Substantial uncertainty surrounds our knowledge of tree stem growth, with some of the most basic questions, such as when stem radial growth occurs through the daily cycle, still unanswered.We employed high‐resolution point dendrometers, sap flow sensors, and developed theory and statistical approaches, to devise a novel method separating irreversible radial growth from elastic tension‐driven and elastic osmotically driven changes in bark water content. We tested this method using data from five case study species. Experimental manipulations, namely a field irrigation experiment on Scots pine and a stem girdling experiment on red forest gum trees, were used to validate the theory.a‐priori predictions. Patterns of stem radial growth varied across case studies, with growth occurring during the day and/or night, consistent with the available literature. Importantly, our approach provides a valuable alternative to existing methods, as it can be approximated by a simple empirical interpolation routine that derives irreversible radial growth using standard regression techniques. Our novel method provides an improved understanding of the relative source–sink carbon dynamics of tree stems at a sub‐daily time scale.Time courses of stem radial growth following irrigation and stem girdling were consistent with  Our knowledge of the daily patterns of tree stem growth is limited. Sap flow sensors and stem dendrometers were used to isolate irreversible stem radial growth. New theory was developed that fully permits to separate elastic from irreversible diameter changes. Under drought conditions, growth was limited to night‐time. Under well‐watered conditions, growth tended to occur throughout the 24 h cycle, with differences from case study to case study. When exet al.et al.et al.et al.a priori. As a consequence, it is difficult both to determine general diel patterns of tree growth and to identify critically limiting variables. An alternative strategy has been to develop empirical fitting schemes calculating various indices of daily and seasonal growth rates to relate them to environmental factors  trees in a field experiment in Switzerland. We hypothesized that irrigation changed the diel pattern of growth. In particular, as a consequence of reductions in cellular turgor and carbon availability, growth would be limited to night‐time in the control plot; however, improvement in water supply and carbon status would extend growth into the daytime in the irrigated plot. The second experiment tested the model by comparing the diel pattern of diameter growth of girdled and control forest red gum  trees in a field experiment with well‐watered trees in Australia. Here, we hypothesized that relative to the growth pattern prior to girdling, increased substrate availability above the point of girdling would result in growth continuing throughout the 24‐cycle (i.e. including the night). Conversely, again relative to the growth pattern prior to girdling, growth below the point of girdling would be limited to only the most favourable hours of the day  using available carbohydrates stored locally  Use a model of radial water transfer  isolates irreversible growth from elastic dimensional changes; (2) provides the mechanistic basis for a simple empirical curve‐fitting approach that will abridge future analyses; and (3) helps to clarify the relative roles of sink versus source limitations facilitating the diel cycle of radial growth of trees. A better understanding of the processes driving tree growth is essential to achieve a better predictive capability of the effects of global environmental change on carbon sequestration and water use by forests. Our results highlight the need to consider the diversity of growing strategies across different biomes and tree functional types.et al.  and elastic (reversible) (E) components:b is now dropped in these derived bark‐only quantities for clarity. Plastic changes can only be positive or zero. Elastic changes can be positive, negative or zero, depending on whether the water content of the bark increases, decreases or remains unchanged. The two components of elastic change in bark thickness dDxylEdt and dDosmEdt are caused by two processes, that is, transpiration‐driven changes in xylem pressure potential (i.e. dDxylEdt) and phloem‐driven and growth‐driven changes in bark osmotic content (i.e. dDosmEdt)  xylem tension. Note that bark thickness is employed as a predictor to derive the tension‐induced changes in bark thickness during the next time interval  thickness over time comprise irreversible plastic changes  and elastic movements (caused by reversible changes in the water content of the bark). The total radial change of the tree bark thickness is defined as the sum of its plastic (irreversible)  is employed to derive the underlying time series of irreversible radial growth. The differential notation here should be interpreted as referring to finite differences over the time scale during which measurements are carried out. Because the system is linear, no errors are made by moving from differentials to finite differences over 10 to 30 min intervals. In essence, one minimizes the differences between dDb'dt and predicted irreversible changes dDPdt by applying Eqn dDPdt≥0 and Db'≥DP always, that is, osmotically driven elastic expansion/contraction cannot include the changes in irreversible diameter growth underneath. Overall, to identify the mean diurnal patterns of tree growth, the dual approach outlined here requires the estimation of five parameters, a, b, γ, m and r. However, if the main interest resides in interpolating seasonal time series and within‐day patterns are not of interest, three parameters need to be estimated, that is, a, b and γ, as will become clear after the Results section. In the approach outlined by Mencuccini et al. . We selected two experimental manipulations which would allow testing the proposed model. For each species, we restricted our analyses to periods when active growth was taking place.et al.Quercus pubescens Willd. individuals , one of the driest inner valleys of the European Alps . The trees were subjected to an experiment involving two levels of controlled soil watering . Controlled irrigation occurred on average every 14 days with an amount of water equal to half the mean monthly rainfall. In the ‘dry‐down’ treatment, irrigation was stopped from 19 January until 2 May when all trees received again equal amounts of water. Watering was repeated on three more days (3–5 May) on the previously mildly droughted trees. Periodic monitoring of leaf water potential conducted throughout the experiment indicated that trees had fully rehydrated two weeks after the end of the dry‐down . On 16 May 2015, all six trees were stem‐girdled. To carry out the girdling, a 5‐cm‐wide collar of bark was removed from the stem 0.7 m above ground. The exposed sapwood was wrapped in wet paper towels and plastic to prevent dehydration. Occurrence of new growth in the exposed windows was periodically checked. Paper towels were rewetted daily. At the time of girdling, total tree height ranged from 8.02 to 9.34 m and basal stem diameter from 78 to 103 mm. Ten days after girdling (26–27 May 2015), all trees were manually defoliated, and subsequently felled and removed from the chambers. The full experiment involved a temperature treatment crossed with the watering treatment on six additional trees, but those trees were not instrumented with sap flow sensors and stem dendrometers. A full description of the experimental setup is given by Barton et al. . Climate at the site is subtropical, mean annual temperature is 17 °C and mean annual precipitation is around 800 mm. n et al. . Whole‐t  were installed along the main stem on each of the six trees. One sensor was positioned below the canopy , the other inside the upper tree canopy . With regard to the girdling experiment, we will refer to the bottom installation as ‘below’ the point of girdling, and the one further up the stem as ‘above’ girdling. Twenty‐four high‐resolution point potentiometers  were al , Pinus radiata (D. Don) and Alnus jorullensis (Kunth). The three trees (one per species) used for this component of the study were 2–3 years old, growing in a glasshouse in Hobart, Tasmania . At the start of the experiment, the trees were 2–3 m tall and were grown in a potting mix consisting of eight parts composted pine bark to three parts coarse river sand. The potting medium contained low phosphorus premium controlled‐release fertilizer (N:P:K—17.9:0.8:7.3). The plants were kept well‐watered throughout the experiment, which lasted for approximately two weeks. Changes in stem thickness at the bark surface and at the sapwood/xylem surface were measured side by side on the stem at 0.1–0.2 m above the soil surface. Contact on the sapwood tissues was achieved by removing a small portion (ca 10 mm circle) of bark and periderm. The radial dendrometer consisted of two high resolution differential variable reluctance transducers  mounted on an alloy bracket 35 mm apart, attached to the stem via a 2.3‐mm‐diameter alloy rod and backing plate. The signal from each transducer was conditioned  and recorded on a data logger every 15 min . Sap flow was measured using the heat ratio technique  was employed to allow for lack‐of‐independence over time among measurements for each day. Testing of significance of fixed effects, random effects and autocorrelation structure followed Mencuccini et al. . Notably, the watering treatment was not even marginally significant (P > > 0.05) and was excluded from the final model. We firstly derived the parameters of Eqn All statistical analyses were carried out in R 3.0.2  but were allowed to vary across the six daily periods and across days. Initial values of the parameters were obtained by drawing values uniformly within pre‐specified boundaries . Using intervals of four hours and a tolerance of 1 × 10−9, the DE algorithm converged in between 1 and 6 h on a quad‐core laptop, depending on time series length. Following convergence, estimates of plastic and osmotically driven elastic thickness changes, as well as parameter estimates were obtained for each time interval and for each day in the time series  to model the relationship between diameter growth and time of day, with treatment as a fixed factor. To test for differences because of overall growth rates between treatments, we tested the significance of the coefficient for the fixed factor (i.e. intercept shift). To test for difference because of the daily rhythm between treatments, we tested whether the model employing the smooth terms calculated separately for each treatment gave a significantly better fit compared to the model with smooth terms calculated jointly for all data using a one‐degree A. jorullensis, P. radiata and E. globulus, grown under identical environmental conditions in a greenhouse in Tasmania. In all three cases, measured bark thicknesses  showed daily fluctuations larger or equivalent in magnitude to the actual growth signal, making the isolation of intra‐day and across‐days growth problematic. In all three cases, the majority of the intra‐day fluctuation was accounted for by the dynamics of the transpiration‐driven changes in bark water storage, as predicted by Eqn dDPdt=0 in Eqn S20 shows that elastic changes at times of no plastic growth can either be positive, negative or zero depending on a critical value of parameter a . Conversely, setting dDosmEdt=0 in Eqn S19 shows that bark osmotic content at the time of the daily minimum in the black ‘growth‐related fluctuations’ curve can vary depending on concurrent plastic growth, a and k  showed a clear periodicity, with minima found in the early afternoon, seedlings of the other two species showed relatively little diurnal dynamics. By way of comparison, for the Scots pine experiment in Switzerland, the diurnal cycle of bark water content driven by changes in bark osmotic content was very pronounced in the control (severely droughted) trees, but much smaller in the irrigated trees  are directly compared in Fig. ed trees . In the periment .A. jorullensis, radial growth was almost continuous through the 24 h cycle. E. globulus showed a similar trend, whereas in P. radiata growth slowed down during the night.Mean daily patterns of irreversible growth for the three trees in the Tasmanian experiment are also given in Fig. P < 0.01) for the irrigated compared to the control plot , with faster rates of growth during daytime in the irrigated plot, compared to a pattern of growth primarily occurring at night in the control plot.Finally, under two experimental conditions , we tested whether our model predicted diel patterns of diameter growth consistent with theoretical predictions. The mean within‐day patterns of thickness increase of Scots pine in a field irrigation experiment in Switzerland are shown in Fig. lot Fig. e. HoweveE. tereticornis before and after girdling in the Australian experiment are shown in Fig. P > 0.05, for both stem positions and both prior to and after re‐watering, cf., P < 0.001), but no difference in absolute rates of growth . The diel patterns dramatically diverged after girdling . Growth above the girdling point increased in magnitude, while growth below the girdling point decreased . In addition, below the point of girdling, thickness increases were close to linear prior to girdling in both periods; however, they only occurred for a few hours at the end of the night and in early morning after girdling . In contrast, thickness increases above the point of girdling peaked during afternoon hours prior to girdling, whereas thickness increases continued almost throughout the 24 h cycle except the early and middle parts of the night after the girdling . Environmental conditions remained relatively comparable throughout the three periods.Within‐day patterns of thickness increase of et al.  is the predicted bark thickness change after accounting for changes in xylem tension. In the second step, a constrained optimization routine is employed to fit the theoretical equation for irreversible growth to the ‘growth‐related fluctuations’ curve to separate out the residual fraction of variation attributable to osmotically driven elastic changes. Importantly, the optimization routine minimizes the difference between ‘growth‐related fluctuations’ and predicted irreversible growth. In other words, it determines the minimum amount of osmotic‐induced elastic changes that need to be invoked to predict zero or positive irreversible growth, given the time series and the assumed osmotic model. While this approach is useful in providing a mean within‐day pattern of irreversible growth, it does not need to be applied at the seasonal time scale, if the main interests are the long‐term growth patterns and not the within‐day variations. It follows from the minimization approach that a simple linear interpolation between the daily minima of the ‘growth‐related fluctuations’ curve effectively gives the seasonal curve for irreversible growth, at the cost of being unable to determine within‐day patterns accurately. This empirical interpolation considerably simplifies the problem of isolating irreversible growth. Isolating growth at a seasonal time scale requires 1) applying Eqn  applyinget al.  model oret al.m of Eqn kFgr,0 gives the irreversible thickness growth for a given flux of carbohydrate to the bark), while parameter r relates to the interplay between irreversible growth and elastic properties affecting the transients. The approach is based on two assumptions  and the daily shape of the sap flow data.The osmotically driven model presented here is based on a solution to the problem of growth as a balance between relative carbon source strength and carbon sink demand. The model cannot quantify source and sink strength in an absolute sense and it has therefore a reduced scope compared to models directly addressing the question of the links between source strength of photosynthesis and sink strength of stem radial growth  plot, but would occur also during day‐time in the irrigated plot. In the et al.  and with much of the available empirical evidence of stem leader growth of trees. The application of the osmotic content‐driven model derived here is not necessary at the seasonal time scale, because a linear interpolation of the minima of the regression function following Mencuccini et al.  sufficesNote 1. Details of the bark water use modelNote 2. Details of the osmotic model.Figure S1. Typical example of convergence of the function employed to derive irreversible growth from the ‘growth component’ curve. The minimum of the function is achieved very quickly as a function of iteration number; however, numerous additional iterations are required to achieve the global minimum.Figure S2. Example of parameter search and convergence as a function of iteration number. Three example parameters are shown.Figure S3. Application of the two models based on Eqns 3 and 6 to the irrigation experiment in Scots pine. Left panels) Application of the coupled models for control (top) and irrigated (bottom) trees. Green curve, raw bark thickness readings from the point dendrometers; black curve, ‘growth component’ curve from which tension‐driven elastic changes have been subtracted from; red curve, plastic irreversible growth. Right panels) Averages across the sampled period of the 24 h cycles of the two elastic curves, that is, tension‐driven bark water content changes (blue curve), and osmotic‐driven bark water content changes (green curve). The bands around the blue and green curves give one standard error around the mean.Figure S4. Same as for Fig. S3, but applied to the stem girdling study on Eucalyptus tereticornis. Plots are given for the two positions (above and below the point of girdling) and two time intervals (before and after girdling)Eucalyptus globulus at Richmond, New South Wales, Australia. This figure replicates data given in Fig. 5 of the main paper but with the data now split to show the two positions separately for the control and the mildly droughted trees. Panels on the top provide environmental information for three periods throughout the summer of 2014 . Panels represent photosynthetically active radiation PAR, air relative humidity RH and soil relative water content SWC . The first period represent the initial phase of the experiment, when three trees were mildly droughted and three were continuously watered. The second period represents the second phase of the experiment when the mildly droughted trees were re‐watered. The third period represents the post‐girdling phase, three days after the girdling had been carried out. The bottom panels give the mean inferred daily patterns of growth, for two positions along the stem (top and bottom) and separately for control (Con) and dry‐down trees (Dry). Growth for the control is given in blue (base) and violet (top); growth for the dry‐down is given in red (base) and pink (top). The ‘top’ stem position corresponds to a point above girdling; the ‘bottom’ position corresponds to a point below girdling. The figure shows that no differences exist between the mildly droughted and the control trees for either position and either period. Only the boundaries of the standard error bands for eachFigure S5. Test of model performance in a field stem‐girdling experiment on Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.


Immune checkpoint‐blocking antibodies have therapeutic activity against relapsed or progressive classic Hodgkin lymphoma (cHL), but Hodgkin Reed‐Sternberg cells can develop resistance to this therapy via multiple mechanisms. To improve the efficacy of immune checkpoint blockade, we need a more precise understanding of the immune escape mechanisms active in individual cHL patients, and this requires a detailed characterization of immune cell populations in the tumor microenvironment. These cell‐cell interactions can now be studied by multiplex immunohistochemistry coupled to digital image analysis. This method should allow the identification of actionable target molecules mediating resistance to immune checkpoint inhibitors in individual cHL patients, thereby favoring the implementation of personalized therapies. A diagnosis of classic Hodgkin lymphoma (cHL) is based on the finding of neoplastic Hodgkin Reed‐Sternberg (HRS) cells within the heterogeneous cellular setting of a lymph node.Programmed cell death ligand 1 (PD‐L1) expression is invariably observed among at least a large fraction HRS cells in nearly all cases of cHL. PD‐L1 expression is driven by gains of chromosome 9p24.1, the locus that includes PD‐L1, PD‐L2, and JAK2. Gains at 9p24.1 directly increase the PD‐1L expression and JAK2 expression. Increased JAK2 may result in a heightened sensitivity of HRS cells to cytokine‐mediated JAK‐STAT signaling and thus even greater PD‐L1 expression due to cytokine‐mediated induction of the protein.Given the particularities of cHL, where a small number of tumor cells reside in close proximity to various types of immune cells, greater information on the spatial distribution of these cells is required. This need is even more important in the immunotherapy era to support therapeutic decision‐making. The choice of therapy for a particular patient should take into account not only the level of expression of the protein targeted by a therapy, but also the location and phenotype of immunosuppressive cells in the TME. In particular, it is important to know which secretory ligands and membrane‐bound molecules are being expressed by immune cells that are in proximity to HRS cells and that may be providing signals that allow HRS cells to resist apoptosis.Hodgkin Reed‐Sternberg cells' ability to process and present antigens may also dictate their susceptibility to immunotherapy. In solid tumors, the response to immune checkpoint blockade requires tumor antigen presentation by HLA class I molecules on cancer cells to cytotoxic CD8 + T lymphocytes. In cHL, however, most tumor cells do not express HLA class I due to loss of beta2‐microglobulin.The identification of immune escape mechanisms should have therapeutic implications because the characterization of these mechanisms in individual cHL patients may guide the choice of immunotherapy or combination therapy. Achievement of this clinically relevant goal requires the detailed study of immunomodulatory proteins expressed by the different cell populations infiltrating the TME. This analysis is now possible using multiplex immunofluorescence or immunohistochemistry coupled to digital image analysis.The fact that HRS cells use different mechanisms to escape antitumor immunity is currently limiting the efficacy of immune checkpoint blockade. The multiplex analysis of the TME could reveal which cells  and proteins  are limiting the efficacy of immunotherapy in individual cHL patients, and these results may guide the choice of a personalized treatment. The personalized combination of monoclonal antibodies, immunomodulators, and checkpoint inhibitors with mechanism‐based therapies may make HRS cells vulnerable to immune eradication. This approach has important basic scientific and translational implications, since it will enable future investigations into the mechanisms regulating immunity to cHL and will extend the perspective of optimizing immunotherapy for relapsed or progressive cHL and probably also for newly diagnosed disease.The authors declare no competing financial interests.


We believe that this is the most densely sampled longitudinal study of CD; previous studies collected samples every 1–3 months.https://github.com/knightlab-analyses/longitudinal-ibd).We collected daily stool samples for up to 6 weeks from 19 CD subjects and 12 controls (see the analysis notebook for cohort description, methods and data, A critical experimental design question for clinical studies is whether a finite budget should best be spent collecting samples from more patients or collecting more serial samples from each patient? Therefore, we created a Random ForestsNovel analyses aggregating features over time and combining both alpha and beta diversity over time using our intensive daily sampling demonstrate that the main benefits are already obtained by collecting between three and five faecal specimens, and no additional benefits are obtained beyond seven serial samples. Similar results are found for monthly sampling. These results highlight the importance of treating CD as a volatile, time-varying condition, even during clinical remission, but provide hope to clinicians in that a relatively small number of samples yield large additional benefits, facilitating patient compliance. This information can be used to design collection of faecal samples for a large prospective cohort of patients with CD for longitudinal studies of host–microbial interactions over time.The methods demonstrated here have not previously been used for microbiome analyses but have been used for other engineering applications, for example, in production lines to predict product specification outcomes in a steel manufacturer’s facility.


Studies of schizophrenia have pointed to the role of glutamate in its pathophysiology. Mice lacking D‐serine show impairments in neurotransmission through NMDA receptors and display behaviors consistent with features of schizophrenia. Yet, socio‐communicative deficits, a characteristic of schizophrenia, have not been reported in serine racemase knockout mice.We use behavioral testing  to examine socio‐communicative behaviors in these mice.Serine racemase mice show abnormal social investigation and approach behavior, and differ from wild‐type controls in the duration and number of vocalizations they emit in the presence of a conspecific. Serine racemase knockout mice were not impaired in a cognitive test (novel object recognition), although they displayed abnormal behavior in the acquisition phase of the task.Serine racemase knockout mice demonstrate abnormalities in socio‐communicative behaviors consistent with an impairment in sociality, a negative symptom of schizophrenia. Studies of schizophrenia have pointed to the role of glutamate in its pathophysiology. Mice lacking D‐serine show impairments in neurotransmission through NMDA receptors and display behaviors consistent with features of schizophrenia. Yet, socio‐communicative deficits, a characteristic of schizophrenia, have not been reported in serine racemase knockout mice. Our findings indicate that these mice show abnormal social investigation and approach behavior. Characterizing changes in glutamatergic neurotransmission has been a major emphasis in efforts to elucidate this disease's underlying pathophysiology  of the University of Minnesota—Twin Cities. Mice were provided food and water ad libitum except during experimental testing, and housing lights were maintained on a 12:12 hr light/dark cycle. All experimental mice were pair‐housed with same‐sex littermates. SR knockout mice were obtained via the Coyle Lab at Harvard University  mice were handled for 3 consecutive days (5 min per day). Test mice (WT or KO) were paired with sibling heterozygous mice. On the test day, each mouse pair was placed into a 20 × 25 × 45 cm arena containing corncob bedding with a nontransparent partition for 5 min. Following removal of the partition, the mice were allowed to interact freely for 10 min. Oral–oral and oral–anogenital interactions were scored manually for each mouse by a genotype‐naïve experimenter using Button Box 5.0 . Ultrasonic vocalizations (USVs) were also recorded during interactions using high‐frequency microphones and Avisoft‐Recorder software .2.4Novel object recognition (NOR) test procedures were similar to those described previously by our laboratory , which varied in color, material, texture, and shape. The location of the novel object in the test chamber, as well as the test order and the novel/familiar object pair, were counterbalanced across conditions. Object investigation during testing was scored by trained group‐naive raters using Button Box . During the acquisition phase of the task, time spent exploring either of the two identical objects placed in the testing apparatus was recorded. The total duration of investigation was calculated for each mouse (exploration score). During the test phase, a normalized novelty score was calculated for each animal by dividing the difference in investigation times for the novel and familiar objects by the time spent investigating the familiar object.Preference for the novel object (and memory for the familiar object) was defined as time spent investigating the unfamiliar object compared to chance during the test phase.2.5T tests were used for analyses of behavioral data.Ultrasonic vocalizations were analyzed with custom‐built R script . Generation and processing of spectrograms were conducted as described previously  than in KO mice . There were no significant differences between the groups in the ratio of time spent investigating the mouse versus empty chamber . The SRKO group did not show this preference . Further analysis indicated no between‐group differences in time spent exploring the novel chamber , but a significant difference in the novel/familiar ratio . Thus, SRKO mice demonstrated decreased novelty‐related exploration in social contexts.During the social novelty phase, within‐group comparisons revealed a strong preference by WT mice for the chamber containing the familiar conspecific . To further investigate whether there was a difference in vocalization type between the two groups, we derived normalized vocalization estimates for each syllable. For this analysis, we first calculated the frequency of occurrence of each syllable within each experimental group and divided this number by the total number of vocalizations within the group. The resulting measures were used in between‐group comparisons of vocalization types  vocalizations, and a higher proportion of JU  and MJ vocalizations .Analyses revealed no differences in oral–oral and oral–anogenital investigation between WT and KO mice. However, a significant between‐group difference was revealed in the duration of vocalizations. Overall, SRKO animals showed longer total duration of vocalizations than WT mice . There were no significant differences between the groups in the animals' relative exploration of the novel versus familiar object in the test phase .There were significant between‐group differences in exploration scores Figure , with SR4Social deficits are a prominent negative symptom of schizophrenia and have a profound impact on quality of life. Given the putative relevance of NMDARs to the pathophysiology of the disorder, their contribution to aberrant social behavior has been studied extensively in rodent models utilizing pharmacological interventions and genetically modified animals  in the SRKO mice. Conversely, the same mice showed a lower proportion of short, nonmodulated vocalizations (NS). Although our understanding of the functional significance of specific patterns of USVs that are prototypical in the mouse is limited, it is interesting to note that variations in these patterns and their relative durations are related to sociality and subject to genetic influences  task, the SRKO mice exhibited greater exploration of the two identical objects. This may be due to an inability to properly form a stable representation in memory of the previously viewed object, or possibly indicative of increased perseverative behavior. In contrast, the SRKO mice did not show deficits in discriminating between the novel and familiar objects during the test phase of the task. This suggests a sparing of long‐term object recognition memory in these mice, consistent with previous findings (DeVito et al., The fact that novel object preferences were intact in the SRKO mice is in itself an important positive control because it confirms that the observed abnormalities in their preferences for a novel conspecific were not due to a deficit in memory consolidation or to a more general aversion to novelty. Rather, the aberrant social behavior seen in both the social approach task and dyadic interaction suggests that SRKO animals manifest a pattern of abnormal social interaction akin to asociality—a prominent negative symptom of schizophrenia. This reinforces the view that depletion of D‐serine recapitulates a range of seemingly disparate schizophrenia‐like behavioral abnormalities in mice (Balu et al., Thus, our findings lend support for the use of SRKO mice in studying mechanisms underlying impaired social functioning in schizophrenia and highlight the potential benefit of exploring the therapeutic utility of SR agonists in the treatment of this disorder's negative symptoms.None declared.


Pan troglodytes verus) living in the Comoé National Park, Ivory Coast (CI). We analyzed all the stick tools and leaf‐sponges found that the chimpanzees used to forage for ants, termites, honey, and water. We found a particular behavior to be widespread across different chimpanzee communities in the park, namely, dipping for water from tree holes using sticks with especially long brush‐tip modifications, using camera traps, we recorded adults, juveniles, and infants of three communities displaying this behavior. We compared water dipping and honey dipping tools used by Comoé chimpanzees and found significant differences in the total length, diameter, and brush length of the different types of fluid‐dipping tools used. We found that water dipping tools had consistently longer and thicker brush‐tips than honey dipping tools. Although this behavior was observed only during the late dry season, the chimpanzees always had alternative water sources available, like pools and rivers, in which they drank without the use of a tool. It remains unclear whether the use of a tool increases efficient access to water. This is the first time that water dipping behavior with sticks has been found as a widespread and well‐established behavior across different age and sex classes and communities, suggesting the possibility of cultural transmission. It is crucial that we conserve this population of chimpanzees, not only because they may represent the second largest population in the country, but also because of their unique behavioral repertoire.Over a 6 month period during the dry season, from the end of October 2014 to the beginning of May 2015, we studied tool use behavior of previously unstudied and non‐habituated savanna chimpanzees . WDTs are significantly different from tools used to gather honey. The use of WDTs is widespread across neighboring communities in the park. Regarding large predators at the site, the presence of lion (Panthera leo) has not been confirmed after 2002, but leopards (Panthera pardus) and spotted hyenas (Crocuta crocuta) are common  to 165 km2 (block A). The forest cover ranged from the 15% in blocks A and E to the 49% in block B.We chose a 900 km2.22 study area with 823 km of reconnaissance walks (recces) and transects. Since the population status and distribution of the Comoé chimpanzees was largely unknown at the beginning of this study, we first concentrated on localizing activity “hot spots” including nesting areas, tool use sites and promising places for camera trapping due to the presence of fresh signs of chimpanzee activity and tool use. In order to maximize the number of chimpanzee‐related observations, we conducted a stratified sampling program focusing on forested habitats , although we also crossed long stretches of savanna  along which we recorded all chimpanzee observations.From October 29th 2014 to May 9th 2015, we sampled the 900 kmSince this research was carried out within the framework of a larger project, which had as one of its main objectives to find chimpanzee communities suitable for long‐term study, we devoted more sampling time 80% vs. 20%) to the communities found in the strata closest to Research Station Comoé. Thus, we dedicated six weeks to sampling block C, 6 weeks to D, 2 weeks to E 2 weeks to G, and only 1 week to block A and one to B , targeting mainly potential tool use sites, but also areas where it would be easy to identify individual chimpanzees, such as natural bridges. Cameras were placed using a systematic design across a grid of sampling cells of 1 kmIn order to identify the individual chimpanzees in the videos, we used double blind recognition: two trained biologists (the first author and P. C. Köster) observed the videos independently. To further corroborate the accuracy of the identifications, we contrasted the results with observation of 50 sample videos by two untrained students. We tested the reliability of our identifications using Cohen's K coefficient to measure the percentage of coincidence between independent identification by the two trained biologists. For the tool‐use videos, we considered both episodes . All the stingless bees found had their hives in tree holes and never in the ground or branch surfaces. Although we systematically checked all the trees containing honey‐bee (Apis mellifera) colonies, we never found any tool or signs of exploitation by chimpanzees.We found water dipping sticks (WDT) in four of the sampling blocks Figure , while wA total of 283 water dipping sticks (WDT) were found at 77 water dipping sites, of which we confirmed revisits by the chimpanzees to 12. Two hundred and eighty nine honey gathering tools (HDT) were found at 38 different sites. Leaf‐sponges were found at 18 sites, six of which also included water dipping sticks. Sponges were found only in three of the sampling blocks, and in block B, only sponges and no water dipping sticks were found (it should be pointed out that in block B sampling effort lasted only 1 week). Sponges were found exclusively in or around those tree‐holes which were wide enough to allow the introduction of a chimpanzee's hand (diameter >8 cm). Stick tools, by contrast, were more frequently found associated with holes too narrow for a chimpanzee's hand (175 WDT in holes with a diameter range between 1.5 and 8 cm), although they were found in wider holes as well (108 WDT in holes with diameter between 8 and 34 cm). We never found sponges associated with honey hives. As we confirmed later with videos, chimpanzees used either sticks or sponges to dip for water from holes that were wide enough. Videos confirmed that sponges were produced using the well‐known techniques of chewing or folding the leaves in the mouth prior to use.Dialium guineense and 12% were in Cynometra megalophyla. These also were among the most frequent species of the nine trees at which honey dipping tools were found , although 16% of honey dipping sites were located in Albizia adianthifolia trees. Holes containing honey do not need to be as impermeable as those containing water, since bees are capable of sealing them with wax. Therefore, beehives can be built in trees were water could not otherwise be retained. Even though all the WDT and sponges were found during the second half of the dry season (from the end of January to May), when the small rivers were dry, there were still water pools remaining alongside riverbeds, and knuckle and footprints revealed that these were used by chimpanzees. Five of the water dipping sites were located alongside the large Comoé River, which always carried abundant water, and chimpanzees were seen drinking from this large river on one occasion. The use of WDTs peaked during the driest part of the season at the beginning of April, and was already in decline at the study's end at the beginning of the rainy season in May  of stick‐tools had been modified only minimally (the branches and leaves had been removed and the tips cut off). However, 74% of all fluid dipping stick‐tools (N = 620) were more extensively modified, with the bark removed and brush tips fashioned on one or both of the ends. As revealed by camera trap videos, chimpanzees produce brush tips by chewing the tip of the stick to loosen the fibers. Sixty‐three per cent of the HDTs had brush tips (N = 188) while 83% of the WDTs had them (N = 232).Both at water and honey dipping sites, we found that 26% . While honey assemblages appeared in most cases to be the product of a single episode  but only in 32.47% of water dipping sites , suggesting that this kind of tool was part of a tool set  more probably in the case of honey dipping than water dipping.Tool assemblages found at honey dipping sites were significantly larger than those found at water dipping sites,  than the ones with brushes. Based on three cases observed in the videos, it is likely that these later tools were selected by juveniles.Tools that we classified as honey‐perforators or probes, based on blunt or frayed tips and the absence of brush tip, were significantly longer than honey collectors, but not significantly thicker Table . In contU = 17217, P < 0.0008). HDTs without brushes  were longer than any of the other tool types . This may be due to the bees choosing deeper holes as a defensive strategy, but we were unable to measure all of the beehive holes, since they were sometimes inaccessible to us. Beehives for which we could acquire accurate height measurements were significantly higher  than water holes on trees . We found a weak positive correlation between water tree‐hole depth and WDT total length . This may result from the fact that chimpanzees sometimes used long tools (N = 11) in shallow holes and short tools in deep wide holes (N = 13).Brush‐tipped HDTs  were significantly longer than brushed tipped WDTs  by repeatedly chewing the tip during use . We found that this latter form of modification was more frequent in WDT (46% of brushes) than HDT (28% of brushes). Seventeen percent of the water dipping brushes were >10 cm long, with a maximum recorded length of 38 cm  were significantly longer than honey collector brushes (mean length of 4.29 ± SD 2.9 cm) 3.2R2 = 0.6001, P = 2.557 e−11) between the length of the brush and the amount of water absorbed.In order to test the relationship between brush length and water absorption, we introduced in the laboratory 50 WDTs, which had been used by chimpanzees, into a container full of water  10 WDTs were selected from each of five brush‐length categories . After dipping, we weighed the water absorbed (see methods). We found a significant positive correlation   were of leaf‐sponge use. This small number of honey‐dipping videos may be explained by the low revisit rate  and the fact that the greater average height of honey sites (6.27 ± SD 4.54 m for beehives vs. 2.15 ± SD 2.73 m for water holes) complicated the installation of our cameras. In the sponge use videos, we recorded two adult males, six adult females, and five juveniles drinking with leaf‐sponges. We recorded one adult male, five adult females, one juvenile, and one infant using WDTs. The honey dipping videos were of one juvenile and two adult females , Lowe's monkey (C. lowei), lesser spot‐nose monkey (C. petaurista), and white‐naped mangabey (C. lunulatus).In addition, we recorded 14 videos of chimpanzees checking waterholes in trees, presumably to see if there was any water left, with three adult males, five adult females, four juveniles, and three infants looking inside the holes or probing into them with discarded stick tools. We never observed in any of our videos a chimpanzee using its hands to gather water from tree holes, while four different monkey species were recorded doing so, baboon (3.4N = 38), we were able to determine that the technique used by the chimpanzees to drink using sponges did not differ from that reported for chimpanzees at other sites: leaves were collected from the immediate vicinity, then introduced in the mouth and chewed (73.5%) or folded (26.5%) to make them absorbent. Next, the chimpanzees dipped the sponge repeatedly into the water, taking it to the mouth, sucking it with the lips or pressing it between the lips and teeth. In three holes of depth exceeding 60 cm and diameter greater than 12 cm, the apes introduced their arm up to their shoulder into the hole to reach the water with the sponge. The water dipping technique included sticks which were always chewed before use, to prepare brush tips, and then repeatedly chewed producing long brushes or fiber‐sponges during the water dipping episode (N = 35). Sticks were torn off from saplings and fashioned by detaching branches, leaves, and bark while the chimpanzees were on the ground at the base of the tree hole. In four cases, videos revealed that tools were prepared at a distance (10–20 m) and transported to the site. In other 12 cases, we found remains of the tool preparation  up to 60 m away. We also found 25 tools that had not been detached from plants present within 10 m of the water dipping site. In 93 of the 111 water dipping bouts recorded on video, the tip was chewed after drinking and before reintroduction into the hole. The chimpanzees would keep the tools inside the tree holes for 2–10 sec, moving them slowly up and down to soak up the water. When repeated more than 10 times, the repeated chewing produced very long brushes or fiber‐sponges   used by the Comoé chimpanzees to obtain water from tree holes during the late dry season. 175 of these WDTs were used in tree holes too narrow for a chimpanzee's hand to be introduced, thus the tools allowed the chimpanzees to obtain water from otherwise inaccessible sources. The tools were found during the late dry season, when other water sources were scarce, which supports our hypothesis.If Comoé chimpanzees develop tool‐use behaviors to confront their dry savanna environment then we expect to find evidence of adaptive tool‐use behavior produced by these chimpanzees particularly during periods of water scarcity.‐The 283 WDTs were found distributed across four different sampling blocks that contain the home ranges of at least four different chimpanzee communities. We also recorded 39 videos of individuals of all sex/age classes using the WDTs. This evidence supports our hypothesis, suggesting that water dipping behavior with stick tools is common and widespread among Comoé chimpanzees.Assuming that tool technology in wild chimpanzees is learned by watching others, then we expect water dipping behavior to be common and widespread across the chimpanzee communities under study.‐U = 17217, P < 0.0008). Sixty‐three per cent of HDT had brush tips, while 83% of WDT had them. Forty‐six per cent of WDTs had long brush tips (>5 cm) versus 28% of HDTs. Thus, WDTs were found to be different in dimensions and structure from HDTs, supporting our hypothesis.The mean total length of 289 HDT found during this study was significantly longer than the mean total length of 283 WDT  are different in dimensions and structure from those made to dip for and harvest honey (HDT).‐R2 = 0.6001, P = 2.557e−11), thus, our hypothesis was supported by the result.We carried out an experiment measuring the water absorbed by a sample of 50 WDTs and found that there is a significant correlation between brush length and water absorved  than the mean brush length of 232 brush‐tipped WDTs , thus supporting our hypothesis.The mean brush length of the 188 brush‐tipped HDTs  was significantly shorter . We suggest, therefore, that many of the honey tools without brush tips could have been used as perforators or probes whereas the tools with brush tips were collectors. If this is correct, then most of the groups of tools found at honey gathering sites were probably tool sets , as most of them had been found freshly used probably following a single episode of honey dipping .We have found that the WDTs produced by Comoé chimpanzees are significantly different in dimensions and absorption capacity from HDTs, which indicates specialization. Further research is needed to determine if the production of brush‐tipped tools, the extrapolation of their use to different contexts and the additional improvement of longer brush‐tipped fiber sponges qualifies for a case of cumulative culture.Comoé chimpanzees have successfully tackled the challenging environmental conditions of their harsh habitat not only by generalizing the use of brush sticks to both honey and water, but developing a new variety of brush tool with a specially modified tip which increases the effectiveness of the tool. It appears to be habitual or customary at Comoé, observed in all age and sex classes, although we need further research to confirm it. We consider the water dipping with brush‐tipped WDT behavior of the Comoé chimpanzees as a new type of tool‐use behavior.From all the data collected up to now we cannot yet conclude if making and using WDTs to drink water is more a choice than an ecological need, habituating the chimpanzees and evaluating the relative importance of water consumption from the different sources will allow us to answer this question in the future. The water‐acquisition technology of Comoé chimpanzees represents a new set of chimpanzee traditions. Our study demonstrates the importance of carrying out detailed studies of threatened and isolated chimpanzee populations. Without these kind of studies, we will lose forever the opportunity to discover fascinating cultural variations and unique adaptations that could provide with important clues to better understand human evolution. Tool use gives chimpanzees a clear advantage in water acquisition with respect to any other sympatric animals. Exclusive access to key resources through tool use could also have spurred increased rates of cultural evolution in our own ancestors. We must preserve and study the Comoé chimpanzees not only because they can provide us with important clues about our evolution, but also because they probably are the second largest population in Ivory Coast and the only known viable population of savanna chimpanzees remaining in the country. We are just scratching the surface in our understanding of this population with its extraordinary behavioral and cultural richness.Additional Supporting Information may be found online in the supporting information tab for this article.Supporting Information Video 1.Click here for additional data file.


A cost‐effective climate change adaptation strategy for the forestry sector is to move seed sources to more northern and higher elevation planting sites as part of ongoing reforestation programs. This is meant to match locally adapted populations with anticipated environments, but adaptive traits do not always show population differences suitable to mitigate climate change impacts. For white spruce, drought tolerance is a critical adaptive trait to prevent mortality and productivity losses. Here, we use a 40‐year‐old provenance experiment that has been exposed to severe drought periods in 1999 and 2002 to retrospectively investigate drought response and the adaptive capacity of white spruce populations across their boreal range. Relying on dendrochronological analysis under experimentally controlled environments, we evaluate population differences in resistance, resilience, and recovery to these extreme events. Results showed evidence for population differentiation in resistance and recovery parameters, but provenances conformed to approximately the same growth rates under drought conditions and had similar resilience metrics. The lack of populations with better growth rates under drought conditions is contrary to expectations for a wide‐ranging species with distinct regional climates. Populations from the wettest environments in the northeastern boreal were surprisingly drought‐tolerant, suggesting that these populations would readily resist water deficits projected for the 2080s, and supporting the view that northeastern Canada will provide a refugium for boreal species under climate change. The findings also suggest that white spruce is sensitive to growth reductions under climate change in the western boreal. The study highlights that population differentiation in adaptive capacity is species‐ and trait‐specific, and we provide a counterexample for drought tolerance traits, where assisted migration prescriptions may be ineffective to mitigate climate change impacts. For resource managers and policy makers, we provide maps where planning for widespread declines of boreal white spruce forests may be unavoidable. The species is also commercially important and comprises roughly a quarter of the Canadian Forest Inventory  we quantify drought resistance, recovery, and resilience of boreal spruce populations from throughout the range; (2) we evaluate drought tolerance traits in the context of long‐term growth performance and survival; and (3) we interpret correlations between drought tolerance and growth metrics in the context of the origin climate of provenances to infer local adaptation. The results are discussed in the context of sensitivity to growth reductions under climate change and possible climate change adaptation strategies to maintain health and productivity of one of the ecologically and commercially most important boreal forest tree species.22.1White spruce seedlings from 43 provenances across Canada were planted at a common garden trial near Calling Lake, Alberta, . Planting stock consisted of containerized seedlings, germinated in 1978 and planted in 1982. This field trial is a randomized complete block design with 5 blocks, 5‐tree row plots with 2.5 m × 2.5 m spacing. Two border rows were planted to minimize edge effects. Tree height and survival were evaluated at age 32, and the diameter at breast height (DBH), approx. 1.3 m above the ground, was measured at age 27.For dendrochronological analysis, we used a subsampling design with 33 of 43 provenances. Where the original sampling design had local clusters of samples, sometimes from near‐identical locations, we selected one provenance to arrive at a well‐distributed geographical and climatic representation of growing conditions throughout the range of the species Figure . Within 2.2Core samples were preserved in plastic straws with slices for air circulation. After air‐drying for two weeks, a total of 324 increment cores were glued in wooden cores mounts (six cores were too broken to reconstruct). Then, cores were prepared using a belt sander with progressively finer grains  and scanned at 6,400 DPI (Epson Perfection V800 Pro). Scans were analyzed with WinDENDRO software, version 2016a (Regent Instruments Canada Inc.). Tree ring series were cross‐dated in WinDENDRO, and errors such as missing rings were corrected prior to generating final cross‐dating statistics with the program COFECHA , we calculated the expressed population signal (EPS) according to Wigley, Briffa, and Jones  for all smooth.spline function of the stats package  and five years after the second drought (2003–2007) as predisturbance and postdisturbance reference periods for calculation of indices for both drought events.2.3princomp  function of the stats package using the correlation matrix. We also generate maps of current and projected water deficits for the range of white spruce with the same ClimateNA v5.21 software package, using gridded data publicly available at http://tinyurl.com/ClimateNA.Climate data of seed source origins were obtained using the software ClimateNA v5.21 . The weather data included daily average, maximum, and minimum temperature (°C) and the daily amount of snow and precipitation (mm). We calculated water deficits on a daily scale, based on a reference evapotranspiration (ET0) estimated with Hargreaves and Samani , at approximately 4 km distance to the planting site, operated by the Government of Canada , B, and Plot(B) denote region, provenance within region, block, and plot within block, respectively.Taking advantage of the randomized complete block design, regional means of drought response metrics and field measurements were estimated with a mixed‐model approach and best linear unbiased estimates (BLUEs) for the average and standard error of each region. For measurements of height and DBH, we evaluated 25 trees per provenance planted in 5‐tree row plots with a linear mixed model with blocks and plots within blocks specified as random effects. Regions were specified as a fixed factor, and provenances nested within regions were also treated as a random effect. The analysis was implemented with the glmer function of the lme4 package  for binomial data with the e Lenth, . The rescor.test function of the R base package  of regional population means were estimated with generalized linear mixed models (GLMMs) for survival rate, and linear mixed models (LMMs) for all other traits Table . Conform3.4Regional population differentiation was also readily apparent in annual growth increments with the local provenances, Boreal Shield and Temperate Mixedwood sources being the best performers during the pre‐ and postdisturbance periods Figure a. During3.5r < 0.5)  Figure . Provena4The general expectation for widespread forest trees that occur over a wide range of environmental conditions is to find genetic population differentiation in growth and adaptive traits. Local sources are expected to have the highest fitness and often also the highest growth rates . These results support the view that northeastern regions of Canada appear to be a likely refugium for boreal forest tree species in a warming climate (D'Orangeville et al., 5The results highlight that population differentiation in adaptive capacity to climate environments can be species‐ and trait‐specific, and we provide a counterexample where assisted migration prescriptions may be ineffective to mitigate climate change impacts in the western boreal portion of the species range. The study also provides direct evidence that northeastern populations of white spruce may be safe from negative climate change impacts for the medium‐term future, with no permanent damage observed on northeastern populations when subjected to drought conditions at the test site that they would be unlikely to experience in their native region even under pessimistic climate change projections by the 2080s. In contrast, the fate of western populations is more difficult to assess based on direct evidence from this study. We observed strong growth reductions, but no permanent damage arising from droughts in a region where the average annual water deficit is around 150 mm. However, this value is predicted to be exceeded by the 2050s throughout the western half of the species distribution. Planning for widespread declines of western boreal white spruce forests may be unavoidable where water deficits limit the species productivity. This may lead to large‐scale mortality as recently observed for lodgepole pine in western boreal forests. Such subcontinental‐scale ecosystem disruptions are likely too large to be effectively mitigated through forest management or reforestation prescriptions.None declared.


Persistent opioid use following total joint replacement (TJR) surgery is common; however, the association between pre‐surgical opioid use and surgery type has not been established. The objective of this study was to determine the association between pre‐surgery opioid use and persistent post‐surgery opioid use in TJR patients compared to other elective surgical patients.This is a retrospective cohort study, of univariate and multinomial logistic regression of linked, de‐identified Medicare Benefits Schedule and Pharmaceutical Benefits Schedule data, adjusted for perioperative opioid use, age and sex. Oral morphine equivalents daily doses (OMEDD) were calculated and opioid use was categorized into three mutually exclusive categories for each observation window: low (0–5 OMEDD), moderate (5–10 OMEDD), high (10+ OMEDD). Persistent opioid use was defined as opioid use between 180 and 270 days after the date of surgery.Persistent opioid use was associated with older age, female gender and pre‐surgery opioid use. There was no increased risk for persistent opioid use for TJR patients compared to other surgical patients. The intensity of pre‐surgery opioid usage is strongly associated with persistent opioid use in all observed surgical patients.Our results suggest that many patients who use opioids prior to surgery will persist in their opioid use following surgery. No association was found between persistent opioid use and TJR surgery, but rather a risk reduction compared to other elective surgeries when associations with opioid use are controlled for. Primary care clinicians and surgeons should monitor the duration and dosage of perioperative opioid use. While pain relief is more commonly associated with total joint replacement patients than other surgical procedures it is not associated with an increased risk when pre‐surgical opioid use is taken into account. Primary care clinicians and surgeons should monitor the duration and dosage of pre‐surgery opioid use regardless of surgery procedure and conservative pre‐operative management of total joint replacement patients with opioids should be discouraged. Total joint replacement (TJR) is a common surgical procedure for treating osteoarthritis of the hip and knee, a leading cause of pain and disability, and the 11th highest contributor to disability, globally.Guidelines for managing hip and knee osteoarthritis have previously recommended the use of opioids as a method of pain management.Post‐surgery, TJR patients are at higher risk of persistent opioid use compared to other frequently performed procedures.A greater understanding of the perioperative use of opioids in TJR patients is central to reducing persistent, contraindicated opioid use. Furthermore, these findings could inform future studies aimed at establishing the benefits and harms associated with the use of opioids in conservative management strategies, and optimum timing for TJR surgery. In this population‐based study, we sought to determine the association between pre‐surgery opioid use and persistent post‐surgery opioid use in TJR patients compared to other elective surgical patients.A linked 10% sample  containing all federally funded medical and hospital services (Medicare Benefits Schedule (MBS))The MBS data set covered healthcare subsidized by the Australian Federal Government and the total fee charged for the surgical procedure. It contains all elective surgeries performed on patients in private hospitals, and those conducted on private patients in public hospitals. It does not capture public patients treated in public hospitals, who are funded through the State departments of health. The PBS data set captures all prescription medication subsidized by the Australian Federal government from 1 April 2012. Prior to this the government did not collect dispensing record information for medicines which fell under the co‐payment threshold.Fifteen procedures with the longest elective surgery wait time were selected as candidates for the analysis based on the 2015 Australian Institute of Health and Welfare's report by procedure for public hospitals 2015.To enable a 180‐day lead up and a 270‐day follow up period to establish whether opioids were used pre‐ and post‐surgery, we identified all first instances of elective surgery between 1 October 2012 and 5 April 2014 (herein referred to as the index surgery). If patients underwent more than one eligible procedure during the study period, we included only the first (index) procedure. We then identified the use of opioids within the sample based on dispensing information and using PBS item codes  S2. Pre‐To quantify the strength of opioid use, total daily oral morphine equivalents were calculated. Oral morphine equivalents are used to standardize the impact of a range of classes and doses of available opioids into a common unit of analysis allowing for direct comparison of a range of medication regimes.P < 0.05 We report this study according to the reporting of studies using observational routinely collected health data checklist.All analyses were performed on Stata Version 14.1 . We fit univariate and multivariate multinominal logistic regression models with robust standard errors to calculate the relative risk of persistent opioid use for the different opioid categories. The multivariate model was adjusted for covariates pre‐surgery opioid use, post‐surgery opioid use, age and gender with the dependant variable being persistent opioid use. Sensitivity analyses were undertaken to test for the importance of prior opioid use in the 180 days before surgery by restricting the analysis to those who were opioid naïve. A sensitivity analysis was also run restricting the population to those who were opioid exposed in the 180 days prior to surgery. To test for the importance of the OMEDD threshold a sensitivity analysis was undertaken with the range substantially widened . Finally, all individual who had a second eligible operation within the follow‐up time were removed from the population and the analysis undertaken. This is in contrast to the main analysis where these individuals were retained but only the first operation included in the analysis. All tests were two‐tailed, and significance was set at This study received ethics approval from the University of Melbourne Human Research Ethics Committee (No. 1749347).The demographic profile and surgical category of 14 354 individuals who underwent at least one eligible elective surgery during the study period are described in Table Figure On univariate analysis age, gender, pre‐surgery opioid use, post‐surgery opioid use and surgical category were significantly associated with persistent opioid use  S4. FromAfter adjustment for model covariates, low persistent opioid use was not associated with ENT and general surgeries. Moderate persistent opioid use was no longer associated with ENT and other surgeries. General surgery remained significant when compared to TJR; however, the direction of the relative risk changed from a risk reduction to a relative risk increase. Compared to TJR, the relative risk for a positive association between urological surgery and persistent opioid use became statistically significant. All surgical types were associated with a significant increased relative risk of high persistent opioid use when compared to TJR Table .n = 13 847), no differences were observed from the main analysis  across all surgical categories. Persistent opioid use was associated with older age, female gender and pre‐surgery opioid use. Controlling for these variables, TJR was associated with either the same, or lower risk, of persistent opioid use when compared to other surgical categories. Our results indicate that the prescribing practices associated with patients, rather than the operation itself, are the key contributing factors to persistent opioid use. This was supported by sensitivity analysis which found no significant relationship between surgical category and persistent opioid use among those who were opioid naïve during the pre‐surgical period.Our results suggest that primary care clinicians and surgeons should monitor the duration and dosage of perioperative opioid use, in line with current guidelines.Conservative management of patients with osteoarthritis is based on well‐founded concerns which include the lifespan of implants. The prevalence of revision surgery after TJR is 12% after 10 years,Our findings pre‐date current Royal Australian College of General Practitioners Guidelines for the management of osteoarthritis which recommend against opioid use. The impact of these guidelines on the prescribing practices of general practitioners, where the majority of prescribing in Australia occurs, has yet to be elucidated. Following the release of the 2009 guidelines an increase in prescribing of potent opioids to future TJR patients was observedGiven the continued role of opioids in the management of pain in the TJR population the benefits of conservative management should be assessed against the potential harms of prolonged opioid analgesic use which include tolerance, dependence, opioid‐induced hyperalgesia, misuse, abuse and risk of accidental overdose.While our findings broadly align with the international literature,A strength of this study is the generation of findings from a representative sample from national level, routinely collected administrative data. This high‐quality data is more accurately able to identify the opioid consumption of each patient than patient‐reported opioid use histories.Several limitations are worth noting. The use of an administrative data set limited the number of covariates we could include in the regression model and we are unable to adjust for unmeasured confounders. The nature of the data set also means that we can only identify correlations rather than causal factors. The inability to identify, and adjust for, comorbidities might confound the association between operative incidence and persistent opioid use. The surgical categories used in this analysis did not allow us to separate opioid use according to, for example minimally invasive versus open surgery. This level of granularity is more suited to a clinical trial, that would likely have a much smaller denominator, or a registry‐based study, which, unlike the current data, are typically focused on a single surgical category (i.e. orthopaedics).Finally, this data represents privately insured patients who received an elective procedure in a public or private hospital. There has been an increase in the share of TJRs provided in private hospitals, from around 55% in 2000 to 70% in 2014.In summary, our results suggest that many patients who use opioids prior to surgery will persist in their opioid use post‐surgery. No association was found between persistent opioid use and TJR, but rather a risk reduction for high intensity opioid use compared to other elective surgeries when associations with pre‐surgery opioid use are controlled for. While we have confidence in this data there are limitations in assigning causality and controlling for potential confounding when analysing observational data. A study with random assignment to a conservative management or accelerated surgical management pathway would allow for a more in‐depth understanding of relative benefits for the alternate methods of management. While the observation nature of the data limits causal inference the strong association between pre‐surgery opioid use and persistent opioid use suggest that further study of this association is warranted.None declared.AppendixS1. Medicare Benefits Schedule surgery item numbers.Click here for additional data file.AppendixS2. Pharmaceutical Benefits Schedule opioid item numbers.Click here for additional data file.AppendixS3. Opioid units dispensed and total oral morphine equivalents (OME's).Click here for additional data file.AppendixS4. Univariate Logistic Regression.Click here for additional data file.AppendixS5. Sensitivity Analysis.Click here for additional data file.


There is discord on the value of the erect abdominal radiograph for diagnosing acute abdominal pathologies. The erect radiograph can be uncomfortable for patients in pain and increases patient radiation dose.PAR) improved diagnostic accuracy for identifying mechanical bowel obstruction and/or paralytic ileus in adults presenting with acute abdominal pain.To determine if including the erect abdominal radiograph in plain abdominal radiography  consultants and two radiology consultants for bowel obstruction and paralytic ileus across two sessions. In session 1, the assessors assessed the supine abdominal radiographs (PAR 1) and clinical details in a randomised order, and session 2, at least 6 weeks later, they assessed the supine and erect radiographs (PAR 2) and clinical details of the randomly re‐ordered cases. Computed tomography was the reference standard. Pair‐wise comparisons of receiver operating characteristic curves were calculated to assess for significant differences in participants’ diagnostic accuracy using MedCalc 16.4.3.AUROC) were 69.7%, 61.0% and 0.642 for PAR 1, respectively, and 80.0%, 53.4% and 0.632 for PAR 2 respectively. For AUROC there were no significant differences (P > 0.05) between PAR 1 and PAR 2. Intra‐rater and inter‐rater agreement improved in PAR 2.Average sensitivity, specificity and area under the receiver operating characteristic curves (PAR for the acute abdomen.There was no statistically significant improvement in diagnostic accuracy when including the erect radiograph in  Plain abdominal radiography (PAR) is often the initial diagnostic imaging tool for patients presenting with acute abdominal pain.The diagnostic value of the EAR may vary depending upon the experience of the interpreting doctor. Doctors with less experience may find it difficult to interpret abdominal radiographs that appear to have normal anatomy but have an unusual bowel gas pattern.We undertook this study to (1) determine if the inclusion of the EAR in PAR improves diagnostic accuracy in identifying mechanical bowel obstruction and/or paralytic ileus in adults presenting with acute abdominal pain and (2) to determine if there is a difference in the interpretations of abdominal radiographs between doctors.This study was undertaken at Flinders Medical Centre, South Australia. Ethical approval was granted by the Southern Adelaide Clinical Human Research Ethics Committee and the Human Research Ethics Committee, University of South Australia.The diagnostic accuracy of two different plain abdominal radiography (PAR) protocols was compared by retrospectively reviewing patient cases who had presented with acute abdominal pain and were clinically suspected of mechanical bowel obstruction or paralytic ileus.P < 0.05).Cases were retrospectively selected from the picture archive and communication system (PACS) and radiology information system (RIS) of Flinders Medical Centre. Consecutive patient cases from 29 November 2016 were sampled in a backwards time direction until 40 cases were retrieved. Case inclusion criteria were that the patient was 18–65 years old, presented to the emergency department (ED) with acute abdominal pain and clinical suspicion of mechanical bowel obstruction or paralytic ileus, have undergone SAR and EAR within 24 h of presentation to the ED and undertaken an abdominal computed tomography (CT) scan within 4 h after PAR. Cases were excluded if the patient was institutionalised, had psychiatric or neurological disorders or had an equivocal CT result. Our sample size of 40 cases was based on an estimation that 20 of the 40 cases would have mechanical bowel obstruction or paralytic ileus. According to a published tableTwo PAR protocols were compared for each case. PAR 1 consisted of only SAR, and PAR 2 consisted of SAR and EAR.Two radiology consultants and two ED consultants, all of whom were experienced in interpreting PAR in the acute setting, participated in this study. Post‐registration experience ranged from 25 to 27 years, and 10 to 13 years for the radiologists and ED consultants respectively. Each assessor independently assessed the two PAR protocols, with a minimum 6‐week interval between starting PAR 2 and completing PAR 1. Clinical information from the request form for each case was included with the images.Cases were presented to the assessors in a randomised order using an electronic survey tool  Fig. . The assThe radiology report for the CT scan from each case was used to categorise each case as ‘positive’ or ‘negative’ and served as the reference standard. The assessors were blinded to the CT results. Each CT scan was reported as per the department protocol, with an available consultant radiologist or radiology registrar producing the report. All reports produced by registrars were checked by consultants with an addendum report issued if necessary. CT has a reported sensitivity, specificity and accuracy of 90–94%, 93–100% and 94–95%, respectively, for the detection of mechanical bowel obstruction,P < 0.05) were made between PAR protocols and assessors using the statistical method of Delong, Delong and Clarke‐Pearson.All statistical calculations were performed using MedCalc 16.4.3 . Two receiver operating characteristic (ROC) curves were generated for each assessor using continuous data from the VAS scale (index test) and binomial data from the CT radiology report (reference test). The first ROC for each participant represented their diagnostic assessments when using only the supine abdominal radiograph (PAR 1). The second ROC represented their diagnostic assessments when using both the supine and erect abdominal radiograph (PAR 2). Area under the receiver operating characteristic curves (AUROC) were calculated for each participant and pair‐wise comparisons (Ten duplicate cases for each PAR protocol were used to test for intra‐rater agreement of each assessor's diagnostic interpretations. Each assessor was given different duplicate cases randomly mixed into the case series. Intra‐class correlation coefficients (ICC) were calculated using a two‐way mixed‐effects model based on a single measure and absolute agreement. Inter‐rater agreement was tested by comparing diagnostic assessments between the assessors for each PAR. Agreement was tested by calculating the ICC using a two‐way mixed‐effects model based on the average of two raters and absolute agreement.The 40 cases included 17 females and 23 males (mean age 49.0 ± 9.42 years). Table P > 0.05) in AUROC between the two PAR protocols. Average sensitivity and specificity were 69.7% and 61.0% for PAR 1, respectively, and 80.0% and 53.4% for PAR 2 respectively (Table Diagnostic accuracy data for each assessor for PAR 1 and PAR 2 are presented in Table Moderate‐to‐excellent intra‐rater agreement (ICC of 0.551–0.939) was achieved for PAR 1 Table . Adding Moderate‐to‐good agreement ICC of 0.413–0.733) between the assessors was achieved for PAR 1, and good‐to‐excellent agreement was achieved for PAR 2 (Table 13–0.733 Both PAR protocols demonstrated low‐to‐moderate diagnostic accuracy for identifying mechanical bowel obstruction and/or paralytic ileus in adults presenting with acute abdominal pain. We found no significant differences in the overall accuracy between the two protocols. This is consistent with other studies which have demonstrated limited value of the EAR.We found no significant differences in overall diagnostic accuracy between the assessors. This is in contrast to the study by Thompson et al.,Intra‐rater and inter‐rater agreement increased when the EAR radiograph was added to the protocol. This improvement was most profound for inter‐rater agreement between the two ED consultants, which more than doubled when the EAR radiograph was added. Factors for this result may include both the doctors’ speciality or years of experience which was different from the radiologists. However, the wide confidence interval for some results indicates that the 10 duplicates cases used to test reliability may not have been enough to give a true indication of reliability.We asked the assessors to rate, on a continuous scale, the definite presence or absence of the conditions rather than to dichotomise their assessment into ‘positive’ or ‘negative’. This reflects radiologic practice, where descriptors such as ‘probable’, ‘unlikely’ or ‘apparent’ are commonly used.Our results do not strongly support the inclusion of EAR in PAR, with the likelihood of additional confirmatory imaging such as CT still being required. This study builds on the existing limited body of evidence investigating the value of the EAR when bowel obstruction or paralytic ileus is suspected. We used CT as a consistent and sole reference standard. Compared to other studies where clinical history was not revealed to interpreting doctors, the assessors in our study reviewed clinical details together with the radiograph, reflecting normal practice.PAR is still used in many practices as the initial imaging modality for patients experiencing acute abdominal pain due to its low cost and wide availability.The retrospective study design and sampling methods are limitations. The criteria of including patients who had both PAR and CT may have created bias to the sample to include more cases referred for CT investigation due to equivocal abdominal radiographs, and more cases with unequivocal CT findings. Patients who were institutionalised, or who had psychiatric or neurological disorders were also excluded and therefore results cannot be applied to these patient groups. Confidence intervals for many of our outcome measurements were wide, raising the possibility that the sample was not large enough to detect true significant differences.Alternative radiographs to the EAR, such as decubitus abdominal or erect chest radiographs, were not considered in this study. This study used CT as the sole and standardised reference standard due to its high accuracy, however, it is not 100% accurate for diagnosing bowel obstruction and paralytic ileus.We minimised the risk of memory recall bias and cross‐referencing between assessors by randomising the order of case presentation for each assessor, and a time interval of at least 6 weeks between interpretation of each protocol.The survey tool restricted the use of ‘windowing’ of images, making measurements which may have been used to facilitate the determination of the degree of bowel loop distension, and a standardised film reading environment. In normal practice, radiographs are viewed on high‐definition computer screens. However, the assessors’ interpretations were unlikely to have been affected as radiologic signs for bowel obstruction and paralytic ileus do not need high resolution.Both PAR protocols demonstrated low diagnostic accuracy for the identification of mechanical bowel obstruction and paralytic ileus in adults presenting with acute abdominal pain raising questions about the value of PAR in this setting. The addition of the EAR to the SAR gave a slight but insignificant increase in diagnostic accuracy, and improved the intra‐rater and inter‐rater agreement, particularly for ED consultants. Radiographers performing PAR in the investigation of mechanical bowel obstruction and paralytic ileus should be aware of the limited value of the erect radiograph, especially in situations where it is technically difficult to achieve, patient tolerance is low and the radiographs are to be viewed by an experienced consultant radiologist.The authors declare no conflict of interest.


Homologous domains embedded in multidomain proteins of different domain architectures (DA) may exhibit subtle, but important, differences in their structure and function. Here, we consider two multidomain proteins, Arf nucleotide binding site opener (ARNO) and G protein‐coupled receptor kinase 2 (GRK2), which have very different DAs, but both contain pleckstrin homology (PH) domains. We analyzed the roles of residues selectively conserved in these subfamilies of PH domains from ARNO and GRK2 proteins. DA‐specific residues in PH domain are found to contribute to structural and functional specialization of ARNO and GRK2 in terms of (a) specific intra‐ and interprotein interactions; (b) specificity for phospholipids; and (c) participation in conformational excursions, leading to various functional forms. Our approach can also be applied to subfamilies of other protein families to identify subfamily‐specific residues and their specialized roles. Arf6ADP‐ribosylation factor 6ARNOArf nucleotide binding site openerDAdomain architectureGRK2G protein‐coupled receptor kinase 2PHpleckstrin homology domainHomologous proteins are known to perform same or similar functions, but their functional levels, specificity to ligands, and regulatory mechanisms may vary among homologues Pleckstrin homology domain is named after the protein pleckstrin, which is found in platelets Pleckstrin homology domain is very versatile in nature and is known to be tethered with many protein domains Arf nucleotide binding site opener protein consists of two structural domains, a Sec7 domain followed by a PH domain as shown in Fig. ARNO protein exists in three structural forms Auto‐inhibited state is maintained by a self‐regulatory mechanism, wherein the major differences between them are the conformations and hinge dynamics There are two isoforms of ARNO that have been crystallized and structure determined in full‐length form GRK2 protein has three structural domains, namely RGS, protein kinase, and PH domain as shown in Fig. In this work, we have classified the PH domain sequences into two subfamilies based on their occurrence in ARNO and GRK2 proteins, which are characterized by different DAs. We recognize residues in PH domain that are selectively conserved in one subfamily compared to the other and explore their structural and functional roles.The two datasets used in the analysis are sequences of homologous ARNO proteins and sequences of homologous GRK2 proteins with their respective DAs conserved within ARNO and within GRK2 homologues.Pfam database http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit) and are removed from the list of sequences for analysis to result in nonredundant datasets corresponding to two different DAs. We safely used a 90% sequence identity cutoff as some of the identical proteins show < 100% sequence identity due to additional few residues in one compared to the other either in N‐terminal end or C‐terminal end or both.A script was needed that uses the DAs of ARNO and GRK2 proteins as a query and searches in the Pfam database to identify homologous proteins with same DA. This script should be such that it seeks to avoid entries with incomplete/partial domains and long insertions between domains that may correspond to a domain that is yet to be recognized and documented in the domain databases. Therefore, we developed our own script to recognize PH domain containing sequences corresponding to two different DAs with the other desired features mentioned above. The Perl scripts developed are provided in Appendix The dataset has been further refined by removing proteins with overlapping domains, incomplete domains, and domains with long insertions.jalviewThe PH domain region of homologous sequences of ARNO and GRK2 was multiply aligned as depicted in Fig. naccess1u29 and 1u27 and visualized using pymolThe conserved residue columns or conservatively substituted residue columns between the ARNO homologs and GRK2 homologs in the master alignment (CSA3) were identified, and its corresponding residue columns in SA1 and SA2 with more than or equal to 85% conservation have been considered as DA‐specific residue columns. A conserved residue column in SA1, but with a semi‐conservative or % residue conservation > 50 in its corresponding residue column with same residue or residue type in SA2 and vice versa was not considered to be DA‐specific residues. Intraprotein and protein–protein interactions were identified, from the Protein Data Bank entries 2r09, 4kax of ARNO and 5ukl of GRK2 proteins and using Protein Interactions Calculator The final dataset consists of 88 homologous sequences of ARNO and 21 homologous sequences of GRK2.The conserved residue columns or conservatively substituted residue columns in the PH domain region of ARNO homologs and GRK2 homologs with the cutoffs described in the Methods have been considered as DA‐ or subfamily‐specific Fig. . HencefoFurther, solvent‐buried residues of PH domain of ARNO and GRK2 were identified as described in Methods and the corresponding aligned residue columns were not considered for further analysis, as buried residues are generally known to be situated in the core maintaining the tertiary structural integrity of the domain.There are 16 of the alignment positions in the PH domain that are conserved or conservatively substituted across the members of ARNO and GRK2. As these are conserved across all the PH domain sequences from ARNO and GRK2 subfamilies, these are not DA‐specific residues. Out of these, 10 of the residues are also buried and hence could be contributing to the structural integrity of the domain fold. Two other residues, Trp‐281 and Trp‐285, maintain the conformation of the inositol binding pocket. Similarly, residues Lys‐273 and Gly‐275 interact with phosphoinositide and its equivalents are also part of the binding site region for phosphoinositide If a residue in an alignment position is conserved only within ARNO members or only within GRK2 members in the datasets, then such residues are considered to be DA‐specific.Using this criterion, 40 DA‐specific residues were identified for ARNO PH domains and 21 DA‐specific residues for GRK2 PH domains. If in an alignment position different residue types are conserved within ARNO (say Ser) and within GRK2 , these residues also qualify as DA‐specific residues for ARNO and GRK2, respectively.The structural and functional roles of DA‐specific residues for both ARNO and GRK2 are provided in Tables As mentioned before, the ARNO protein exists as three different conformers and Arf6 protein can interact with ARNO protein only to its partially active form, whereas it is inhibited in inactive form. This switch between inactive and partially active forms is maintained mainly by PH domain and its proximal regions in the protein Six DA‐specific residues  are involved in domain–domain interactions in the inactive form of ARNO, whereas two DA‐specific residues (Asp‐266 and Ile‐379) are involved in partially active form of ARNO Table ; Fig. S2One of the major features in determining the ARNO functional forms is the C‐terminal helix. The C‐terminal helix undergoes change in its orientation between the functional forms of ARNO. In inactive form, this helix interacts closely with PH domain and maintains a steric hindrance for Arf6 interaction for its interaction with PH domain. In the partially active form of ARNO, this C‐terminal helix undergoes a change in its orientation, thereby permitting Arf6 to interact with PH domain The conformation and interactions of some of the residues play a role in the helix orientation as shown in Fig. The PH domain of ARNO binds to the cell membrane by mediating interaction with phospholipid head and helps to recruit other proteins to the membrane. The splice variant 2G of ARNO protein is known to interact with Ins  P4 There are nine DA‐specific residues involved in interaction with Ins  P4 in 2G variant and seven DA‐specific residues from 3G variant to interact with Ins  P3. Although both phosphoinositides bind in the same binding pocket, with six DA‐specific residues common between them, it does not imply that the DA‐specific residues are playing same roles. The two phosphoinositides bind in slightly different orientations, resulting in differences in interaction pattern. Despite being the part of same binding pocket, the DA‐specific residues interact with the phosphoinositides in different ways, explaining their vital role in providing specificity toward phosphoinositides.Figure The partially active form of ARNO in its auto‐inhibited state allows Arf6 to interact with activator site. Upon accessing the activator site in PH domain and its proximal elements, it relieves the auto‐inhibited state of ARNO through allosteric activation. ARNO, upon interaction with Arf6, gets activated and turns from auto‐inhibited state to fully active state Arf6 interaction with PH domain seems to bring allostery‐mediated conformational change in ARNO. As mentioned already, re‐orientation of C‐terminal helix is noticed in the transition between inactive and partially active forms of ARNO. C‐terminal helix undergoes further change in orientation between partially active form (auto‐inhibited state) and active form of ARNO as shown by superposition of the two conformers of ARNO Fig. A. Along Loss of main chain–main chain hydrogen bond between Asp‐382 and Ser‐378 and main chain–side chain hydrogen bond between Asn‐291 and Ile‐379 seem to be related to re‐orientation of the helix  P2 and PtdIns  P2 are known to bind GRK2 PH domain. It is known that GRK2 PH domain has two binding pockets for phosphoinositide, where the loop1–loop3 forms one and the loop1–loop5 forms the other Pleckstrin homology domains are known to have a highly conserved fold and a general property of phospholipid binding and localization to the membrane by associating with βγ subunits of heterotrimeric G proteins. In the current work, two of the subfamilies of homologous PH domains were defined on the basis of their occurrence in two different DAs in two multidomain proteins, ARNO and GRK2. Though there are several residues that are conserved completely across these two subfamilies of PH domain, DA‐specific residues were identified in the two sets of PH domain sequences. We identified potential reasons for the DA‐specific conservation. We note that many of these residues in ARNO and GRK2 PH domains are involved in intraprotein interactions with domains and domain–domain linkers outside PH domain. This observation is consistent with the fact that ARNO and GRK2 have very different DAs. Further, some of the DA‐specific residues are found to form variable intraprotein interactions facilitating structural excursions among inactive, active, and partially active forms of ARNO. Some of the DA residues are also identified to confer specificity to specific phospholipids.Our work provides a convenient general framework to understand the residue determinants of subfamily specific functional and structural features for subfamilies in other protein domain families too.The authors declare no conflict of interest.NN performed all the analyses of sequences and structures and wrote computer programs. RS and NS conceived the idea and designed the project. NN wrote the first draft of the manuscript. All the three authors agreed on the final version of the manuscript.Fig. S1. Multiple SA of PH domain regions of ARNO and GRK2 sequences.Click here for additional data file.Fig. S2. Two different conformers of ARNO in auto‐inhibited state.Click here for additional data file.Fig. S3. Phosphoinositide binding with ARNO PH domain.Click here for additional data file.Fig. S4. ARNO interaction with Arf6.Click here for additional data file.Fig. S5. Residues that confer phosphoinositide interaction in PH domain of GRK2.Click here for additional data file.Appendix S1. Perl scripts to search the Pfam sequence database for multi‐domain proteins of specific domain architecture.Click here for additional data file.