N = 14,037, 49% female, 84% White) using the UK population‐representative Millennium Cohort Study. Findings support Patterson's coercion model, providing evidence for reciprocal within‐family relations between parenting practices and child behaviors. They suggest the importance of addressing parenting difficulties in families where children present with socioemotional difficulties in order to help prevent the accumulation of additional issues.Patterson's coercion model describes a gradual escalation in maladaptive parent–child transactions whereby externalizing behaviors lead to increases in maladaptive parenting and vice versa. The current study investigates the role of (predominantly mother‐reported) harsh parenting practices in the within‐person development of conduct problems, hyperactive/inattentive behaviors, and emotional problems. A random‐intercept cross‐lagged panel model was fit across ages 3, 5, and 7 ( ADHDattention deficit hyperactivity disorderBICBayesian information criterionCFIcomparative fit indexCLPMcross‐lagged panel modelCTSConflict Tactics ScaleMCSMillennium Cohort StudyMLRrobust maximum likelihood estimatorOSFOpen Science FrameworkRI‐CLPMrandom‐intercept cross‐lagged panel modelRMSEAroot mean squared error of approximationSDQStrengths and Difficulties QuestionnaireTLITucker Lewis indexSocioemotional difficulties in childhood are one of the best predictors of adverse developmental outcomes, including later diagnoses of mental health disorders, academic‐underachievement, and delinquency  from the UK population representative Millennium Cohort Study (MCS). Considering that early‐ to middle‐childhood is a period in which many developmental changes occur that may have implications for the processes implied by Patterson's coercion model, we allowed for time‐varying effects from age 3 to age 5 as well as from age 5 to age 7 in our model as it may be possible that the effects of harsh parenting vary by age. We hypothesized that, in line with Patterson's coercive model of parenting  harsh parenting tactics , in the development of socioemotional difficulties across early‐ to middle‐childhood as this is the period in which harsh parenting behaviors are most commonly used and at which socioemotional problems tend to emerge. Specifically, we investigate whether these parenting practices show bidirectional relations with two different externalizing behaviors (conduct problems and hyperactive/inattentive behaviors) as well as emotional problems. To analyze these within‐family relations, we fit a RI‐CLPM across ages 3, 5, and 7 using data . This reduced to 79.2%  at the age 5 wave and 72% at the age 7 wave . Since eligible families continued to be invited to participate in future waves of the MCS (unless they indicated they wanted to permanently withdraw from the study), some of the families that did not participate at the age 5 wave rejoined the study at the age 7 wave. To account for non‐random dropout and its complex sampling design, the MCS provides attrition weights as well as stratification and clustering variables that should be incorporated into analyses to correct the sample to be representative of the population. For further details, see the MCS cohort profiles . For demographic characteristics , self‐completion questionnaires included the Strengths and Difficulties Questionnaire (SDQ) and the Straus Conflict Tactics Scale (CTS). These were completed by the primary caregiver (98% mothers).Socioemotional problems were measured using the parent‐reported version of the SDQ Goodman,  The SDQ Disciplinary parenting practices were assessed using six items from the CTS Straus, . The CTShttps://osf.io/7g9dk/.In order to appropriately operationalize the within‐family processes implied by Patterson's model of coercive parenting, a RI‐CLPM was fitted. The RI‐CLPM disaggregates within‐ from between‐person effects by including random intercepts for each repeatedly measured variable. These are then allowed to covary. Cross‐lagged and autoregressive effects are defined between the residuals (reflecting deviations from the person/family‐specific means), giving insights into within‐family processes  disciplinary parenting practices mediated cascades from hyperactive/inattentive behaviors and conduct problems to emotional problems, no evidence for effects of hyperactive/inattentive behaviors on emotional problems via parenting was found. However, results indicated that the combined effect of harsh parenting and withdrawal tactics mediated a cascade from conduct problems at age 3 to emotional problems at age 7, while neither harsh parenting nor withdrawal tactics were significant mediators on their own. Bootstrapped analyses further confirmed these findings and additionally suggested that harsh parenting tactics may have a small mediating effect in cascades from conduct problems to emotional problems. For parameter estimates of the MLR analysis as well as bootstrapped confidence intervals of indirect effects, see Table ef was calculated from the fully standardized solutions for each mediating effect as a sensitivity analysis. The full calculations are available in Appendix e and f for the path from age 3 conduct problems to age 7 emotional problems via age 5 harsh parenting was β = .19. That is, the paths from an omitted confounder to the mediator and outcome would both need to be as large as β = .19. In the context of within‐person effects in RI‐CLPMs this is a reasonably large effect  harsh parenting practices in the development of socioemotional problems in early‐ to middle‐childhood. Following Patterson's coercion model, we hypothesized that conduct problems and hyperactive/inattentive behaviors would share reciprocal relations with harsh parenting tactics. This hypothesis was partially supported with hyperactive/inattentive behaviors sharing bidirectional relations with harsh parenting tactics from age 5 to age 7. However, contrary to our hypothesis, conduct problems did not exhibit reciprocal relations with harsh parenting tactics: only conduct problems at age 3 led to increased harsh parenting at age 5. We also hypothesized that harsh parenting tactics would mediate developmental cascades from conduct problems and hyperactive/inattentive behaviors to emotional problems. This hypothesis was partially supported by the combined effect of harsh parenting and withdrawal tactics mediating a cascade from conduct problems at age 3 to increased emotional problems at age 7, thus pointing toward the value of synthesizing different developmental cascade theories. Taken together, results indicated that the reciprocal relations between harsh parenting and child behavior differ across the studied time‐lags, thus, suggesting that the relations between parenting and child behavior are influenced by developmental changes occurring during early‐ to middle‐childhood. Examining the role of (predominantly mother‐reported) withdrawal tactics in socioemotional development within an exploratory framework, we also observed developmental differences in their effect. While results suggested that withdrawal tactics may be beneficial for reducing emotional problems and hyperactive/inattentive behaviors during the preschool years, we found that they may exacerbate externalizing behaviors from age 5 to age 7. Withdrawal tactics were also seen to increase as a response to externalizing behaviors. Finally, we also explored whether there were sex differences in the relations between (predominantly mother‐reported) disciplinary parenting practices and socioemotional difficulties, finding no evidence for differences by child sex.While a number of previous studies have found support for reciprocal relations between hyperactive/inattentive behaviors and maternal parenting behaviors  harsh parenting, while harsh parenting was associated with subsequent increases in hyperactivity/inattention but not in conduct problems. The absence of an effect of hyperactive/inattentive behaviors symptoms at age 3 on escalations in harsh parenting may reflect that hyperactivity is generally accepted as normative at that age Harpin, , and thuIn contrast to hyperactive/inattentive behaviors, conduct problems were not influenced by (predominantly mother‐reported) harsh parenting tactics, suggesting that during the preschool years, such tactics do not lead to an increase in behaviors such as throwing temper tantrums. Results of the current study also refute arguments for smacking and other forms of harsh parenting which are typically based on a logic that it is necessary to use harsh parenting techniques to effectively discipline children especially when they are young and potentially do not understand verbal explanations YouGov, . In factExamining the effect of withdrawal tactics on externalizing and internalizing behaviors, results of the current study suggested that such tactics may have differential effects over development. Specifically, from age 3 to age 5, (predominantly mother‐reported) withdrawal tactics were associated with a reduction in hyperactive/inattentive behaviors as well as emotional problems, suggesting that during the preschool years such parenting strategies do not have a negative on effect socioemotional child development but may even reduce some unwanted behaviors. However, this effect was reversed from age 5 to age 7, indicating that withdrawal tactics may lead to increased socioemotional difficulties in older children. These differential effects are particularly interesting in the context of recent discussions on the benefit of withdrawal tactics such as “time‐outs” as these used to be a go‐to technique for managing bad behavior  harsh parenting practices and emotional problems by synthesizing Patterson's coercion model and the dual failure model. Similar to the findings on hyperactivity/inattention, we found evidence for reciprocal relations between harsh parenting and emotional problems from age 5 to age 7 but not from age 3 to age 5. Hence, these results suggest that Patterson's coercion model  can alsoResults of the current study suggested that all studied mental health domains and (predominantly mother‐reported) parenting practices showed within‐person stability (as indicated by positive autoregressive effects) across ages 3 to 5, such that, for instance, harsh parenting practices at age 3 were associated with increases in harsh parenting practices at age 5. However, this was not the case between ages 5 and 7 where only emotional problems and hyperactivity/inattention showed within‐person stability. This suggests that, for conduct problems and parenting practices, the age span of 5 to 7 represents a transition period in which parenting behaviors and conduct problems change relative to individuals’ previous behaviors. This could potentially be due to children spending more time in school, which likely leads to an adaptation in parenting strategies and to changes in conduct behaviors as children's social circles grow and peer and teacher relationships become increasingly more important.Overall, results of the current study suggest that the reciprocal relations between parenting practices and child behavior are not stable across early‐ to middle‐childhood but that they are affected by developmental changes that occur during this period. Current developmental theories were not specific enough in guiding us to develop highly specific a‐priori hypotheses about developmental differences; thus, an important future research direction is to ensure that cascade and transactional models, such as Patterson's coercion model, more explicitly take into account developmental changes that have relevance for the transactions they address and more clearly specify any expected developmental differences.Finally, exploration of sex differences suggested that the relations between (predominantly mother‐reported) disciplinary parenting practices and socioemotional difficulties did not differ by child sex. This suggests that while boys comparatively more often exhibit conduct problems and girls more often exhibit emotional problems . This could have led to over‐ or underestimates of problem behaviors and use of parenting tactics and likely introduced shared‐rater bias to our models. Thus, future studies should replicate our findings using multi‐informant measures. Considering that most of the literature on the reciprocal relations between parenting practices and children's socioemotional development primarily focuses on maternal‐reported parenting, studies focusing specifically on the effect of paternal parenting behaviors are also strongly needed  harsh parenting practices and hyperactive/inattentive behaviors as well as emotional problems. Findings not only highlight that parenting practices such as smacking, or shouting may have detrimental effects on children's mental health but also that children presenting with behavioral issues may place additional strain on maternal parenting behaviors. Consequently, it is crucial for interventions aiming to reduce the occurrence of socioemotional problems, and particularly the co‐occurrence of emotional and conduct problems, to focus on the whole family system and specifically on parenting behaviors. Furthermore, considering that harsh parenting is still used, more attention should be paid to public health campaigns that can inform parents about the potential harmful effects of such parenting practices on children's socioemotional development and equip them with alternative, more adaptive parenting tools. Finally, the findings of this study support recent changes in legislation that ban smacking in Scotland and Wales and suggest that similar legislations should also be implemented in countries such as England where smacking is still permitted as a reasonable punishment.The authors have no conflicts of interest relevant to this article to disclose.The MCS was approved by the London Multicentre Research Ethics Committee and is funded by the UK Economic and Social Research Council. Written consent was obtained from all participating parents at each sweep.Supplementary MaterialClick here for additional data file.


Mature temperate woodlands are commonly dominated by ectomycorrhizal trees, whereas understory plants predominantly form arbuscular mycorrhizal associations. Due to differences in plant–fungus compatibility between canopy and ground layer vegetation the ‘mycorrhizal mediation hypothesis’ predicts that herbaceous plant establishment may be limited by a lack of suitable mycorrhizal fungal inoculum.We examined plant species data for 103 woodlands across Great Britain recorded in 1971 and in 2000 to test whether herbaceous plant species richness was related to the proportion of arbuscular mycorrhizal woody plants. We compared the effect of mycorrhizal type with other important drivers of woodland plant species richness.We found a positive effect of the relative abundance of arbuscular mycorrhizal woody plants on herbaceous plant species richness. The size of the observed effect was smaller than that of pH. Moreover, the effect persisted over time, despite many woodlands undergoing marked successional change and increased understorey shading.This work supports the mycorrhizal mediation hypothesis in British woodlands and suggests that increased abundance of arbuscular mycorrhizal woody plants is associated with greater understory plant species richness. Within woodlands, a greater diversity of tree species has been shown to buffer the negative effects of drought  mechanisms  seedlings, an EM host, were less colonised by EM fungi when growing near AM type trees. In other words, the sources of EM fungal inoculum were limited in soil in which AM type hosts were dominant. Similarly, the successful colonisation of AM‐dominated grasslands  could be excluded from forest areas dominated by EM type trees due to a lack of AM fungal inoculum. Kovacic et al. (Pinus ponderosa) compared with dead pines and observed a higher abundance of AM type understorey plants beneath dead rather than live pines. Similarly, Barni & Siniscalco , which is currently in decline due to the invasive emerald ash borer (Agrilus planipennis) and Chalara ash dieback (Hymenoscyphus fraxinus). Therefore, if herbaceous plant species richness is related to AM tree cover, this may have important consequences for woodland ecology and management in Britain.Most broadleaved woodlands in Great Britain are dominated by EM rather than AM hosts  that shades out the understory and can lead to a reduction in diversity. A noticeable exception to this trend was the 1987 storm in the southeast of the UK, which introduced open areas and resulted in increased herbaceous plant species richness . Tree species in each plot are recorded separately with diameter at breast height (DBH) and number of stems in each DBH class. Additionally, soil organic matter content (SOM) and soil pH (pH) were measured from a 5 × 15 cm soil sample removed from the centre of each plot. The assignment of mycorrhizal type of the trees and woody shrubs was made after thorough scrutiny of sources cited in available trait databases . The subset of AM type trees and shrubs was extracted and the AM overstory cover was calculated. The relative abundance of AM type trees and shrubs (RelAm: dimensionless ratio) was then the AM cover divided by the total cover. To estimate the inoculum potential of each plot, we use the correlation between shoot and root biomass. In a meta‐analysis of over 786 studies a positive linear correlation was found between shoot and root biomass in woodlands  and SOM (% dry matter lost on ignition) were also extracted from the data.The species richness (α‐diversity) for the ground flora was calculated for each 200 met al., lme4 package in R  and hawthorn (Crataegus monogyna) are two of the most common tree species found in British woodlands, and assignment of mycorrhizal type was considered weak for these plants. We therefore conducted a sensitivity analysis in which the mycorrhizal type was varied between AM and EM for hawthorn and AM and unknown for holly. In each instance the modelling process described above was repeated.Site was fitted as a random intercept with pH, shading, SOM, RelAm and year as fixed effects. In a small number of cases (five sites in year 1 and six sites in year 2) there were strong correlations between explanatory variables when examined within groups (Spearman correlation > ¦0.80¦). These sites were removed from the analysis, which reduced the between variable correlations to ¦0.26¦ Fig. . As the  Barton,  was usedet al., Spatial autocorrelation was tested by examining spline correlograms of the fitted model Pearson residuals , indicating that unknown site‐specific factors explained variation in understorey richness in addition to the fixed effects. Sensitivity analyses revealed that the models were not sensitive to changes in the mycorrhizal status of Ilex aquifolium or Crataegus monogyna, and changes in the mycorrhizal type did not alter the variable set in the model with the lowest AIC (Table The AIC ‘best’ model (lowest AIC) of understory herb α‐diversity contained pH, RelAm, SOM, year and the interaction between year and pH Fig. ; detailsIC Table .et al., We asked whether the proportion of AM type trees and woody shrubs affected herbaceous plant species richness in British broadleaved woodlands. We found that, in agreement with the mycorrhizal mediation hypothesis  should be directed to the Click here for additional data file.


Hydrothermal vents are rare deep‐sea oases that house faunal assemblages with a similar density of life as coral reefs. Only approximately 600 of these hotspots are known worldwide, most only one‐third of a football field in size. With advancing development of the deep‐sea mining industry, there is an urgent need to protect these unique, insular ecosystems and their specialist endemic faunas. We applied the IUCN  Red List criteria to assess the extinction risk of vent‐endemic molluscs with varying exposure to potential deep‐sea mining. We assessed 31 species from three key areas under different regulatory frameworks in the Indian, West Pacific, and Southern Oceans. Three vent mollusc species were also examined as case studies of different threat contexts  to explore the interaction of local regulatory frameworks and IUCN Red List category assignment. We found that these assessments were robust even when there was some uncertainty in the total range of individual species, allowing assessment of species that have only recently been named and described. For vent‐endemic species, regulatory changes to area‐based management can have a greater impact on IUCN Red List assessment outcomes than incorporating additional data about species distributions. Our approach revealed the most useful IUCN Red List criteria for vent‐endemic species: criteria B and D2. This approach, combining regulatory framework and distribution, has the potential to rapidly gauge assessment outcomes for species in insular systems worldwide. Article impact statement IUCN Red List assessments robustly compare extinction risk from deep‐sea mining at understudied hydrothermal vent habitats. The International Union for Conservation of Nature (IUCN) Red List of Threatened Species is the most comprehensive and rigorous tool for assessing the extinction risk of species worldwide  with iron‐infused scales to giant vesicomyid clams with red blood, vent molluscs span five taxonomic classes and display an amazing array of diversity  and extent of occurrence (EOO) provide important additional context. To clarify specific IUCN Red List terminology relevant to criteria B and D2 in the assessment of extinction risk of vent species, we have provided brief explanations of our application approach.location as a distinct area where a threatening event can rapidly affect all individuals in the area; thus, the size of a location depends on the area covered by the threatening event  grid  which is liable to continue unless remedial measures are taken” . We determined continuing decline and plausible threats based on deep‐sea mining regulations. A plausible likelihood of mining commencing exists in the areas of exploratory contracts signed by the ISA or in the EEZ of a nation that has not implemented a deep‐sea mining moratorium. By contrast, effective protection from mining  means that there is neither a continuing decline nor a plausible threat present. Such regulations are only considered effective where the threat is sufficiently mitigated by their implementation, for example, MPAs that include specific legislation preventing seabed mining , a classification to identify species that are likely extinct but for which confirmation is required , our approach ensures consistency between vent Red List assessments and enables the comparison of extinction risk across vent species and regions. Consistency in IUCN Red List assessments is imperative to support objective decision‐making for species conservation. Ensuring this consistency is a core value of the IUCN Red List  and AOO (12 km2) exceed the respective 100 and 10 km2 thresholds for CR under criteria B1 and B2. Consequently, B. manusensis was assessed as EN rather than CR , can affect IUCN Red List assessments (Akçakaya et al., Cyrtodactylus geckos have recently been described from karstic areas in Myanmar (e.g., Grismer et al., An approach based on our location‐threat matrix could, therefore, be applicable to other insular habitats, including terrestrial and freshwater systems, although this is limited to cases that have equivalency to the key features of the vent Red List: species endemic to a particular habitat; habitat occurs in spatially small, insular patches; limited mobility of species between habitat patches; and potential threat would cause eradication of all local habitat. This is potentially the case in many other threatened insular habitats including, for example, regions that are the targets of terrestrial mining. A high number of The IUCN Red List criteria can be applied successfully and correctly to understudied taxa in a habitat entirely new to the red‐listing process. With our approach, we were able to identify and summarize the variables that most affected Red List assessment outcomes for hydrothermal vent‐endemic species in our location‐threat matrix. The location‐threat matrix translates the IUCN Red List framework into a simple, preliminary tool to evaluate the extinction risk of vent‐endemic species, a tool we also believe will provide insight to conservationists studying species in similarly insular systems worldwide.Appendix S1. Vent Red List assessments published up to 2020. IUCN Red List category abbreviations: CR, critically endangered; EN, endangered; VU, vulnerable; NT, near threatened; LC, least concern.Click here for additional data file.


Revealing how formation protocols influence the properties of the solid‐electrolyte interphase (SEI) on Si electrodes is key to developing the next generation of Li‐ion batteries. SEI understanding is, however, limited by the low‐throughput nature of conventional characterisation techniques. Herein, correlative scanning electrochemical cell microscopy (SECCM) and shell‐isolated nanoparticles for enhanced Raman spectroscopy (SHINERS) are used for combinatorial screening of the SEI formation under a broad experimental space . This novel approach reveals the heterogeneous nature and dynamics of the SEI electrochemical properties and chemical composition on Si electrodes, which evolve in a characteristic manner as a function of cycle number. Correlative SECCM/SHINERS has the potential to screen thousands of candidate experiments on a variety of battery materials to accelerate the optimization of SEI formation methods, a key bottleneck in battery manufacturing. Scanning electrochemical cell microscopy (SECCM) enables combinatorial electrochemical assessment of a broad experimental space for solid‐electrolyte interphase (SEI) formation on Si electrodes for Li‐ion batteries, which is correlated to the heterogeneous and dynamic SEI chemistry through co‐located shell‐isolated nanoparticles for enhanced Raman spectroscopy (SHINERS). SHINERS provides access to many surface materials and morphologies,[Li‐ion batterieshologies,18, 192O4,4SEI understanding is, however, limited by the low‐throughput of conventional characterisation approaches, which make it impractical to study a large experimental space. Developing combinatorial strategies for high‐throughput exploration of SEI formation and properties is thus essential to accelerate the discovery of optimal SEI formation protocols, which is a key bottleneck in manufacturing of Li‐ion batteries. Combinatorial electrochemistryIn this work, we report a powerful combinatorial screening method to characterise battery materials and its application to study SEI formation and properties on Si negative electrodes for Li‐ion cells. This combinatorial method is made possible by the high‐throughput spatially‐resolved nature of SECCM and correlative chemical analysis of the SEI composition by SHINERS. By screening a broad experimental space, we reveal the dynamics of the SEI formation on Si electrodes under different conditions. This novel combinatorial approach is widely applicable to other interfacial processes that control performance in battery materials beyond the SEI with the potential to screen thousands of candidate experimental conditions in a short time, which opens new avenues to significantly accelerate experimental research in battery materials.+, Figure S2), 5 different cycles of charge/discharge  and 2 different electrolytes (1 M LiPF6 in propylene carbonate (PC) or ethylene carbonate/ethyl methyl carbonate (EC/EMC)) was studied . The cut‐off voltages were chosen to explore two state‐of‐charge (SOC) conditions: at low SOC (+0.05 V) where the SEI should be formed without severe mechanical changes on Si, and at high SOC (−0.13 V) where an overpotential below 0 V is reached, which can occur when operating under fast charging conditions.2 shell‐isolated nanoparticles (SHINs)  on top of the already formed SEI on the Si surface.The combinatorial correlative approach is illustrated in Figure 6 in PC. Figure Epa) of ca. +0.50 V is assigned to the phase transfer from LixSiy to amorphous Si. Delithiation current densities slightly increased upon cycling  due to the generation of more accessible amorphous Si,Epa was consistent over cycling . The appearance of only one delithiation process reinforces the idea of working under low SOC, with only one LixSiy phase formed, in contrast to macroscale experiments where two delithiation processes are usually observed.[The first set of combinatorial experiments was performed in 1 M LiPFcQ)  oxide (PEO) type species which agrees with previous SHINERS studies.−1 region that are assigned to C=O/CH2 modes. The main SEI forming reactions from the solvent are represented in Figure S14. Interestingly, a loss of Raman signal at 10 cycles was consistently detected and this correlated with increased background emission . High levels of emission have been previously assigned to LiPF6 decomposition.6 to the electrode surface, this is in‐line with the Raman spectra, which shows fewer contributions from SEI products. After cycle 10 the emission again decreases indicating that SEI formation, as observed by Raman, correlates with decreased LiPF6 decomposition. This result signifies that the initial SEI formed on Si is not sufficiently passivating to prevent further reaction of LiPF6 to “PxFyOz” type species. Elemental mapping  underlines the lack of phosphorus species in the SEI, showing that the degraded salt species is highly mobile. SEI dynamic formation is associated to the SEI breathing effect,Cathodic current densities decreased upon cycling but the rate of decay in cathodic charge  Figure C was depn Figure F. Most dd Figure G, correlQa) variation upon cycling . Note that anodic processes could be a combination of delithiation and Li stripping under high SOC. a/QcQ ratio , suggesting that Li+ is still lost on side reactions as the SEI might be unable to provide full passivation,[The two SOC conditions also led to a different behaviour of the anodic charge , and was also relatively consistent upon cycling . There was a stronger effect of the solvent under high SOC conditions  and associated with a shift in Epa .The second set of combinatorial experiments was carried out using 1 M LiPFs Figure A were qus Figure B. CurrenQc trend , but with a rough morphology and locally heterogeneous thickness . SHINERS analysis again reveals the SEI dynamic formation and evolution with EC/EMC, as a consistent set of SHINERS bands was not obtained for all charge/discharge cycles , which conceals some bands, with some recovery at the 15th cycle. Main SEI components detected (see Table S2 for assignment of main Raman bands) under these conditions were lithium methylene carbonate (LMC), PEO and ROCO2Li and RCOOLi species . The spectra are inconclusive on the specific formation of either lithium ethylene monocarbonate (LEMC) or lithium ethylene dicarbonate (LEDC) that have been previously observed on materials such as Sn.st cycle due to differences in solubilities of initial reduction products, with EC/EMC products being more soluble as barely any bands are observable. After further cycling, the main species detected by SHINERS are polymeric (PEO‐type), which have lower solubility and measured differences between the SEIs from PC and EC/EMC are minor.The d Figure C provides Figure F. Increae Figure G indicataQ trend upon cycling  has been demonstrated. Through this novel correlative SECCM/SHINERS approach, the dynamics of the SEI formation on Si electrodes was revealed, which is shown to be particularly dependent on experimental variables such as cycle number. The Raman analysis supports the important observation that the SEI formed upon cycling on Si negative electrodes is continuously evolving coupled with instability towards the LiPFSHINERS were placed onto an already formed SEI, thereby the outer SEI layer (i.e. SEI/electrolyte interface), which is more polymeric in nature, is detected. Future work will investigate the combination of SECCM with SHINERS already positioned onto the electrode surface, where they would preferentially enhance the electrode/SEI interface, thereby accessing information on the inner inorganic and organic layers, with use of a Kerr Gate to suppress fluorescence effects.This combinatorial approach has the potential to provide huge datasets of correlated electrochemical and chemical information to facilitate the understanding of complex interfacial phenomena in battery materials. Screening thousands of candidate experimental conditions would drive the discovery of high‐performing ones, which could be later assessed by more conventional battery techniques. This development opens new avenues to significantly accelerate the experimental throughput in battery research being widely applicable to different materials.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


We report a rare case of ovarian carcinoma in which a mediastinal recurrence was resected 16 years after the initial operation. A 72‐year‐old woman underwent hysterectomy with adnexectomy for stage IIIC ovarian serous carcinoma after neoadjuvant chemotherapy. Six courses of adjuvant chemotherapy were administered. Three years after surgery, left supraclavicular lymph node metastasis occurred, and radiotherapy and two courses of chemotherapy were administered. Six years before presentation, a metastasis at the right cardiophrenic lymph node was resected, and six courses of chemotherapy were administered. During follow‐up, a retrosternal tumour was found. The metastatic lesion in contact with the diaphragm was thought to result from pleuroperitoneal communication, and it increased in size. Although high‐grade serous carcinoma is aggressive, its sensitivity to chemotherapy may suppress early recurrence, contributing to good outcomes, but with late recurrence. Multidisciplinary therapy including surgery is required for improved long‐term prognosis for mediastinal metastasis of ovarian serous carcinoma. We report a case of ovarian serous carcinoma in which a mediastinal recurrence was resected 16 years after the initial operation. Recurrence of ovarian cancer often occurs in the abdominal cavity, but there are only a few reports of its recurrence in the thoracic cavity and mediastinum. Here, we report a case of ovarian serous carcinoma in which a mediastinal recurrence was resected 16 years after the initial operation.A 72‐year‐old Japanese woman presented with an abnormal shadow on chest computed tomography (CT). Sixteen years previously, peritoneal dissemination was observed when laparotomy was performed based on a diagnosis of ovarian cancer. After four courses of chemotherapy consisting of carboplatin and paclitaxel, hysterectomy with adnexectomy was performed. The pathological finding was stage IIIC serous cystadenocarcinoma. Six courses of adjuvant chemotherapy composed of the regimen mentioned above were administered. Three years after surgery, left supraclavicular lymph node metastasis occurred, and the patient was irradiated (70 Gy/35 fractions) after two courses of chemotherapy composed of carboplatin and paclitaxel. Six years before presentation, a metastatic lesion at the right cardiophrenic extrapulmonary area suspicious of lymph node metastasis was resected under the complete video‐assisted thoracic surgery, and six courses of chemotherapy also composed of carboplatin and paclitaxel were administered at another hospital.During follow‐up, a tumour that kept increasing in size was found in the inferior retrosternum of the anterior mediastinum, and the patient was referred to our department. Chest CT revealed a tumour sized 52 × 21 mm that was not detected 6 years previously (Figure Serous carcinoma is the most common histological type of ovarian cancer, accounting for 40% of all cases of ovarian cancer. A recent study showed that it originates from the fallopian tube epithelium and secondarily involves the ovary.To our knowledge, there are only seven reports of mediastinal metastasis from ovarian cancer other than mediastinal lymph nodes metastasis, including our case, and all seven were cases of serous carcinoma Table . The repInoue et al. reported a case of ovarian serous carcinoma that metastasized to the anterior mediastinum 29 years after the initial treatment.Although high‐grade serous carcinoma is aggressive, it is responsive to chemotherapy. Conversely, chemotherapy was not associated with improved survival for low‐grade serous carcinoma.Dr Hiroyuki Miura and Dr Shinichi Goto helped in the conception and design of the work and the acquisition and analysis or interpretation of data for the work. Dr Jun Miura drafted the work and revised it critically for important intellectual content. Dr Tomoko Yamamoto diagnosed this cancer pathologically. All authors contributed to the final version of this manuscript and approved it to be published.None declared.Appropriate written informed consent was obtained for the publication of this case report and accompanying images.


How mycoheterotrophic plants that obtain carbon and soil nutrients from fungi are integrated in the usually mutualistic arbuscular mycorrhizal networks is unknown. Here, we compare autotrophic and mycoheterotrophic plant associations with arbuscular mycorrhizal fungi and use network analysis to investigate interaction preferences in the tripartite network.We sequenced root tips from autotrophic and mycoheterotrophic plants to assemble the combined tripartite network between autotrophic plants, mycorrhizal fungi and mycoheterotrophic plants. We compared plant–fungi interactions between mutualistic and antagonist networks, and searched for a diamond‐like module defined by a mycoheterotrophic and an autotrophic plant interacting with the same pair of fungi to investigate whether pairs of fungi simultaneously linked to plant species from each interaction type were overrepresented throughout the network.Mycoheterotrophic plants as a group interacted with a subset of the fungi detected in autotrophs but are indirectly linked to all autotrophic plants, and fungi with a high overlap in autotrophic partners tended to interact with a similar set of mycoheterotrophs. Moreover, pairs of fungi sharing the same mycoheterotrophic and autotrophic plant species are overrepresented in the network.We hypothesise that the maintenance of antagonistic interactions is maximised by targeting well linked mutualistic fungi, thereby minimising the risk of carbon supply shortages. Complete individuals of mycoheterotrophic plants were dug out and, around each, three root tips of autotrophic plants were collected. In addition, we randomly sampled five autotrophic plant root tips from each quadrant of each plot aiming to better represent the local belowground mutualistic community. In total, we collected 60 root samples of mycoheterotrophic plants and 220 autotrophic plant root samples. For the autotrophic plants, 123 samples could be identified by DNA sequencing (please refer to subsequent paragraphs). Ninety‐nine samples among 28 autotrophic, and 45 samples among the five mycoheterotrophic plant species had Glomeromycotina reads. After removing samples with < 500 Glomeromycotina reads, we retained a total of 77 samples of 21 autotrophic species and 27 samples of the five mycoheterotrophic species. A detailed list of mycoheterotrophic and autotrophic plant species collected in this study can be found in Supporting Information Table As mycoheterotrophic plants are relatively rare and often have patchy distributions , using the KingFisher Flex Magnetic Particle Processor . Autotrophic plant species were identified by sequencing the markers s et al. . Plant i, et al.  in two s  of the fungal community associated with a plant species summarises the level to which the fungi in this community are phylogenetically related , where Cij is the number of shared plants between fungus i and j, and min is the smallest number of associated plants between fungus i and j . The ecological similarity of a pair of fungi represents their similarity in interactions through shared plants. We calculated similarity matrices between the fungi linked to autotrophic and mycoheterotrophic plants separately, using two different measures. The first measure is the Jaccard index, which corresponds to the number of plant species with which both fungi interact divided by the total number of plant species with which they interact, and the second is the overlap measure: motif , we built on the existing theoretical knowledge of trophic interrelationships and selected the diamond‐shaped module among those that were part of the theoretical research agenda on food webs. This module was described as ‘apparent competition’ and has been found to be overrepresented, and therefore highly relevant, in real food webs  and five mycoheterotrophic  plant species (please refer to Table The 100 rarefied binary matrices included 26 plant species (21 autotrophs and five mycoheterotrophs) and 110 ± 1.47 fungi. Of these, mutualistic networks had 100 ± 1.51 fungi (reduced from 115 fungi before rarefaction) and antagonistic networks had 61 ± 3.14 fungi (reduced from 96 fungi before rarefaction). In all the generated matrices, autotrophic and mycoheterotrophic plants shared 51 ± 3.16 fungi, which represents 55% and 92% of total fungi present in autotrophic and mycoheterotrophic, respectively Fig. .r = 0.20 ± 0.06, P = 0.003 ± 0.005) and mutualistic  networks  for autotrophic plants, in which Acacia sp. and Aspidosperma sp. had the highest psv and Sapindaceae the lowest, throughout which, the mycoheterotorphic plants are distributed , and also when accounting for the phylogenetic signal of the fungi . We also observed that the fungi with the highest normalised degrees, both in the mutualistic and antagonistic network, were all members of the Glomeraceae family , and also when pruning the dataset to only include the overlapping fungi . Repeating the analysis on resampled matrices that drew three random individuals per species resulted in an overrepresentation of the module in 99% of the cases when including all the fungi in the dataset , and in an overrepresentation of the module in 24.4% of the cases when including only the overlapping fungi in the dataset . Using the overlapping fungi dataset, the repeated analysis for multiple rarefaction depths indicates that, despite the number of diamond‐shaped modules increases with increasing rarefaction depth, it does not affect the result that pairs of fungi share a mycoheterotrophic and an autotrophic plant more often than expected by chance  on network metrics  should be directed to the Click here for additional data file.


To investigate the diagnostic value of an owner‐completed canine osteoarthritis screening checklist to help identify previously undiagnosed osteoarthritis cases, and assess their response to carprofen treatment by monitoring pain and functional mobility.Dogs (n=500) whose owners reported ≥1 positive response to the osteoarthritis checklist were examined to identify dogs with previously undiagnosed osteoarthritis. Eligible dogs (n=133) were evaluated for pain and video mobility analysis by Helsinki Chronic Pain Index and visual analogue scale scores, respectively, following carprofen treatment, administered for 30 days (n=95) or up to 120 days (n=38). Dogs were filmed at clinics performing activities , and scored at days 0, 30 and 120 using visual analogue scale by an independent blinded expert.A diagnosis of osteoarthritis was confirmed by a veterinarian in 38% (188 of 500) of dogs. Balance of sensitivity and specificity across the original group of nine screening questions was optimised to approximately 88 and 71%, respectively, after elimination of three questions. Pain measured by Helsinki Chronic Pain Index and functional mobility improved over time in response to treatment with carprofen. Mean ability scores for activities significantly improved between days 0 and 30 for walking, jogging, sitting/lying and walking down stairs, and days 0 and 120 for sitting/lying and walking up stairs.More osteoarthritis cases were identified in study dogs than previous prevalence estimates, indicating the screening checklist's potential to help identify for further evaluation cases that could otherwise remain undiagnosed. Improvements in function were demonstrated after carprofen treatment. This was accomplished by monitoring pain and mobility through ease of performing defined functional tests of activities of daily living in dogs with previously undiagnosed OA, in response to treatment intervention with carprofen.The study design was an open‐label, blinded assessment of clinically relevant outcome measures collected in a field setting. Ten veterinary practises in the United States, ranging from the East North Central division of the Midwest  and South Atlantic division of the South  were recruited for participation. Study enrolment proceeded from autumn 2014 to spring 2015. All dog owners signed informed consent forms before participation in the study and the participating veterinarians adhered to best‐practise standards for providing care, specifically associated with the treatment, monitoring and management of OA in dogs.Dogs that were presented to the veterinary practises for routine preventive care or evaluation of lameness/stiffness and were over 1 year of age, that were not receiving any treatments  for OA, were eligible. Dogs already diagnosed with OA and receiving NSAID therapy, corticosteroid therapy or with serious endocrine disorders were excluded.A nine‐binary response (yes/no) question owner‐completed OA checklist Table  was deveet al. Each veterinary clinic was asked to identify up to 20 dogs that met these criteria during their daily practise. The total enrolment goal was 20 dogs per clinic in each of the 10 clinics . Dogs confirmed as new (previously undiagnosed) cases of OA by a veterinarian based on the owner‐completed checklist, clinical history and examination were considered eligible as long as they were dogs of any pure or mixed breed, of any size or sex, and older than 1 year of age, considered to have OA‐associated pain and appropriate candidates for at least 30 days of carprofen administration by the veterinarian, based on pre‐treatment laboratory work  and physical examination. Confirmation of OA diagnosis was based on history, clinical examination and radiographs (as required), consistent with the typical approach in veterinary general practise settings  and location of limbs with OA.Enrolled dogs were prescribed carprofen at the labelled target dose of 4.4 mg/kg (2.0 mg/lb) bodyweight, administered orally every 24 hours or divided into two daily doses administered every 12 hours, for at least 30 days. The drug dosage was not changed at any time during the study. Two of the 10 clinics agreed to continue treatment to 120 days.On the day of OA diagnosis (before administration of carprofen), following 30 days (±5) of treatment (in the 30‐day group), and again following 120 days of treatment (in the 120‐day group), practise personnel collected videos of the dogs performing five activities of daily living: walking on a flat surface , jogging on a flat surface , rising from a sitting/lying position (side view), ascending stairs (front and rear views) and descending stairs (front and rear views). Written instructions for recording the videos (Table Pain assessment was performed by the pet owner at baseline (day 0), 30 days and 120 days (if relevant) by completion of the HCPI questionnaire. Owners rank pain on a scale of 0 to 4 with greater severity of pain represented as a higher pain score, for a total of 11 different questions. The expert evaluator was blinded to the results of the HCPI questionnaire.diagt in Stata 12.0 .Data from the checklist questions (answered as yes or no) were analysed to determine their accuracy in identifying incident cases of OA in canine patients, in comparison to veterinary assessment. OA diagnosis performed by the veterinarian as standard was considered to refer to the true disease status. Sensitivity, specificity, accuracy, predictive positive value and negative predictive value and their 95% confidence intervals (CI) were computed for each question, as well as for answering yes to at least one of the nine questions of the checklist and to at least one of the six questions with sensitivity >40%, using the command in vivo activities to ensure the use of animals was consistent with sound scientific practises, the 3Rs  and range] for VAS ability scores were computed overall and by time of observation. Associations between time of observation  and ability scores for walking and jogging were tested using linear mixed models fitted with a Gaussian distribution, identity link and residual pseudo‐likelihood in SAS . Time was modelled as an independent variable  and ability scores for walking and jogging were modelled as the dependent variables (in separate models). A random intercept for clinic was included, as well as a random residual component with a first‐order autoregressive heterogeneous covariance structure [also Toeplitz and unstructured covariance structures were tested and information criteria (Akaike's Information Criteria (AIC) and Bayesian Information Criteria (BIC)) were used to determine best model fit], to account for the design structure (dogs within clinics and repeated measures at the dog level) of the study. A similar procedure was followed to test associations between time of observation with outcomes pertaining to sitting and lying, walking up stairs and walking down stairs. Due to the lack of normality and homoscedasticity of these models' residuals, generalised linear mixed models were fitted using a Beta distribution, logit link, residual pseudo‐likelihood and Newton‐Rapson with Ridging optimisation algorithm. Dependent variables consisted of ability scores, which were transformed to continuous proportions from 0 to 1 . Zeros were assigned a value of 0.005. The same random structure as described above was fitted in these models. Model assumptions were tested in all models and residuals were investigated at the different hierarchical levels using graphical tools. Mean ability scores and their 95% CI were computed. P values <0.05 were considered statistically significant. The Tukey–Kramer procedure was used to prevent inflation of type I error due to multiple comparisons. For this one‐arm, open‐label study, a sample size of 133 provides a power >80% to detect an increase of a mean ability score of at least five points, considering a significance level of 5%, SD of 20, one baseline measurement and two follow‐up measurements (and 70% correlation between measurements).Descriptive statistics of the study population and tabulation of demographic variables by OA diagnosis are presented in Table Table A total of 399 video scores were collected from 133 dogs with a range of three to 36 cases per clinic. Data on the location of OA were available for 68% (90 of 133) of dogs, with the majority  affected bilaterally in the hind limbs  previously undiagnosed cases of OA, confirmed by physical or radiographic examination, from 500 previously undiagnosed dogs attending non‐acute healthcare visits, or specifically presented because of stiffness or lameness. The proposed checklist represented a starting point for discussion with owners and further veterinary investigation. We report prevalence (38%) at almost double that of previous, widely cited prevalence estimates of 20% for OA in the canine population Johnston . In addiet al. Screening tests are not considered diagnostic but are used to identify a subset of the population that should undergo further investigation in order to accurately establish the presence or absence of disease. Use of a simple, well‐targeted questionnaire, based on simple questions of canine behaviours indicative of mobility impairment, proved effective in identifying for further evaluation cases OA that could otherwise remain undiagnosed. The sensitivity and specificity of the nine‐question screening checklist were approximately 99 and 37%, respectively. After elimination of three questions, sensitivity and specificity were approximately 88 and 71%, respectively. Identification of previously undiagnosed OA cases allows veterinarians to initiate a treatment intervention to manage pain and improve quality of life. To our knowledge, owners completing the checklist in the present study had not been previously educated about OA in dogs. A previous study using a DJD screening checklist in cats reported that accuracy of the checklist was substantially higher when completed by informed owners who were aware of DJD and associated pain , and continued treatment up to 120 days  resulted in additional improvement in certain activities. The activities that improved with continued treatment up to 120 days were rising from sitting/lying and walking up stairs. Our assumption is that these are more painful than walking and trotting in a straight line or walking down stairs. These activities measured are relevant as they are encountered by most dogs in their daily home environment. The most difficult activities of going up stairs and rising from sitting/lying appear to take longer to improve. In addition, location of limb data was available for 90 dogs and only five dogs had OA diagnosed in one front limb only. Going up stairs and rising from sitting/lying involves more strength and weight on joints in the hindlimbs and in 60% of the patients OA was identified in the bilateral hindlimbs. Most dogs where location of the limb data was available included hind limbs and or back/spine which would make going up stairs and rising from sitting/lying more difficult. The high prevalence of bilateral hindlimb OA in this study population would also make recognition of OA in these cases more difficult than cases where only one limb is involved showing more obvious clinical signs of lameness.et al. A limitation of this study consisted of the lack of a complete set of videos from all veterinary clinics, which left evaluators unable to score a full set of activities for all dogs. Forty percent of videos were not scored due to the absence of some views required for scoring, but all dogs had at least three activities available for VAS scoring. In one clinic, no stair activities were videotaped, contributing largely to the missing scores and fewer dogs analysed in these activities. There were also fewer dogs in each activity recorded at day 120 as only two veterinary clinics chose to participate for 120 days and more dogs were lost to follow‐up in these clinics. If more dogs had videos collected at 120 days, a more complete picture of the degree of this drug's effect on ability over this longer time span may have been seen. Additional studies could be improved by using a more advanced model by including the nested effect of dogs to the clinics. In other words, the structure of dogs nested in the clinic should be considered. To improve the quality of the videos, it is now widely recommended to take the videos in slow motion. The surfaces that the videos were recorded differed in hardness that may have impacted the mobility issues seen but the surface hardness was consistent for each individual dog in each individual video. Another limitation is that radiographic confirmation was not available for all dogs in this study. The location of the limbs involved was confirmed for 90 dogs but not for all dogs. We cannot be certain that all dogs had decreased ability to perform activities due to OA only. Diagnosis was based on history, clinical examination, and radiographs (as required), as typically performed in veterinary general practise settings  significantly improved over a 30‐day period in dogs newly diagnosed with OA and treated with carprofen. For the two activities of sitting/lying and walking up stairs, a positive treatment response was maintained between 30 and 120 days, and this improvement was significant for climbing stairs. These results highlight the benefits of long‐term treatment and regular monitoring in a clinical setting for dogs with OA‐associated impairment of ability in performing activities of daily living. Video recording these functional activities of daily living combined with pain assessment tools and re‐evaluating after therapy may assist with ongoing treatment decisions and discussions with dog owners about response to therapy.This study was funded by Zoetis.AW, DB and DMA are full‐time employees of Zoetis. NC was remunerated for the advanced statistical analysis. BDXL was remunerated for the time taken to score the videos. AP provided independent contracting services for Zoetis.


Stable isotope analysis of teeth and bones is regularly applied by archeologists and paleoanthropologists seeking to reconstruct diets, ecologies, and environments of past hominin populations. Moving beyond the now prevalent study of stable isotope ratios from bulk materials, researchers are increasingly turning to stable isotope ratios of individual amino acids to obtain more detailed and robust insights into trophic level and resource use. In the present article, we provide a guide on how to best use amino acid stable isotope ratios to determine hominin dietary behaviors and ecologies, past and present. We highlight existing uncertainties of interpretation and the methodological developments required to ensure good practice. In doing so, we hope to make this promising approach more broadly accessible to researchers at a variety of career stages and from a variety of methodological and academic backgrounds who seek to delve into new depths in the study of dietary composition. Homo sapiens demonstrates a dietary flexibility and adaptiveness not seen among other hominins  are usually used to infer trophic position as the enrichment of 15N of the total proteinaceous tissue is generally 3‰–5‰ over that of the diet (Smith and Epstein δ13C) has been primarily used as an ecological source tracer because 13C enrichment during trophic transfer is small, usually less than 1‰, but δ13C varies predictably among different plant groups  and environmental settings  can vary significantly with location, season, and environment. For example, the δ15N range between nitrogen-fixing plants and those fertilized by seabird guano can be as large as 20‰ in plant tissues  under Creative Commons license.Pertaining to the carbon skeletons, the nonessential amino acids can be grouped according to their association with biosynthetic pathways. The glycolytic amino acids are synthesized from metabolic intermediates  of the glycolytic pathway (in the cytosol) and the tricarboxylic acid cycle  amino acids are synthesized from intermediates of the Krebs cycle (in the mitochondria). Glucose and glycerol are sourced to the glycolytic pathway, and fatty acids and short-chain fatty acids are sourced to the tricarboxylic acid cycle. Tricarboxylic acid products can also function as intermediates for Ala via routing to phosphoenolpyruvate and pyruvate. The catabolism of excess amino acids either occurs via gluconeogenesis or ketogenesis. Gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors such as the glucogenic amino acids (marked with a 1) and ketogenesis is the metabolic pathway for producing ketone bodies by breaking down fatty acids and ketogenic amino acids (marked with a 2). A large group of amino acids can be catabolized by both processes (marked with a 3). The broken lines indicate transportation of metabolites across the cell membrane. Images from macrovector . In hominin studies, the most applied TDF value for glutamic acid to phenylalanine (Glx–Phe) spacing is 7.5‰, although the degree to which this value is affected by turnover rates, nutritional demands and the macromolecular composition of the diets remains elusive. A feeding trial with mice showed that higher protein led to smaller TDFGlx–Phe, probably because proteins were used as an energy source instead of lipids  did not change significantly, indicating that these two amino acids may provide a more precise estimate of trophic position when the diet quality is unknown  value, is much lower in terrestrial than aquatic primary producers. However, it is becoming increasingly evident that it is not habitat type , but the degree of vascularization  that determines the β value in photoautotrophs  for vascular autotrophs and 3.3‰ (SD = 1.8‰) for nonvascular autotrophs  are considerably less variable than the βGlx–Phe values in vascular plants. Since vascularization greatly affects trophic position estimates, it is important for paleodietary and paleoecological reconstructions to rely on additional archeological and biogeochemical evidence to define the most realistic β values. The following expression can be used to estimate trophic position, provided that both TDF and β values are well defined:The protein source of consumers will significantly influence the accuracy of estimates of the trophic position of amino acid stable isotope ratios. Based on lleagues , 2010 fiT and S signify trophic and source amino acids, respectively. In terms of carbon, essential amino acids are powerful tracers of basal resources in part because δ13Cessential amino acid values in most animals match those in source protein with little or no isotopic offsets, in contrast to the nonessential amino acids where the offsets are typically much greater  is often the choice for exploring δ13Camino acid variability and patterns in a data set because it is an unsupervised technique  that seeks to maximize variability among samples while reducing the number of dimensions. Linear discriminant function analysis (LDA) is a supervised technique that seeks to maximize variability among the predefined groups or classes with the goal of predicting specific protein sources. The number of samples in each linear discriminant group should supersede the number of δ13Camino acid variables, and the number of samples representing a food source should be greater than the number of essential amino acids. Overall, many amino acid variables as possible are desired to maximize dietary information. An important distinction between PCA and LDA is that only the latter technique assesses the variability of each δ13Camino acid variable relative to the group mean. To achieve the same for PCA, it is necessary to factor out δ13C baseline variability by mean centering the data.Given that amino acid stable isotope ratios involves δ13Camino acid offsets can also differentiate consumers and predict major protein sources, but it has less predictive power than PCA and LDA, because it relies on three to four amino acids only. Archeologists have traditionally used paired amino acid offsets in conjunction with δ13C bulk or δ13Camino acid baselines to identify consumption of high freshwater, marine, terrestrial C3, and terrestrial C4 protein sources. The offset between glycine and phenylalanine has been used to separate the high terrestrial C3 and high freshwater protein groups from the high terrestrial C4 and high marine protein groups, and the offset between valine and phenylalanine has been used to separate aquatic and terrestrial resources  or liquid chromatography–combustion–isotope ratio mass spectrometry (LC–C–IRMS) to determine amino acid stable isotope ratios. These methods each come with analytical advantages and drawbacks. Of the two systems, LC–C–IRMS produces the most reliable data, but it is only suited for carbon isotope analysis, the required sample amounts are very high, and some essential amino acid peaks cannot be baseline separated . In terms of bones, because ribs are remodeled at a much faster rate than femurs, the former will represent a more recent dietary history prior to death than the latter . In contδ13Cessential amino acid offsets (Δtissue–diet) can fall outside the 1‰ analytical uncertainty range. Notably, Δtissue–diet for valine ranged between 1.3‰ and –2.1‰ . Degradation and residues from conservation treatments can also introduce exogenous materials to tissues, but this is less of an issue for amino acid stable isotope ratios than for bulk stable isotope ratios because it is possible to extract and isolate amino acids bound in tissue proteins. According to a wool degradation study, soil microbial degradation of keratin appears not to be protein selective, which means that δ13C and δ15N values of measured bone collagen . Although bulk bone collagen δ13C and δ15N analyses have been a staple of paleodietary investigations for four decades  and herbivores  have largely confirmed Neanderthals as high trophic level carnivores at sites such as Les Cottés and the Grotte du Renne, in France. However, they have also highlighted dietary contributions of plants in locations such as Spy Cave in Belgium , which, in turn, can lead to erroneous estimations of trophic positions  and their potential food sources represented by a mixture of archeological and modern rat, chicken, plant food, and different marine resources  estimated that the islanders relied much more on marine dietary resources than was previously thought, highlighting the finely tuned environmental adaptations and social resilience of humans on an island previously simplistically discussed as a classic case study of human overexploitation and societal collapse  from archeological studies using fauna as proxies for marine, terrestrial C3, and terrestrial C4 protein sources, and two human populations from C3- and C4-dominated biomes, respectively. One population was from Herculaneum  and is documented as living on a diet composed primarily of animal–plant terrestrial C3 foods and, to a lesser extent, marine fish , are skewed toward marine protein sources, which may reflect that fish protein contribution is much greater than overall fish caloric contributions  plot with the herbivore C4 faunal group, but with an offset along LD2 owing to the aforementioned Δtissue–diet or the fact that nonfaunal protein sources, such as millet grains, have slightly different δ13Cessential amino acid fingerprints than the C4 grasses eaten by the herbivorous fauna. It is also noteworthy that the elongated ellipse of the C4 fauna group along LD1 indicates that some specimens relied more on C3 plants than others. The LDA cannot adequately explain the diets of the two outliers: It is unlikely that the Nancheng individual predominantly relied on marine proteins given the site's distance from the ocean, and δ13Camino acid baseline values of the Herculaneum individual bins it with the other individuals from the site.A cornerstone for inferring past human diets is analyzing food sources that accurately represent past hominin diets. As was mentioned earlier, the breadth of archeologically relevant training data is still limited. Another constraint is the relatively low number of essential amino acid variables reported in archeological studies to date. Despite these limitations, much can be gleaned from already published um Rome, 9 CE and δ13Camino acid based dietary estimates, we compiled published δ13Camino acid data of humans from archeological sites applying the criteria that the data contained at least four essential amino acid variables . In addition to the Herculaneum and Nancheng individuals, we compiled data from four other sites: The first two sites are Jabuticabeira II, Piacaguera, and Galheta IV , in Brazil , where humans mostly subsisted on a maize-dominated diet ecology, the joint application of these compound-specific isotopic methodologies offers the opportunity of pinpointing trends of dietary reliance, perhaps even to individual resources in some cases, and determining metabolic patterns and nutritional deficiencies. For these methods to reach their full potential and to be used in a proper and uniform manner, it is essential that a wider, global multidisciplinary audience is aware of both their promise and current limitations.Bulk δ13Camino acid and δ15Namino acid approaches should be tempered by a need for consistency in preparation, measurement, and correction for added carbon during derivatization and for the use of reference standards and samples . More open, thorough discussions on the topic of analytical issues are essential if growing δ13Camino acid and δ15Namino acid data sets in archeology are going to be comparable and useful for future meta-analyses and building understandings of how modern variation can be related to archeological problems.As with many novel methodological tracer approaches, the enthusiastic application of  samples . Interlaδ13Camino acid and δ15Namino acid variability. With regards to δ13C, the nonessential amino acids are more complex to interpret than the essential amino acids, but they do open the door for the development of a series of additional biomarker approaches to diet reconstruction, such as inferring macromolecular composition  might influence δ13Camino acid and δ15Namino acid variability. As we demonstrated in the present article, amino acid stable isotope ratio analyses of hominin remains, where preservation conditions allow, hold a vast potential for advancing and nuancing our understanding of past human–environment interactions. A deep understanding of our past is important, because increasing social complexity and implementation of new technologies in human societies have not only shaped the diet of own species but have also altered the habitats and resource base of other species  can provide a point of exploration as to how spacing of biac028_Supplemental_FilesClick here for additional data file.


Neurological features have now been reported very frequently in the ongoing COVID‐19 pandemic caused by SARS‐CoV‐2. The neurological deficits associated features are observed in both acute and chronic stages of COVID‐19 and they appear to overlap with wide‐ranging symptoms that can be attributed to being of non‐neural origins, thus obscuring the definitive diagnosis of neuro‐COVID. The pathogenetic factors acting in concert to cause neuronal injury are now emerging, with SARS‐CoV‐2 directly affecting the brain coupled with the neuroinflammatory factors have been implicated in the causation of disabilities in acute COVID‐19 and patients with Long‐COVID syndrome. As the differentiation between a neural origin and other organ‐based causation of a particular neurological feature is of prognostic significance, it implores a course of action to this covert, yet important neurological challenge. COVID‐19 has challenged clinical experts from diverse disciplines in the management of patients with end‐organ COVID‐19 complication‐related clinical features, which are at times seen to be more disabling than lethal. Central nervous system (CNS) manifestations have been reported now with lesions affecting the brain in particular in the acute phase of COVID‐19Evaluation of the COVID‐19 patients with neurological complaints using current modalities like electroencephalograms and neuroimaging2 and PCO2 assessment with radiological examination of the lungs for opacities and scarring can serve as a useful guide in the differential diagnosis of symptomatic patients with neurological features, as alteration of these gases may explain the vague presentations  that are the basis of neurological features in COVID‐19.In the future, pieces of evidence elucidating neuronal injury caused by SARS‐CoV‐2, with unequivocal evidence of viral budding from neurons undergoing cell death, is expected to further clarify the direct role of SARS‐CoV‐2 in the causation of neurological manifestation of COVID‐19. It remains obscure why many Long‐COVID patients continue to exhibit neurological features,In conclusion, regardless of the mechanisms underlying the neuronal damage, the finding of neurological complications of COVID‐19 in the acute phase and Long‐COVID syndrome is alarming. A distinction of the focal neuronal zone(s) affected causing a specific neurological problem should be identified whenever possible. The healthcare professional should be mindful of differential diagnoses considering the roots of the origins of the adverse syndromic neurological outcomes in COVID‐19 and Long‐COVID. It is also worth considering that in COVID‐19 and Long‐COVID, a slow smoldering inflammation or apoptosis in neurons set by SARS‐CoV‐2 could be covert symptomatically in the early course of the disease that later becomes evident in the coming months to years. The remote consequences of this could set up a background for neurodegeneration on which data are emerging in patients with Long‐COVID and can impose an enormous burden on an already weakened healthcare system because of the ongoing pandemic. Identification of biomarkers in CSF and serum that can alert when the damages are occurring at molecular levels in the CNS is important and urgently needed.The author has no conflict of interest to declare and no funding was obtained for this article.


However, despite thousands of published articles on S. aureus skin infections, we still do not fully understand how infections resolve in vivo. Furthermore, the consensus is that most researchers most often infect mice using high doses of bacteria in planktonic or free form (summarised in Ref. S. aureus (500 CFU) and attach it to an agar bead to model a foreign‐body infection. This infection model formed a biofilm which is common for S. aureus during clinical infections and surprisingly recruited equal numbers of both neutrophils and monocytes to the infection site. While neutrophils were responsible for capturing and killing S. aureus, the functional role for recruited monocytes was not known.2S. aureus. By tracking the monocyte fate, we visualised the monocytes maturing to macrophages and identified these monocyte‐derived cells moved into the injured site well beyond the time‐course of bacterial clearance. In our study, we used complimentary methods of genetically modified mice, which lacked the chemokine receptor CCR2 involved in monocyte recruitment, as well as anti‐CCR2 depletion which depleted monocytes from circulation, to interrogate the functional role of recruited monocytes. In these monocyte‐deficient mice, bacterial clearance was equally efficient regardless of presence of absence of monocyte raising the question what are monocyte‐derived macrophages doing at the wound.Our group had previously investigated the recruitment, fate and function of various immune cells during a sterile injury using a small thermal injury model in the liver.3Imaging these wounds at 14 days post‐infection revealed that the wounds of monocyte‐deficient mice were hypervascularised, and wounds were unable to heal at 30 days post‐infection indicative of a aberrant healing and a perpetual wound in monocyte‐deficient mice. The delayed healing phenotype at 30 days post‐infection with a thick collagen capsule was evident in the CCR2‐deficient mice. Furthermore, many of the CCR2‐deficient wounds were still hypervascularised at 30 days post‐infection. This was an interesting observation since patients with chronic, hypertrophic scars or keloid scars are often hypervascularised compared to normal skin.We pursued this mechanism of dysregulated angiogenesis and delayed healing and found that leptin, a hormone involved in satiety and metabolism, was over‐produced by adipocytes in the wounds of monocyte‐deficient mice. Leptin was able to directly induce angiogenesis in the wounds of wild‐type mice by acting on leptin receptor positive endothelial cells. The angiogenic response was able to be reduced in monocyte‐deficient mice by administrating a leptin antagonist, as well as the counteracting hormone, ghrelin. Unexpectedly, the recruited monocytes were a source of ghrelin at the site of infection and by performing bone marrow transfers of ghrelin‐deficient bone marrow into wild‐type recipients, we showed that immune‐derived ghrelin was critical for angiogenesis and healing.4The results of this study may extend to cancer. Excessive angiogenesis is observed in solid tumours, where tumour progression from benign to malignant tumours can often be associated with an angiogenic switch from quiescent to proliferative vasculature.S. aureus skin infections, and this can be done by collecting tissue biopsies of skin infections and performing immunostaining for monocytes and ghrelin. We would predict that patients with increased levels of leptin, for example obese patients or patients with metabolic disorders might be more likely to have less efficient wound healing. Indeed, it is well known that patients with chronic diseases and co‐morbidities are at a higher risk for S. aureus infections.S. aureus bead infection model would be to determine whether co‐morbidities such as diabetes or obesity would further delay bacterial clearance and/or tissue repair.Our findings that monocyte‐derived ghrelin is critical for regulating angiogenesis in a mouse model of infection may be translatable to the human context considering human monocytes have been shown to express ghrelin at the mRNA levelThe authors declare no conflict of interest.


During voluntary muscle contractions, force output is characterized by constant inherent fluctuations, which can be quantified either according to their magnitude or temporal structure, that is, complexity. The presence of such fluctuations when targeting a set force indicates that control of force is not perfectly accurate, which can have significant implications for task performance. Compared to young adults, older adults demonstrate a greater magnitude and lower complexity in force fluctuations, indicative of decreased steadiness, and adaptability of force output, respectively. The nature of this loss‐of‐force control depends not only on the age of the individual but also on the muscle group performing the task, the intensity and type of contraction and whether the task is performed with additional cognitive load. Importantly, this age‐associated loss‐of‐force control is correlated with decreased performance in a range of activities of daily living and is speculated to be of greater importance for functional capacity than age‐associated decreases in maximal strength. Fortunately, there is evidence that acute physical activity interventions can reverse the loss‐of‐force control in older individuals, though whether this translates to improved functional performance and whether lifelong physical activity can protect against the changes have yet to be established. A number of mechanisms, related to both motor unit properties and the behavior of motor unit populations, have been proposed for the age‐associated changes in force fluctuations. It is likely, though, that age‐associated changes in force control are related to increased common fluctuations in the discharge times of motor units. This examination first necessitates a description of the measurement and quantification of force fluctuations; with the latter being of critical importance given that this review is the first to address changes in both the magnitude and complexity of force fluctuations. We then provide empirical evidence regarding age‐associated changes in force fluctuations and their functional implications, before discussing whether this is an inherent component of the aging process  or coefficient of variation (CV).Force fluctuations are typically assessed during submaximal isometric (or sometimes anisometric) contractions at an imposed target force.complexity”.Advances in analytical techniques have led to the recognition that fluctuations in muscle force are neither random nor independent but rather possess a statistically irregular temporal structure or “Complexity measures are derived from the field of non‐linear dynamics and include those related to information theory , which quantify the apparent regularity or randomness of an output, and those related to fractal geometry, which quantify long‐range correlations within an output.physiologically complex. As such, metrics such as DFA, which can estimate the temporal fractal scaling and differentiate the noise color of an output, are necessary to fully characterize physiologic complexity. The DFA α exponent theoretically ranges from ~0.5 to ~1.5 and differentiates outputs that are random , possess statistically self‐similar fluctuations  or are Brownian in nature .Complexity in muscle force has typically been quantified using approximate entropy (ApEn),3There is strong evidence that older adults (aged > 60 years) exhibit a greater magnitude of force fluctuations than young adults (aged ~20–30 years), which is interpreted as a decrease in force steadiness , in older adults during contractions ranging from 5%–40% MVC.3.3Many ADLs require either the maintenance and/or modulation of a specific force.During isometric,During isometric tasks, older adults exhibit lower complexity compared to young adults.3.4Many ADLs involve simultaneous performance of cognitive and motor tasks.Voelcker‐Rehage et al.4An impaired ability to control force will result in a neuromuscular response that is insufficient to withstand a perturbation or adequately compensate when performing a task.4.1Balance, locomotion and manual dexterity represent three fundamental motor skillsr = 0.455) between the CV of plantarflexion force during contractions at ≤5% MVC and the CV of foot center of pressure displacement . This relationship was observed for both young and old adults, though with the old adults exhibiting significantly greater CVs of both force fluctuations and center of pressure displacements. Similarly, a correlation between plantarflexion force variability (at 20% MVC) and postural sway has been observed in older women when standing on an unstable surface.Kouzaki and ShinoharaIncreased postural sway and sensorimotor variability have been proposed to be major risk factors for falls in older adults.With regards to locomotion, a correlation between muscle force accuracy (defined as the difference between the exerted and target forces) during eccentric knee extensor contractions at 50% MVC and measures of mobility in older adults with a history of falling has been observed.For manual dexterity tasks, moderate correlations have been observed between performance on a pegboard task and index finger abduction, bi‐digit pinch and wrist extensor forces across both young and old adults.4.2r = 0.48), but not with strength.As many ADLs have both motor and cognitive components, the greater variability exhibited by older adults during dual‐task conditions could have significant functional effects. An innovative study by Lodha et al.Other ADLs involve the maintenance of low levels of force for prolonged periods of time5To determine whether aging processes, inactivity processes or a combination of both contribute to increased torque complexity requires investigation of physically active/exercising older people. The fact that the detrimental age‐associated changes in force control appear to be reversible, at least to a certain extent suggests that this is not solely an aging phenomenon. Acute interventions such as skilled movement trainingPractice of a skilled motor task has been demonstrated to improve both force steadiness and manual dexterity. In Ranganathan et al.,With regards to strength training, a decrease in the magnitude of force fluctuations in the first dorsal interosseous of older adults has been observed during slow concentric and eccentric contractions following 2 weeks of light load training, consisting of lifting and lowering a load of 10% MVC.Older males (aged 70–80) who underwent 6 weeks of upper body strength training  in just one limb, decreased the CV (9.9 ± 13.1 to 6.0 ± 6.2%) and increased the SampEn (0.16 ± 0.13 to 0.27 ± 0.19) of tri‐digit pinch force in the trained limb.Studies on strength training in the lower body have, however, found more equivocal results. Bellew,Forms of exercise other than structured resistance training can also improve force control. Tai Chi is a low‐ to moderate‐intensity activity that involves a series of slow, fluid movements of the body with the aim of enhancing balance and stability. Christou et al.Taken together, these results indicate that indices of muscle force steadiness and complexity fall under the classification of variables that are age‐dependent but malleable by exercise.6As motor units transduce synaptic input from the central nervous system into muscle force, changes in their properties and the input to them are responsible for force fluctuations. Aging is characterized by a number of detrimental effects on the motor unit, including a net loss of motor units, changes to the morphology and properties of existing motor units, and altered input from peripheral, spinal and supraspinal centers6.1Both simulation and experimental data have demonstrated that weaker muscles exhibit greater force variability.The loss‐of‐force complexity with advancing age has been speculated to relate to the remodeling of motor unit populations,Simulation studies, have, however, indicated that increases in the amplitude of motor unit twitch forces have a negligible effect on force fluctuations.A further speculated mechanism is a difference in motor unit discharge properties between young and old adults. Simulation studies have indicated that varying the CV of motor unit discharge has a more pronounced effect on force fluctuations than reducing the number of motor units.6.2Motor neurons receive both independent and common synaptic input from a multitude of sources.R2 = 0.5, P < 0.01), and between force variability and common input fluctuations . Furthermore, differences in the magnitude of force fluctuations in the wrist extensors between voluntary and evoked contractions indicate that variance in common synaptic input is greater for older adults than young adults.Common modulation of motor unit activity can be assessed by either determining the level of synchronization between the discharge times of motor units (a time‐domain measure) or by performing a coherence analysis (a frequency‐domain measure).2 dopamine receptors and lower concentrations of serotonin.2 receptor increases force variability during low‐and moderate‐intensity elbow flexion contractionsOne source of synaptic input received by motor neurons arises from neuromodulatory pathways from the brainstem.7The evidence presented above has demonstrated that it is possible to reverse decrements in force control exhibited by older adults with various types of acute exercise training. Of greater interest and significance, though, is whether the decrement in force control in later life can be attenuated (or even prevented in the first place) and, therefore, whether the accompanying decrease in functional performance can be attenuated.The necessity to be physically active in order to maintain health and physical function throughout the lifetime is well established.Lifelong physical activity (both endurance and resistance training) has been demonstrated to slow the progression of age‐associated effects on muscle output ,Given the maintenance of other aspects of physiological and muscle function in physically active adults, the lack of research on lifelong physical activity and force control is a pertinent issue. It is, therefore, imperative that future research on aging and force control seeks to establish whether differences in force control exist between lifelong physically active adults and age‐matched sedentary individuals . Such research will, for the first time, elucidate the effects of aging on force control, and the mechanisms underlying the loss‐of‐force control, independently from those of inactivity. Moreover, tracking lifelong physically active older adults over a number of years, alongside age‐matched controls, would be novel and provide insight into the inherent aging process.A further area of focus for future research is the type of training intervention used in longitudinal studies. Given that muscle strength is not responsible for the age‐associated loss‐of‐force control and that training‐induced improvements in force control can occur independently of increases in strength,8There has, in recent years, been increasing research interest into how and why muscle force control decreases with age. Such research has demonstrated that the age‐associated loss of muscle force control is not only characterized by an increase in the magnitude of force fluctuations but also by a loss of complexity in force fluctuations. Importantly, this loss‐of‐force control is predictive of poorer performance of the fundamental motor skills, that is, balance, locomotion and manual dexterity, inherent to activities of daily living. Recent research has provided the strongest evidence yet for the mechanistic basis of the age‐associated loss‐of‐force control: namely, an increase in common synaptic input to motor neurons across the lifespan. There are, however, still many unanswered questions relating to aging and force control and, as such, there is a tremendous opportunity to perform studies that determine: (1) the source of the age‐related increase in common synaptic input to motor neurons; (2) whether physical activity interventions to reverse age‐associated changes in force control can also influence functional performance; and (3) whether lifelong physical activity has a protective role against the age‐associated loss‐of‐force control.The authors received no funding for this work.The authors report no conflict of interest.