratings are reported and discussed in the Supplemental Materials. Perceptual judgements for each stimulus are available at Supplementary Information.


Somatopause refers to the gradual declines in growth hormone (GH) and insulin‐like growth factor‐1 throughout aging. To define how induced somatopause affects skeletal integrity, we used an inducible GH receptor knockout (iGHRKO) mouse model. Somatopause, induced globally at 6 months of age, resulted in significantly more slender bones in both male and female iGHRKO mice. In males, induced somatopause was associated with progressive expansion of the marrow cavity leading to significant thinning of the cortices, which compromised bone strength. We report progressive declines in osteocyte lacunar number, and increases in lacunar volume, in iGHRKO males, and reductions in lacunar number accompanied by ~20% loss of overall canalicular connectivity in iGHRKO females by 30 months of age. Induced somatopause did not affect mineral/matrix ratio assessed by Raman microspectroscopy. We found significant increases in bone marrow adiposity and high levels of sclerostin, a negative regulator of bone formation in iGHRKO mice. Surprisingly, however, despite compromised bone morphology, osteocyte senescence was reduced in the iGHRKO mice. In this study, we avoided the confounded effects of constitutive deficiency in the GH/IGF‐1 axis on the skeleton during growth, and specifically dissected its effects on the aging skeleton. We show here, for the first time, that induced somatopause compromises bone morphology and the bone marrow environment. Induced somatopause during aging causes a drop in GH/IGF‐1 signaling, inhibits radial bone expansion and associates with increased bone marrow adiposity. At the bone tissue level, induced somatopause leads to progressive declines in osteocyte lacunar density and connectivity.  ALSacid labile subunitB.Arbone areaBMDbone mineral densityBV/TVbone volume/total volumecGHRKOconstitutive GHRKOCn.Ncanaliculi numberCt.Thcortical bone thicknessDMP‐1dentin matrix protein‐1FGF23fibroblast growth factor‐23GHgrowth hormoneGHRGH receptorIGF‐1insulin‐like growth factor‐1IGFBPIGF‐binding proteiniGHRKOinducible global‐GHR knockoutIHCimmunohistochemistryIL6interleukin‐6iLID mouse modelinducible liver‐IGF‐1 deleted0Jpolar moment of inertiaLac.Nlacunae numberLac.Vlacunae volumeLCNlacunar‐canalicular networkM.Armarrow areamCTmicro computed tomographyRCArelative cortical bone areaSOSTsclerostinT.Artotal cross‐sectional areaTb.Ntrabecular bone numberTb.Thtrabecular bonethicknessTMDtissue mineral densityTRAPtartrate acid phosphatase1In vitro studies have shown resistance to somatotropic stimulation in osteoblasts from old versus young subjects  and the insulin‐like growth factor‐1 (IGF‐1) are key endocrine factors regulating body composition, acquisition of peak bone mass, and maintenance of bone mineral density (BMD) through development and aging , 22 (iGHRKO6‐22), and ~30 (iGHRKO6‐30) months of age (at the time of death)  mice, a model previously validated  Figure . Here, w22.10) (~54%) in iGHRKO male mice  across all ages  analyses revealed that induction of somatopause after peak bone acquisition (at 6 months of age) resulted in failure of bones to undergo the normal radial expansion at the periosteal surfaces. Male iGHRKO mice exhibited significantly less total cross‐sectional area (T. Ar) across all time points (~26%), suggesting that radial expansion was reduced due to lower periosteal bone apposition Figure . Accordie Figure . Bones os Figure . Howevers Figure . Both ses Figure .periosteal = (T. Ar/π)1/2), and the endosteal (inner) radius from the M. Ar 1/2) were calculated from mCT data of 12‐ and 22‐month‐old mice. Differences in the periosteal radii indicated the average amount of periosteal formation, while differences in the endosteal radii indicated the average amount of endosteal bone resorption. Control males and females showed comparable amounts of periosteal bone formation and endosteal bone resorption. In contrast, male iGHRKO mice had increased periosteal apposition and endosteal loss, while female iGHRKO mice had reduced both processes  was compared with those of constitutive GHRKO (cGHRKO) in male and female mice Figure . As prevTrabecular bone volume  declined with age Figure A, and wa2.26‐30 females  did not change significantly with age in either male or female control mice  that were exacerbated with induction of somatopause in male iGHRKO mice at all ages, and at 30 months in iGHRKOs Figure . Reductis Figure . Reductie Figure . Inductie Figure .3) in the compact bone was measured as an overall index of osteocyte lacunar connectivity, and canaliculi per lacunae  measured to assess changes at the unit cell level  and by ~1.5‐fold in aged male mice (that did not reach significance). Induction of somatopause did not affect the accumulation of these “acellular” foci of compact bone . H&E stain revealed increased bone marrow adipocytes in both male and female iGHRKO mice, detected at 12 and 22 months of age Figure . IncreasFinally, accumulation of senescent cells has emerged as one of the characterizations of the aging fat pads , and significantly reduced Ct. Th . In control females, we observed increased endocortical resorption and periosteal formation with age, but significant cortical thinning was seen only between 22 and 30 months of age. Somatopause in females, resulted in the inhibition of endocortical resorption (M. Ar) and periosteal expansion (T. Ar). The fact that the effects of somatopause on bones in female iGHRKO mice manifested later in life may relate to their increase in lifespan and decreased senescence in their bone tissue. Alternatively, we speculate that it may relate to their sex steroid state. It is well established that mice aged around 9–12 months typically experience irregular estrous cycle (estropause) that shifts into anestrous state (around 20–24 months) ‐specific Cre (DMP‐GHRKO), which also showed slender bones during development and maturation , play significant roles in bone tissue quality or local material properties  can result in bone tissue compositional changes. As such, accumulation of mineral in lacunae (micropetrosis) with aging can significantly compromise bone strength, leading to decreased energy absorbing and dissipating capacities of the bone tissue  GHRKO was previously described , cortical bone area , cortical cross‐sectional thickness , and bone mineral density . We used 3D analysis of the 2‐mm mid‐diaphyseal region  to calculate total cortical bone porosity (%). Following “despeckle and shrink‐wrap” steps in 3D to remove noise, we wrap the region of interest tightly within the boundaries of the object, isolated the marrow cavity, and calculated all the closed and open pores included in the cortical envelope. Trabecular bone parameters were taken at the femur distal metaphysis in a 2.5‐mm region below the growth plate and included bone volume fraction , trabecular thickness , trabecular number , and bone mineral density . Data reconstruction was done using NRecon software , data analysis was done using CTAn software , and 3D images were done using CT Vox software .Micro‐CT was done in accordance with the American Society for Bone and Mineral Research (ASBMR) guidelines  at the mid‐diaphysis, immediately distal to the third trochanter, using a low‐speed diamond‐coated wafering saw (Buehler). Specimens were dehydrated and embedded undecalcified in poly‐methyl methacrylate (PMMA). Two hundred‐micron sections were adhered to plastic slides using Eukitt's mounting medium and used for Raman Microspectroscopy: We used the Thermo‐Fisher DXR2 confocal Raman microscope, with 785‐nm wavelength excitation at 50X/0.75 BD objective, a 50‐µm entrance slit provided a spectral resolution of 50–3300 cm−1 and a 0.96‐µm diameter spot size. This spot size was small enough to avoid including osteocyte lacunae visible on the bone surface in the measurements, allowing us to capture Raman spectra in interlacunar bone areas that contained only matrix and canaliculi.−1), carbonate band (1070 cm−1), and Amide I band (1620–1700 cm−1). Mineral/matrix ratios were obtained by dividing the integrated band areas of phosphate to amide I. Carbonate/phosphate ratios were calculated by dividing carbonate band areas by phosphate band areas. Mineral crystallinity information was determined by taking the inverse of full width at half maximum (FWHM) for the phosphate band .Spectra were collected from middle of the cortex of the anterior quadrants of the bone, as in our previous studies  at the mid‐diaphysis, immediately distal to the third trochanter, using a low‐speed diamond‐coated wafering saw (Buehler). Specimens were dehydrated and embedded undecalcified in poly‐methyl methacrylate (PMMA) mixed with Pylaklor Pink S351 (Pylam Products) fluorescent dye. Two hundred‐micron sections were adhered to plastic slides using Eukitt's mounting medium and used for imaging. Confocal laser scanning microscopy image stacks were obtained with a Zeiss LSM 800 with 561 nm excitation and a 40× oil immersion objective. Images stacks were taken to capture the entire cortical thickness (~74–306 μm), and were 200 μm radially and 30 μm thick . Two images each from the anterior and posterior cortex of each animal were analyzed using an automatic custom software written in MATLAB  using methods similar to previous studies on the morphology of the LCN  were stained with H&E and TRAP . For immunostaining, sections were treated with 3% hydrogen peroxide to block the endogenous peroxidase. The antigen retrieval was done by antigen unmasking solution following the instruction provided by the manufacturer . The sections were incubated with 5% bovine serum albumin for 30 min at room temperature, then with primary antibodies to Cathepsin K , p16 , p53 , FGF23 , SOST , overnight at 4 °C. Incubation with HRP‐conjugated secondary antibody  was done for 1 hour at RT. Immunoreactivity was detected using the horseradish peroxidase‐3,3′‐diaminobenzidine system  followed by counterstaining with hematoxylin (Sigma). The images were acquired by Aperio CS2 Scanner (Leica Biosystems) and analyzed by Fiji ImageJ .4.6p<0.05. For the comparison between groups, data were analyzed by three‐way analysis of variance (ANOVA) (GraphPad Prism version 9.0) with age, sex, and induction of somatopause as independent variables. In cases where only one age was analyzed (IHC), two‐way ANOVA was applied, and this was indicated in the figure legends. Statistical tests for LCS parameters were performed in RStudio  running R  using the rstatix package [R package version 0.6.0. https://CRAN.R‐project.org/package=rstatix]. For measurements taken repeatedly in a scan , an average was obtained for each of the four scans, the scan averages were averaged to obtain a value for the animal, which was used in statistical tests. First, an analysis of variance was performed, with age, sex, and induction of somatopause as independent variables. This was followed by post hoc Tukey honestly significant difference tests as needed to determine subgroup differences by age and somatopause.Data presented as mean ± SEM, and significance accepted at The authors declare no conflict of interest. All authors have discussed the results and approved the final version of the manuscript. SY is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study conception and design: SY, JJK, AB, MBS. Acquisition of data: MD, SDO, GY, SBP, LDL, AB. Analysis and interpretation of data: SY MD, GY, LDL, MBS. Manuscript preparation: SY and MBS.All authors concur with the submission. The material submitted for publication has not been previously reported and is not under consideration for publication elsewhere.Fig S1‐S6Click here for additional data file.


Allium sativum L.). The treatments consisted of five clove sizes: 1–1.49 g, 1.5–1.99 g, 2–2.50 g, 2.51–2.99 g, and 3–3.5 g. An improved variety “Tseday” was used as a planting material. The experiment was laid out as a randomized complete block design in a factorial arrangement and replicated for three times. The results revealed that clove size significantly influenced all growth and yield parameters of garlic. Planting of 3–3.5 g cloves reduced days to emergence by 11 and 6.33 days and days to maturity by 28.33 and 18.00 days, respectively, as compared to planting of 1–1.49 g and 2–2.5 g cloves. This treatment also increased total bulb yield by 25.88% and 15.58%, respectively, as compared to planting of 1–1.49 g and 2–2.5 g cloves. In addition, this treatment significantly increased most of the growth and bulb yield components. Hence, it can be concluded that planting of 3–3.5 g cloves could be recommended to enhance early emergence, good vegetative growth, and total bulb yield of garlic.Garlic is an important cash crop in many regions of Ethiopia. However, the yield of the crop is constrained by several factors. Among these, inappropriate clove size is one of the major agronomic practices that can decrease the yield of the crop. Hence, a field experiment was conducted during the 2020/21 main cropping season at Debre Berhan University, College of Agriculture and Natural Resource Sciences, demonstration and research site to evaluate the effect of clove size on growth and bulb yield of garlic ( Allium sativum L.) belongs to Alliaceae family, and it is believed to be originated in Central Asia  and spread to other parts of the world through trade and colonization [Allium next to onion and used as a seasoning in many foods and as condiment [Garlic . A distance of 1 m between the plots and 1.5 m between blocks was maintained. A garlic cultivar called “Tseday” was used for the experiment. Cloves of different sizes were planted on June 15, 2020, during the rainy season according to the standard planting density of 10 × 30 cm with 20 plants per row. Fertilizer was applied at the rate of 92 P2O5 kg ha−1 and 105 N kg ha−1 in the form of DAP and urea with equivalent amount of 200 and 150 kg ha−1, respectively, where all the recommended rate of DAP was applied at the time of planting to all plots uniformly. The applications of urea were splitted into one-third during planting, one-third at active vegetative growth (three weeks after plant emergence), and one-third six weeks after plant emergence, as side dressing. Weeding, chemical sprays, and harvesting were done according to the recommendation for the crop as described by Getachew and Asfaw [Experimental plots were ploughed, thoroughly harrowed, and leveled and ridges of about 20 cm high were prepared. The plot size was 3.6 mTen sample plants from each plot were randomly tagged from the middle four rows and data were recorded on the growth parameters of garlic. Vegetative parameters related to growth (growth parameters) were measured on the sample plants: plant height (cm), leaf length and width (cm), and shoot dry mass (g). Additionally, two extra parameters have been recorded: (i) days to 50% emergence of shoots and (ii) 75% of maturity of plants were recorded. Yield components were measured after harvest from each treatment, namely, neck diameter (cm), bulb length and diameter (cm), average bulb weight (g), and total dry biomass (g).Total bulb yield was calculated from total plants harvested from the central four rows of each plot and expressed on a hectare basis. To determine bulb dry matter, cloves from five bulbs randomly selected were chopped into small pieces with the help of stainless steel knife and mixed thoroughly, and the exact weight of each sample was determined and recorded as fresh weight. The samples were placed in paper bags and dried in an oven at 70°C until a constant weight was obtained. Each sample was immediately weighed using digital sensitive balance and recorded as dry weight. Percentage of dry matter content for each sample was calculated by the following formula:Data obtained were subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) of the SAS statistical package version 9.2. All significant pairs of treatment means were compared using Duncan's multiple range test (DMRT) at 5% level of significance.P < 0.001) affected by clove size (Days to emergence was significantly (ove size . PlantinP < 0.001) influenced days to maturity (Clove size significantly (maturity . PlantinP < 0.001) affected by clove size (Plant height was significantly (ove size . The lonP < 0.01) affected by clove size (Leaf length was significantly (ove size . The lonP < 0.001) affected by clove size (Leaf width was significantly (ove size . The widP < 0.01) affected by leaf number (Clove size was significantly (f number . The higP < 0.01) affected by clove size (Shoot dry mass was significantly (ove size . The higP < 0.001) affected by clove size (Neck diameter was significantly (ove size . The widP < 0.001) affected by clove size (Bulb length was significantly (ove size . The lonP < 0.001) affected by clove size (Bulb diameter was significantly (ove size . The widP < 0.001) affected by clove size (Clove number was significantly (ove size . The higP < 0.01) affected by clove size (Average clove weight was significantly (ove size . The higP < 0.001) affected average bulb weight (Clove size significantly (b weight . The higP < 0.001) affected by clove size (The percentage of dry matter content was significantly (ove size . The higP < 0.001) affected by clove size  affected by clove size (Total bulb yield was significantly (ove size . The higOur findings suggest that garlic growth and yield are directly related to the size of cloves planted. Therefore, planting cloves of the appropriate size can improve garlic production. Clove size showed significant effects on all growth and yield parameters of garlic. The results of the present study indicated that planting of 3–3.5 g cloves significantly reduced days to emergence and maturity and provide the highest yield of garlic. Hence, it can be tentatively recommended that production of the highest yield of garlic is achieved by using 3–3.5 g cloves. As a result, incomes of smallholder garlic producers could be significantly improved if the recommended clove size is applied to garlic crops cultivated in the study area.


There are some endoscopic resection (ER) methods for neuroendocrine tumors (NETs), however, which method is most useful remains unclear. This study aimed to compare the outcomes of different ER techniques, such as conventional endoscopic mucosal resection (cEMR), endoscopic submucosal dissection (ESD), and endoscopic submucosal resection with a ligation device (ESMR‐L) for rectal NETs.We retrospectively analyzed 96 consecutive patients with 102 rectal NETs of less than 10 mm in diameter who underwent ER between January 2001 and December 2019 at Hiroshima University Hospital. We compared the clinical outcomes of each ER method , divided according to the treatment periods, and evaluated the risk factors for vertical margin (VM) positivity in relation to clinicopathological and endoscopic characteristics.As for the mean procedure time, ESD took significantly longer to perform than the other methods. The histological complete resection rate was 80% (48/60) for cEMR, 85.7% (18/21) for ESD, and 100% (21/21) for ESMR‐L, and the VM positive rate was 20% (12/60) for cEMR, 14.3% (3/21) for ESD, and 0% (0/21) for ESMR‐L, with no significant difference. However, the tumor‐front‐to‐VM distance was significantly longer in the ESMR‐L group than in the cEMR and ESD groups. cEMR and ESD were both significant risk factors for VM positivity. No perforation or local recurrence was observed in all methods.ESMR‐L is the most useful ER method for small rectal NETs. The number of colorectal neuroendocrine tumors (NETs) successfully diagnosed, particularly rectal NETs, has been increasing due to the widespread use and quality improvement of colonoscopy techniques.There is an international consensus that rectal NETs of less than 10 mm in diameter are eligible for endoscopic resection (ER) because of their low lymph node metastasis (<2%), distant metastasis (<0.7%),Various ER methods are utilized for the resection of rectal NETs, including conventional endoscopic mucosal resection (cEMR), endoscopic submucosal dissection (ESD), and modified EMRs, such as pre‐cutting EMR, cap‐assisted EMR, and endoscopic submucosal resection with a ligation device (ESMR‐L). Previous reports demonstrated that ESMR‐L achieved a complete resection rate not inferior to ESD.In this study, we compared the outcomes of cEMR, ESD, and ESMR‐L for rectal NETs of less than 10 mm in diameter. We also measured and compared the tumor‐front‐to‐vertical margin (VM) distance in the resected specimen for each ER method to assess the efficacy of histologically complete resection.Figure This study enrolled a total of 106 consecutive patients who underwent ER for 112 rectal NETs between January 2001 and December 2019 at Hiroshima University Hospital. Data related to patient and lesion characteristics, procedure outcomes, pathological results, and postoperative clinical course were recorded. We excluded cases with lesions of 10 mm or larger in diameter , and finally analyzed a total of 96 patients with 102 rectal NETs of less than 10 mm in diameter Figure 1, 2. NETs were diagnosed via endoscopic or histopathological examination. The indications for ER for NETs were as follows: no obvious ulceration, tumors located within the submucosal layer, and no local or distant metastasis.Figure The resected specimens were stretched, pinned on the flat styrofoam board, fixed with 10% formalin solution, and then evaluated under a microscope. Histological diagnoses, such as histopathological type, World Health Organization (WHO) grade, depth of invasion, horizontal margin (HM) or VM, and lymphovascular invasion were evaluated. VM positivity was defined as the presence of tumor invasion at the vertical resection margin. Lymphovascular invasion was evaluated by hematoxylin and eosin staining. However, depending on the situation, immunostaining  was performed, as a reference for diagnosis. Immunohistochemical staining for neuron‐specific enolase, synaptophysin, and chromogranin A was also performed to confirm the diagnosis. Ki‐67 was used to assess tumor proliferation and classify the WHO grade of the tumors based on 2019 WHO classification of tumors of the digestive system.We evaluated the clinicopathological characteristics, such as age, sex, tumor size, tumor location, and clinical outcomes of NETs, including en bloc resection rate, histologically complete resection rate, procedure time, procedure‐related complications (delayed bleeding and perforation), pathological diagnosis, and existence of recurrence for each ER method to compare their efficacy and safety. We also evaluated the risk factors for VM positivity using the following variables: sex, age, tumor size, tumor location, presence of depression, ER method, en bloc resection rate, and pathological diagnosis (lymphovascular invasion and WHO grade). En bloc resection was defined as the tumor being entirely endoscopically resected in one piece. Histologically, complete resection was defined as en bloc resection without positive HM or VM of the resected specimen. The patients underwent colonoscopy and CT at 6 and 12 months after ER, and every year thereafter, to determine the presence of local recurrence and metastasis.This study was performed in accordance with the principles of the Declaration of Helsinki in compliance with good clinical practice guidelines and local regulations. The study was approved by the Institutional Review Board Ethics Committee of the Hiroshima University Hospital (No. E‐1637).t‐test, and the Wilcoxon rank‐sum test with Holm–Bonferroni method were used to compare the qualitative and quantitative variables. Statistical significance was set at p < 0.05.The data are presented as mean ± standard deviation (SD) or median (range). Pearson's chi‐square test, Fisher's exact test, Student's All statistical analyses were performed using the JMP software version 14 .p < 0.01). There were no significant differences in sex, age, tumor size, tumor location, presence of depression, or other pathological diagnoses (venous invasion and WHO grade) among the three groups.Table p < 0.01). En bloc and histologically complete resection rates were 100% (102/102) and 85.3% (87/102), respectively. Histologically complete resection rate in each ER method was 80% (48/60) for cEMR, 85.7% (18/21) for ESD, and 100% (21/21) for ESMR‐L. The VM positive rate was 20% (12/60) for cEMR, 14.3% (3/21) for ESD, and 0% (0/21) for ESMR‐L, with no significant difference. However, the tumor‐front‐to‐VM distance in ESMR‐L (641.5 ± 763.8 μm) was significantly longer than that in cEMR (188.9 ± 199.1 μm) and ESD (202.8 ± 125.4 μm) (p < 0.05). There were no significant differences in complications among the three groups. No local recurrence was observed during the follow‐up period .Clinical outcomes of each ER method are presented in Table p < 0.05).We analyzed the risk factors for VM positivity Table . Of the Among the 15 patients who were VM positive, 12 patients were followed up endoscopically upon their own request, one patient underwent additional surgery, and two patients were lost to follow‐up. All patients who were followed up had no local recurrence or metastasis during the follow‐up period .Our study revealed that ESMR‐L is the simplest and most reliable ER method for rectal NETs of less than 10 mm in diameter. Histologically, the complete resection rate of ESMR‐L is 100%, which was equally impressive as the results shown in several previous studies (93.3‐100%).One of the most important reasons for the superior outcomes of ESMR‐L is its low VM positive rate. Previous reports revealed that the VM positive rate was 22.6–50% in cEMR,ESMR‐L was performed to resect the submucosal layer by suction using a ligature device to ensure a safe margin. After the submucosa was sufficiently suctioned into the transparent cap, a ligature band was placed, and the snare was resected just below the band; this theoretically provides a deeper and wider vertical resection margin.No perforation occurred in any of the cases in this study. In a previous report, 0–2.5% (one case) of perforation was reported with ESMR‐L, indicating that it is safe and can be treated using ER methods.In the analysis of VM positivity, the only significant risk factor was the non‐selection of ESMR‐L as an ER method. Various experienced endoscopists were able to achieve a high histologically complete resection rate of ESMR‐L, even if they were not familiar with the procedure. Therefore, ESMR‐L could be the first choice as an ER method for small NETs, regardless of the endoscopist's experience. In performing ESD, it is essential to pay attention to the aforementioned points and make an effort to dissect just above the muscle layer with a clear field of view.After ER, there were two major management courses: follow‐up with or without additional treatment, including surgery. The decision to select either was based on the pathological diagnosis, particularly the presence of risk factors for lymph node metastasis, of rectal NETs resected by ER. Additional surgery was recommended for cases judged to be at high risk of metastasis; however, there was insufficient consensus on its criteria.Among the 15 VM positive cases, two cases in the cEMR group and one case in the ESD group underwent additional surgery. Currently, there is no consensus on surveillance after ER for NETs. Some guidelines recommend surveillance by colonoscopy and CT every 12 months after local resection, while others state that there is no justification for recommending such surveillance.This study has some limitations. First, this was a retrospective study conducted in a single center and the sample size was relatively small. Second, although ER methods were selected according to each period without selection bias, the development of treatment strategies and devices may have influenced the results. Third, each ER method was selected for each treatment period and was not conducted by precisely uniform operator's endoscopic experience. Fourth, immunostaining was not performed for all cases, resulting in a bias in the rate of lymphovascular invasion by ER methods. Finally, the surveillance period is not enough to analyze the long‐term outcome, including prognosis and recurrence.In conclusion, in the technical aspect, ESMR‐L is considered to be the simplest and most reliable ER method for rectal NETs of less than 10 mm in diameter.The authors have no conflict of interest to declare.None.


Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed  and fruit  characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae. arabicum gene annotation using 294 RNA‐seq libraries and 136 307 full‐length PacBio Iso‐seq transcripts, increasing BUSCO completeness by 11.6% and featuring 5606 additional genes. Analysis of orthologs showed a lower number of genes in Ae. arabicum than in other Brassicaceae, which could be partially explained by loss of homeologs derived from the At‐α polyploidization event and by a lower occurrence of tandem duplications after divergence of Aethionema from the other Brassicaceae. Benchmarking of MADS‐box genes identified orthologs of FUL and AGL79 not found in previous versions. Analysis of full‐length transcripts related to ABA‐mediated seed dormancy discovered a conserved isoform of PIF6‐β and antisense transcripts in ABI3, ABI4 and DOG1, among other cases found of different alternative splicing between Turkey and Cyprus ecotypes. The presented data allow alternative splicing mining and proposition of numerous hypotheses to research evolution and functional genomics. Annotation data and sequences are available at the Ae. arabicum DB (https://plantcode.online.uni‐marburg.de/aetar_db). Aethionema arabicum using long‐read transcript sequencing provides a plethora of full‐length isoforms and an important resource for Brassicaceae evolution studies.Improved gene annotation of  Aethionema arabicum has become an important model system for unraveling the control and development of seed and fruit traits because it displays an unusual phenomenon, diaspore (fruit/seed) heteromorphism. It produces two types of fruits, a bigger and dehiscent one that produces two to six mucilaginous seeds (M+) and an indehiscent one, which only contains one non‐mucilaginous seed (M‐)  or Cyprus (CYP) , rapeseed (Brassica napus) and mustard (Brassica rapa), as well as the model plant Arabidopsis thaliana , in flowering plants (At‐ε), in eudicots  and in part of the Brassicales including the sister group of Brassicaceae (Cleomaceae) (At‐β), and importantly, all Brassicaceae species including Ae. arabicum share the At‐α whole genome duplication (WGD) , which affects seed dormancy  and 2872 unordered scaffolds  of the Aethionema Arabicum genome assembly versions are described using capital version letters (V), annotation versions are described using lower‐case v. To generate the gene annotation v3.1, the Ae. arabicum genome sequence V3 was used as reference, i.e., the same genome version as in gene annotation v3.0  from Swiss‐Prot  were used to support the gene prediction  of the assembly and 49.5% of the non‐gap genome sequence, i.e., twice the fraction found in A. thaliana using a similar approach  were generated. Total RNA of each ecotype was split by fragment length in three parts for Iso‐seq, using 15.74 sequencing passes on average, yielding transcripts with 1533 bp length and 95.3% quality on average  from a pool of total RNA from seeds and leaves of two ge Table . The Traet al., et al., et al., et al., MAKER requires assembled transcripts as expression evidence input. A total of 294 RNA‐seq samples sequenced with Illumina technology were used to test assembled transcriptomes fed to MAKER. Most of them were very short single‐end (SE) reads (50 bp) with a minimum length of 30 bp after pre‐processing. Hence, three short‐read assemblers were tested using their genome‐guided mode: Trinity . During the evaluation of the annotation, it was observed that tandem gene duplications and promiscuous protein domains often caused problems, fusing multiple genes into one or splitting genes into several parts. Manual inspection of the results followed by analysis with BedTools intersect , 99.7% in both cases. The final gene set was named according to the previously introduced nomenclature , followed by the number of the LG (linkage group 1 in the example), and ‘G’ for gene followed by the gene number (Gene 10 in the example). Gene numbers start with 10 for the first gene at the 5′ end of each LG or scaffold and increase by 10 for every gene found in the 3′ direction as in the convention for A. thaliana. This allows to use the free numbers between tens if needed in future annotations. Once the names were modified in the GFF file of the final v3.1 annotation, putative transposable elements (TEs) were identified and sequence files were created for cDNA (transcript sequences), gene‐coding sequences (CDSs) and proteins  contains tools to search annotations and genes by keywords, to download sequences and annotations from a list of genes, to look up older gene versions and A. thaliana orthologs and to perform BLAST searches. Its code and tools are based on previous databases such as OliveTreeDB . Additionally, a lookup table with previous versions, A. thaliana genes, overlapping PacBio full‐length reads and other annotations is available in the Ae. arabicum DB download section.A genome browser with all evidence tracks used in this study and the expression data of the light experiments from Mérai et al.  is availAe. arabicum, such as single nucleotide polymorphisms (SNPs) and RNA‐seq and BS‐seq data, are available in CoGe . In total, 3549 genes in v2.5 and 3365 in v3.0 were affected by any of these issues  were found using BLAST versus the v3.1 protein set with a query coverage and identity percentage equal to or higher than 50%. Only six genes had no hits, and 247 had a query coverage or identity percentage below 50%. Using BedTools intersect, 17 639 genes (97.9%) overlapped on the same strand between versions 3.0 and 3.1, with 50% or more of the length of the smaller gene. Only 368 genes from v3.0 did not overlap, and 11 overlapped less than 50%. Protein sequences of v3.0 have an average length of 445 amino acids. The proteins of v3.0 that did not overlap with those of v3.1 have an average length of 334 amino acids, which is more than 100 amino acids lower than average. MAKER did not show good support of proteins or expression data for these 368 potentially incorrectly predicted genes.Of the 23 160 genes in v3.1, 5606 did not overlap with the genes of v3.0 . Of these genes, 2878 (51.3%) are newly predicted and were not present in v3.0, and 2728 (48.7%) correspond to the fixed version of broken genes in v3.0 (81.1% of the 3365 genes broken in v3.0). Of these genes, 1945 (71.3%) were recovered as one‐to‐one relationships . In the remaining cases, multiple genes of v3.0 were joined to generate one gene model in v3.1, while one gene of v3.0 was split in multiple genes in v3.1. For example, nine genes of v3.0 including sequence and GFF format errors were recovered in a single gene with nine exons in v3.1, Aa31LG9G14150  were analyzed with OrthoFinder , sister to the Brassicaceae, as outgroup  identified by OrthoFinder Figure  showed cAe. arabicum 235 exclusive OGs were identified, including 1205 species‐specific genes and 1268 genes not assigned to orthogroups . Other Brassicaceae such as C. rubella have a similar number of exclusive OGs (244), while A. thaliana has a higher number of unassigned genes (1687). Carica papaya  has a higher number of exclusive genes (3559), exclusive OGs (844) and unassigned genes (6200) than any Brassicaceae  derived from the At‐α WGD in 38.87% of the cases based on the classification by  lower number than in A. thaliana (with 10 390). The lower number of genes in Ae. arabicum in comparison with the other Brassicaceae could be partially due to more genome fractionation (duplicate gene loss or diploidization) in the lineage leading to Aethionema, compared to the crown group after the last polyploidization event (At‐α) , 157 genes were annotated with F‐box domains, which, among several functions, participate in seed germination and dormancy in interaction with plant hormones such as gibberellin (GA) or abscisic acid (ABA) , that is, transcription factors (TFs) and transcriptional regulators (TRs), were identified using TAPscan . Based on this analysis, Ae. arabicum has on average 308 TAPs less than other Brassicaceae and 321 TAPs more than C. papaya . These TFs have been reported to participate in seed germination  , AGL12‐, StMADS11‐ (SVP‐), FLC‐, TM3‐ (SOC1‐), OsMADS32‐, TM8‐, AGL15‐ and AGL17‐like genes, have quite variable or more subtle functions in diverse aspects of plant development, ranging from root to fruit development, or have even unknown functions . However, the genomic region where the two FUL‐like genes in Ae. arabicum are encoded are identical for several thousand bases, which might represent an assembly artifact. Remarkably, the FUL and AGL79 orthologs were not identified in the v2.5 annotation of Ae. arabicum, even though expression of the FUL gene had previously been shown in the literature , which play key roles in ABA‐mediated seed dormancy, were studied.Recent work by Punzo et al.  demonstr et al., . Consistet al., et al., A. thaliana  in cereals encode a TF that is a crucial component of the ABA signaling pathway and one of the master ABA‐related regulators of seed maturation and dormancy  in A. thaliana encodes only the A1 and B1 domains and accumulates predominantly near completion of seed maturation. It is postulated to downregulate the seed maturation program   , resulting in transcripts with tissue‐specific functionality  and barley (Hordeum vulgare) results in transcripts not encoding full‐length VP1 proteins and, as a result, a reduction in primary dormancy and sensitivity to pre‐harvest sprouting  to a similar antisense transcript in A. thaliana, although a function for this transcript is not known.1 Figure , and thiet al., et al., et al., et al., Ae. arabicum, we observed full‐length transcripts with introns in the NCED6 TUR accession , Aethionema arabicum V1.0, Sisymbrium irio and Leavenworthia alabamica (http://mustang.biol.mcgill.ca:8885/), Camelina sativa (https://www.ncbi.nlm.nih.gov/assembly/GCA_000496875.1), Boechera stricta v1.2 (Phytozome v12), Eutrema salsugineum v1.0, Capsella rubella v1.0 and Arabidopsis lyrata v1.0 (Phytozome v11), Brassica oleracea (Ensembl 39), Brassica rapa (Ensembl 33), Thellungiella parvula (http://thellungiella.org/data/), Arabidopsis thaliana (TAIR10) and Arabis alpina (https://www.ncbi.nlm.nih.gov/nuccore/JNGA00000000.1).To build libraries of consensus sequences that are representative of repeated sequences, the TEdenovo pipeline  are hard‐masked to avoid portions of TEs to be incorrectly included in annotations of neighboring protein‐coding genes, and low‐complexity repeats (simple) are soft‐masked .The repeat set produced for the Aethionema arabicum total RNA from germinating seeds and leaves of young plants of ecotypes TUR and CYP was extracted using the CTAB protocol  curves than any set of genes tested . Sense and antisense transcripts were detected using Phusion polymerase (Thermo Fisher Scientific) with the following primers: forward (5′‐CGGTCCAGACAACGCTAAAT‐3′) and reverse (5′‐AAGACGTCGGAACATCAGGT‐3′).For RNA samples, 30 mg of seeds of i et al. . RNA wasi et al. . Then, 2AS events were calculated with Astalavista  and 290 experiments of 50‐bp SE reads from multiple tissues and several experimental conditions are available at NCBI SRA . PacBio Iso‐seq reads for the Ae. arabicum with orthologs and TAPs in Brassicales. LG and GT analyzed MADS‐box genes. ZM and OMS contributed RNA‐seq data, managed Illumina sequencing and analyzed eRF1 locus and seed dormancy genes.NFP and SAR conceived the work, supervised it and wrote the manuscript with support of all the authors. NFP and TM performed the bioinformatics analysis. FM did the repeat masking of genome V3. JOC, NFP, ZM and GLM analyzed seed dormancy genes. NFP, MES and SAR analyzed TAPs and compared The authors declare no conflicts of interest.Figure S1. Repetitive content across Brassicaceae.Figure S2. Alternative splicing in MAKER isoforms.Figure S3. Alternative splicing in PacBio full‐length isoforms.Figure S4. Phylogenetic relationships of the Brassicales species included in OrthoFinder and TAP analysis.Figure S5. Count of transcription associated proteins (TAPs) of Ae. arabicum in comparison with other Brassicales.Figure S6. Phylogeny of Type II MADS‐box genes from Ae. arabicum and other representative flowering plant species.Figure S7. PIF6 alternative splicing isoforms in Ae. arabicum.Figure S8. ABI3 alternative splicing isoforms in Ae. arabicum.Figure S9. ABI4 isoforms and expression in the Ae. arabicum DB genome browser.Figure S10. Strand‐dependent cDNA synthesis and PCR analysis for sense and antisense strands of ABI4 in TUR and CYP.Figure S11. DOG1 alternative splicing isoforms in Ae. arabicum (A) and A. thaliana (B) shown in the Ae. arabicum DB and TAIR genome browsers, respectively.Figure S12. NCED6 isoforms in the Ae. arabicum DB genome browser.Figure S13. Annotation edit distance curves for several training sets for SNAP and Augustus.Table S1. PacBio sequencing statistics.Table S2. Characteristics of PIF6 transcripts and encoded proteins in Ae. arabicum.Table S3. Characteristics of ABI3 transcripts and encoded proteins in Ae. arabicum.Dataset S1. List of genes in v3.1 not found or broken in v3.0 and OrthoFinder‐specific genes.Dataset S2. Classification of Ae. arabicum MADS MIKCC‐type genes and TAP version and Brassicales species comparisons using TAPscan.Click here for additional data file.


Abutilon theophrasti).How plants cope with increases in population density via root plasticity is not well documented, although abiotic environments and plant ontogeny may have important roles in determining root response to density. To investigate how plant root plasticity in response to density varies with soil conditions and growth stages, we conducted a field experiment with an annual herbaceous species , under fertile and infertile soil conditions, and a series of root traits were measured after 30, 50, and 70 days.Plants were grown at low, medium, and high densities (13.4, 36.0, and 121.0 plants mRoot allocation increased, decreased, or canalized in response to density, depending on soil conditions and stages of plant growth, indicating the complex effects of population density, including both competitive and facilitative effects.Root allocation was promoted by neighbor roots at early stages and in abundant resource availability, due to low‐to‐moderate belowground interactions among smaller plants, leading to facilitation. As plants grew, competition intensified and infertile soil aggravated belowground competition, leading to decreased root allocation in response to density. Root growth may be more likely restricted horizontally rather than vertically by the presence of neighbor, suggesting a spatial orientation effect in their responses to density. We emphasized the importance of considering effects of abiotic conditions and plant growth stages in elucidating the complexity of density effects on root traits. The research revealed the complexity of root response to density, which may increase, decrease, or canalize, depending on the strength of above‐ and belowground interactions, which varied with soil conditions or growth stage. For plants, increasing density can result in variations in multiple resources and intraspecific interactions  Neither above‐ nor belowground conspecific competition alters root allocation Cahill, ; (b) intAbiotic environmental factors may determine plant response to density in several ways  Higher density reduces root mass allocation, but has mixed effects on root morphological traits; (b) compared to fertile soil, infertile soil intensifies root responses to density; and (c) responses of root traits to density intensify and then weaken as plant growth continues.Here, we conducted a field experiment by subjecting plants of an annual species 22.1Abutilon theophrasti Medicus  is native to China and India but now spreads worldwide. It is an annual weedy species, erect with stout stems, growing to a height of 1–1.5 m. Through rapid growth, it can reach reproductive maturity within 90 days and complete its life cycle in ~5 months . Seeds were collected from local wild populations near the research station in late August 2006 and dry stored at −4°C. We used a split‐plot design, with soil conditions as the main factor and density and block as a subfactor. Two large plots were assigned as two (infertile and fertile) soil conditions; each was divided into nine 2 × 3 m subplots and randomly arranged with three treatments of densities and blocks. Seeds of We established the infertile soil conditions as a plot using the original soil (aeolian sandy soil) of an experimental field at the station that had been used annually for many years. In contrast, fertile soil conditions were established by covering the other large plot with 5–10 cm virgin soil transported from a nearby meadow with no cultivation history (meadow soil), with contrasting nutrient contents of the two soil conditions . Morphological root traits were not measured at 30 days of growth due to small plant sizes. Each individual plant was then separated into roots, stems, petioles, leaves, reproductive organs, and branches (if any), enveloped, respectively, oven‐dried at 75°C for 2 days, and weighed.All statistical analyses were conducted using SAS statistical software . All traits were used including shoot mass, root mass, total biomass, and root:shoot ratio, and morphological traits of main root length, main root diameter, lateral root number, and lateral root length. To minimize variance heterogeneity, all data were log‐transformed before statistical analyses. Three‐way ANOVA and ANCOVA were used to evaluate overall effects of growth stage, soil conditions, and population density and their interactions on all traits, with total biomass nested in growth stage as a covariate in three‐way ANCOVA. Within each soil condition at each stage, effects of density were analyzed with one‐way ANOVAs for total mass and one‐way ANCOVAs for all the other traits, with total mass as a covariate. For a given trait, it was considered to exhibit apparent plasticity whenever plant size  accounted for significant variation in its response to density, and any variation in its expression that was independent of total biomass was considered an indication of true plasticity   but not for those in infertile soil  and lateral root number (p = .002) at 50 days, whereas medium and high densities enhanced shoot mass at 70 days (p < .05); however, there was no response to density in these traits in fertile soil at either stage , whereas main root diameter was decreased by high density in infertile soil only  into deeper rather than upper soil layers ; Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Resources (lead); Software (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Lei Li: Project administration (supporting); Writing‐review & editing (supporting). Dao‐Wei Zhou: Conceptualization (supporting); Funding acquisition (supporting); Methodology (lead); Supervision (supporting).


Acute and overuse injuries affect millions of high school athletes annually and a better understanding of differences between these injuries is needed to help guide prevention, treatment, and rehabilitation strategies. This study compares acute and overuse injuries using a nationally representative sample of high school athletes.Injuries among United States high school athletes participating in 5 boys’ sports  and 4 girls’ sports  reported in the High School RIO™ surveillance system during the 2006-07 through 2018-19 school years were classified as acute or overuse. National estimates and injury rates were calculated.Of 17 434 646 estimated injuries, 92.0 % were acute and 8.0 % were overuse. The acute injury rate was higher than the overuse injury rate among both male  and female  athletes. The overuse injury rate per 10,000 athlete exposures among female athletes (1.8) was slightly higher than among males (1.4). The rate of acute injury compared with the rate of overuse injury was higher during competition  than practice . Boys’ football contributed the most acute (42.1 %) and overuse (23.7 %) injuries among the 9 sports. Among female sports, girls’ soccer contributed the most acute  and overuse  injuries. The lower extremity was most commonly injured in acute (48.9 %) and overuse (65.9 %) injuries. Ligament sprain (31.7 %) and concussion (21.0 %) were the most common acute injury diagnoses, while muscle strain (23.3 %) and tendonitis (23.2 %) were the most common overuse injury diagnoses. Compared with acute injuries, overuse injuries were more likely to result in time loss from sports participation of < 1 week among both boys and girls and across most sports. Acute injuries were more likely than overuse injuries to cause a time loss of 1–3 weeks or medical disqualification from sports participation.Acute and overuse injuries display many differences that provide opportunities for data-informed athlete preparation, treatment, and rehabilitation, which may reduce injuries and improve injury outcomes in high school athletics. Approximately 7.9 million athletes participated in high school sanctioned sports during the 2018–2019 school year, which is the third highest annual number of participants reported since the first National Federation of State High School Associations survey was implemented in 1971  and 4 girls’ sports  using data obtained from RIO™. Specifically, this study investigated differences in frequency and rates of injury as well as injury characteristics  between acute and overuse injuries while also considering the role that gender plays in these differences. Study findings fill a gap in our knowledge about these injuries that will help guide prevention, treatment, and rehabilitation strategies.Study data were obtained from the national high school sports-related injury surveillance system, RIO™. RIO™ data collection methods have been detailed previously  occurred due to participation in an organized high school practice or competition, (2) required medical attention, and (3) resulted in a time loss from participation of at least 1 day beyond the day the injury occurred, or any fracture, concussion, or dental injury, regardless of whether it restricted an athlete’s participation. An AE was defined as one athlete participating in one practice or one competition.This study included athletes injured while participating in 9 sports  from the 2006-07 through 2018-19 school years. Data from the 2005-06 school year were not included because the database did not include “overuse/chronic” as an option for a basic injury mechanism during that year. Six sports were considered gender-comparable based on similar rules or play tactics: boys’ and girls’ soccer, boys’ and girls’ basketball, and boys’ baseball and girls’ softball  head/face/neck, (2) trunk, (3) upper extremity, (4) lower extremity, and (5) other. The “other” body site category included injuries to male genitalia and body parts listed as “other” in RIO™. Injury diagnoses were grouped by their overall diagnosis. For example, injuries coded as ligament sprain, ligament sprain (complete tear), ligament sprain (incomplete tear), and ligament sprain (unknown severity) were grouped as ligament sprain (any severity). “Other unspecified” was used to describe an injury diagnosis reported by ATs as “other,” which prompted the ATs to provide more information for an unlisted or unusual diagnosis in RIO™. The “other specified” category included all diagnoses that were selectable at data entry but were not among the top 10 most common diagnoses.Data were analyzed using IBM SPSS Statistics for Windows, Version 26  and SAS 9.4 . Sampling weights provided by RIO™ were used to calculate national estimates. Descriptive statistics included national estimates and proportions with 95 % confidence intervals (CIs). Poisson regression was used to estimate the rate ratio (RR) using the rate of overuse injury as the denominator. Injury rates and RRs were expressed as the ratio of unweighted injury counts per 10,000 AEs. All other calculations, including injury proportion ratios (IPRs), used weighted data. The level of significance was α = 0.05 for all analyses.An example of a RR calculation follows:An example of an IPR calculation follows:n = 16 039 385) of these injuries were acute, while the remaining 8.0 % were overuse (n = 1 395 261). Overall, male athletes sustained 69.8 % of acute, 55.9 % of overuse, and 68.7 % of total injuries. However, for the gender-comparable sports, female athletes accounted for most acute (54.6 %), overuse (56.1 %), and total (54.8 %) injuries.From the 2006-07 through 2018-19 school year, 52 285 injuries were reported, which represent an estimated 17 434 646 injuries (95 % CI: 17 311 184–17 558 108) among high school athletes nationally, who participated in the 9 sports included in this study  and overuse (23.7 %) injuries among the 9 sports, boys’ baseball contributed the fewest (3.4 %) acute injuries and boys’ wrestling the fewest overuse injuries (3.5 %). Among female sports, girls’ soccer accounted for the most acute  and overuse  injuries, while girls’ volleyball accounted for the least acute injuries  and girls’ basketball the least overuse injuries .The number of acute injuries increased slightly with increasing number of years in high school, with 23.3 % of acute injuries occurring among freshmen, 25.3 % among sophomores,25.4 % among juniors, and 26.0 % among seniors; however, this increase was not statistically significant (p > 0.05). The number of overuse injuries showed no relationship with year in school, with 25.8 %, 24.5 %, 25.4 %, and 24.3 % occurring in freshman through senior years, respectively.Most acute (48.9 %) and overuse injuries (65.9 %) were to the lower extremities. The head/face/neck accounted for 25.6 % of acute injuries and 0.9 % of overuse injuries  was approximately 13 times  higher than the rate of overuse injury   and boys’ baseball had the lowest rate of acute injuries , while girls’ soccer had the highest rate  and both boys’ basketball and boy’s wrestling had the lowest rate of overuse injuries . Compared with overuse injuries, the likelihood of acute injuries was highest for boys’ football , followed by boy’s wrestling  and boys’ basketball .When comparing only the four female sports, soccer had the highest rate of acute injury  and highest RR of acute to overuse injury . Volleyball had the lowest rates of acute  and overuse  injury, and softball had the lowest RR of acute to overuse injury .The rate of acute injury per 10,000 AEs was higher during competition (43.8) than practice (11.9) , while practice had a higher rate of overuse injury (1.7) than competition (1.4) . The risk of acute injuries compared with that of overuse injuries was higher during competition  than during practice .Most (90.3 %) acute injuries were new injuries, while 81.6 % of overuse injuries were new . Recurrent injuries accounted for 9.7 % of acute injuries, while 18.6 % of overuse injuries were recurrent .Compared with overuse injuries, acute injuries were less likely to result in time loss from sports participation of < 1 week overall . This finding held true for both boys and girls and for all sports, except boys’ baseball  Table . OverallAmong all sports, the likelihood of medical disqualification from acute injury was higher than that from overuse injury for girls’ basketball , girls’ softball , and girls’ soccer . All other sports demonstrated no difference in the proportion of medical disqualification from acute versus overuse injury.This is the first study to compare acute and overuse injuries using a nationally representative sample of high school athletes participating in 5 boys’ sports  and 4 girls’ sports . There was an average of 1 341 127 high school sports-related injuries per year, equating to an average of 153 injuries every hour, associated with the sports included in this study. In addition to the monetary toll an injury may take, which can be upwards of $700 per injury in direct medical costs, athletes can be subject to long-term physical and psychological sequelae  athletes for > 3 weeks of time loss. These observed differences are most likely a result of the nature of these types of injury; while overuse injuries are the result of repetitive microtrauma with inadequate rest time for repair, acute injuries can result from greater forces resulting in injuries that may require longer healing time ; therefore, both acute and overuse injuries may be underreported because not all prevent an athlete from returning to play. Previous research showed that 50.8 % of overuse and 29.8 % of acute injuries among athletes from one NCAA Division 1 program resulted in no time loss  and 4 girls’ sports  studied. They also resulted in greater time loss from sports participation than overuse injuries and occurred at a much higher rate during competition than practice. Overuse injuries were more likely to be recurrent injuries than acute injuries, while acute injuries were more often new injuries. Attention to these differences between acute and overuse injuries can help inform improved athlete preparation (such as conditioning and strength training), treatment, rehabilitation, and schedules allowing for adequate rest time, which may reduce injuries and improve injury outcomes in high school athletics.


Fusobacterium nucleatum has been detected in 8%‐13% of human colorectal cancer, and shown to inhibit immune responses against primary colorectal tumors in animal models. Thus, we hypothesized that the presence of F. nucleatum might be associated with reduced T cell density in colorectal cancer liver metastases (CRLM). We quantified F. nucleatum DNA in 181 CRLM specimens using quantitative PCR assay. The densities of CD8+ T cells, CD33+ cells (marker for myeloid‐derived suppressor cells [MDSCs]), and CD163+ cells (marker for tumor‐associated macrophages [TAMs]) in CRLM tissue were determined by immunohistochemical staining. Fusobacterium nucleatum was detected in eight (4.4%) of 181 CRLM specimens. Compared with F. nucleatum‐negative CRLM, F. nucleatum‐positive CRLM showed significantly lower density of CD8+ T cells (P = .033) and higher density of MDSCs (P = .001). The association of F. nucleatum with the density of TAMs was not statistically significant (P = .70). The presence of F. nucleatum is associated with a lower density of CD8+ T cells and a higher density of MDSCs in CRLM tissue. Upon validation, our findings could provide insights to develop strategies that involve targeting microbiota and immune cells for the prevention and treatment of CRLM. Fusobacterium nucleatum was detected in eight (4.4%) of 181 colorectal cancer liver metastasis tissue. We found that the presence of F. nucleatum was associated with lower CD8+ T cell density and greater densities of both myeloid‐derived suppressor cells and tumor‐associated macrophages. We hypothesized that the presence of F. nucleatum might be associated with lower T cell density in CRLM tissue. A better understanding of the relationship between F. nucleatum and the immune microenvironment in CRLM could open new opportunities to target the gut microbiota and immunity for the prevention and treatment of CRLM. To test our hypothesis, we examined the presence of F. nucleatum in relation to the densities of CD8+ T cells, MDSCs, and TAMs in human CRLM tissues.The liver is exposed to gut microbiota through the enterohepatic circulation.22.1We analyzed 181 FFPE tissue specimens from consecutive patients with CRLM who underwent curative resection at Kumamoto University Hospital between November 2001 and January 2018. A prospectively maintained database was used to identify the patients; clinical data were collected by reviewing each patient’s medical record. Patients were excluded if they underwent noncurative resection and/or if they had extrahepatic metastasis. Written informed consent was obtained from each subject, and the study procedures were approved by the Institutional Review Board of Kumamoto University.2.2In total, 94 patients received preoperative chemotherapy. In accordance with the approach in previous reports, preoperative chemotherapy was given to patients with initially unresectable or marginally resectable disease (including patients with concomitant extrahepatic disease in a conversion setting) and to patients with disease that was considered highly malignant  in a neoadjuvant setting.2.3The objective of surgery was to remove all detectable lesions with a tumor‐free margin. The type of hepatectomy was based on preoperative findings, intraoperative ultrasonography findings, and the liver functional reserve. Nonanatomical partial hepatectomy was preferred if permitted by the tumor location. Portal vein embolization was undertaken if the tumors were unilobar and the future remnant liver was of an insufficient size. Radiofrequency ablation in combination with hepatectomy was used to treat unresectable tumors or those located deep within the remnant liver to spare the liver parenchyma, as previously described.2.4After treatment, all patients received regular follow‐up examinations of imaging studies and an estimation of tumor markers such as serum carcinoembryonic antigen and carbohydrate antigen 19‐9. When recurrence was observed, surgical treatment in combination with chemotherapy was preferred if the overall strategy was considered to be potentially curative.2.5F. nucleatum DNA was determined by qPCR. The nusG gene of F. nucleatum and the reference human gene SLCO2A1 were amplified using custom‐made TaqMan primer/probe sets (Applied Biosystems) as previously reported.F. nucleatum forward primer, 5′‐TGGTGTCATTCTTCCAAAAATATCA‐3′; F. nucleatum reverse primer, 5′‐AGATCAAGAAGGACAAGTTGCTGAA‐3′; F. nucleatum FAM probe, 5′‐ACTTTAACTCTACCATGTTCA‐3′; SLCO2A1 forward primer, 5′‐ATCCCCAAAGCACCTGGTTT‐3′; SLCO2A1 reverse primer, 5′‐AGAGGCCAAGATAGTCCTGGTAA‐3′; and SLCO2A1 VIC probe, 5′‐CCATCCATGTCCTCATCTC‐3′. Assays were carried out in a 384‐well optical PCR plate. Amplification and detection of DNA were undertaken using a LightCycler 480 Instrument II (Roche) under the following reaction conditions: initial denaturation at 95°C for 10 minutes, followed by 45 cycles of denaturation for 15 seconds at 95°C and annealing/elongation for 60 seconds at 60°C. The quantity of F. nucleatum DNA in each specimen was calculated as a relative unitless value normalized to the quantity of SLCO2A1 using the 2−∆∆Ct method, as previously described.Genomic DNA was extracted from FFPE tissue specimens of CRLM using a QIAamp DNA FFPE Tissue Kit (Qiagen). The amount of 2.6+ lymphocytes in the tumor margin areas using a digital microscope  and hybrid cell count software , and found a significant correlation between the manual count and digital count , suggesting that manual counting methods of immune cells in the current study would be reasonable.The FFPE tissue was serially sectioned at 3‐5 µm, dewaxed, deparaffinized in xylene, and rehydrated using a graded alcohol series. After the deparaffinization of tissue blocks, antigen retrieval was carried out in antigen retrieval solution using a steamer autoclave at 121°C for 15 minutes or by boiling in a microwave oven for 15 minutes. For CD8, CD33, CD163, FOXP3, Ki‐67, interleukin‐6 (IL‐6), and tumor necrosis factor‐alpha (TNFα) staining assays, the samples were incubated with mouse anti‐CD8 , mouse anti‐CD33 , mouse anti‐CD163 , mouse anti‐FOXP3 , Ki‐67 , IL‐6 , and TNFα , respectively. To evaluate immune cells , positive cells in the “tumor margin area” were counted in three separate fields at 200× magnification and their average values were calculated. We evaluated immune cells at the invasive margin of CRLM tissues because previous studies have shown that T cells infiltrate predominantly in the invasive margin of CRLM tissues, and that higher densities of T cells at the invasive margin of CRLM are correlated with better patient survival.To evaluate inflammatory cytokines (IL‐6 and TNFα), intensity and area of immune activity were each scored 1‐4. Staining intensity was scored according to the following criteria: 1, negative, no staining; 2, weak, light brown; 3, moderate, yellow brown; and 4, strong, brown. Proportion of positive cells was graded as follows: 1, 1%‐25% positive tumor cells; 2, 26%‐50% positive tumor cells; 3, 51%‐75% positive tumor cells; and 4, 76%‐100% positive tumor cells. The staining index  was calculated by totaling the staining intensity and area.2.7For multiplex immunohistochemical staining, an Opal 4‐Color Fluorescent IHC Kit (PerkinElmer) described above was used with anti‐CD8, anti‐CD33, and anti‐CD163 Abs. In accordance with the manufacturer’s instructions, the CD8 staining protocol was optimized using Opal 470 Fluorophore (red); CD33 and CD163 staining protocols were then optimized using Opal 520 Fluorophore (green). Finally, VECTASHIELD mounting medium with DAPI (Vector Laboratories) was used to stain nuclei.2.8U test. Clinical features were compared between groups using the χ2 test. The Kaplan‐Meier method and log‐rank test were used for survival analysis. Overall survival was defined as the time from the date of surgery to the date of death. Relapse‐free survival was defined as the time from the date of surgery to the date of diagnosis of recurrence.All statistical analyses were carried out using JMP software, version 10 (SAS Institute). Densities of immune cells in CRLM tissue were compared between groups using the Mann‐Whitney 33.1F. nucleatum DNA in CRLM tissue specimens were measured by qPCR assays. Fusobacterium nucleatum was detected in eight (4.4%) of 181 specimens . The presence of F. nucleatum in CRLM tissue was not significantly associated with age, sex, primary tumor location, timing of CRLM diagnosis, tumor size, tumor number, levels of serum carcinoembryonic antigen and carbohydrate antigen 19‐9, or the use of preoperative chemotherapy. When Kaplan‐Meier analysis was carried out by F. nucleatum status, F. nucleatum‐positive cases tended to have a poor prognosis for both overall survival  and CD163+ (a marker for TAMs) cells between F. nucleatum‐positive CRLM specimens and F. nucleatum‐negative CRLM specimens. Figure + cells. Compared with F. nucleatum‐negative CRLM specimens, F. nucleatum‐positive CRLM specimens showed significantly greater density of CD33+ cells  between F. nucleatum‐positive CRLM specimens and F. nucleatum‐negative CRLM specimens. There was no significant correlation between F. nucleatum status and density of FOXP3+ cells /PD‐1 ligand (PD‐L1),F. nucleatum‐positive CRLM patients. In our study, there was no significant difference in the density of TAMs or expression of inflammatory cytokines according to F. nucleatum DNA status, in part because the tumor immune microenvironment in the liver could be different from the colorectum. Further investigation is needed to examine the underlying mechanisms.In the F. nucleatum DNA in eight (4.4%) FFPE specimens from patients with CRLM. In previous reports, F. nucleatum DNA was detected in FFPE specimens from 8.6% and 13% of CRCs in JapanFusobacterium species, including F. nucleatum and other species, were detected in approximately 50% of Fusobacterium species‐positive primary CRC specimens by qPCR. Fusobacterium nucleatum‐positive CRCs have been associated with a high level of microsatellite instability.F. nucleatum in CRLM tissue. Our findings need to be validated by further studies.We detected F. nucleatum and its immunosuppressive effects; these factors could have important implications for the development of CRLM prevention and treatment strategies that involve targeting microbiota and immune cells.Oral health, diet, antibiotics, and pro‐ and prebiotics have been shown to influence the composition of intestinal microbiota.F. nucleatum in CRLM tissue. However, technical artifacts, if any, would have presumably biased our results towards supporting the null hypothesis. We recognize that another limitation of our current study is the lack of a widely accepted, standardized evaluation of antitumor immune response against human CRLM. Hence, we evaluated CD3+ T cells in addition to CD8+ T cells at the invasive margin of CRLM. However, we did not observe a significant association between density of CD3+ T cells and patient survival in CRLM. The current study suggests that density of CD8+ T cells at the invasive margin of CRLM are associated with antitumor immune response against human CRLM, although these findings need to be validated by further studies. Further studies are also needed to examine expressions of interferon‐γ, Perforin, granzyme B, and the immune checkpoint molecules  on T cells. Another limitation was that our study used a cross‐sectional design. Hence, we cannot exclude the possibility of reverse causation. Although it is possible that immune cells could eradicate F. nucleatum, experimental evidence indicating an immunosuppressive effect of F. nucleatum on T cell activity formed a basis for our specific hypothesis. Because no experimental system can perfectly recapitulate the complex nature of human tumors or the human immune system, analyses of human cancer tissue are useful for elucidating the relationship between microbiota and immunity in cancer.We acknowledge some limitations in our study. Because FFPE tissue specimens were used, routine histopathology procedures might have influenced the results of qPCR assays for the detection of F. nucleatum is associated with a lower density of CD8+ T cells and a higher density of MDSCs in CRLM tissue. Following validation, our findings could provide insights to develop strategies that involve targeting microbiota and immune cells for the prevention and treatment of CRLM.In conclusion, the presence of The authors declare no conflict of interest.This study was approved by the human ethics review committee of the Graduate School of Medicine, Kumamoto University and the study was carried out in accordance with the Helsinki Declaration of 1964.Supplementary MaterialClick here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.