Understanding pressure pathways and their cumulative impacts is critical for developing effective environmental policy. For coral reefs, wide spread bleaching resulting from global warming is occurring concurrently with local pressures, such as increases in suspended sediments through coastal development. Here we examine the relative importance of suspended sediment pressure pathways for dredging impacts on corals and evidence for synergistic or antagonistic cumulative effects between suspended sediments and thermal stress. We show that low to moderate reductions in available light associated with dredging may lead to weak antagonistic (less than expected independently) cumulative effects. However, when sediment loads are high any reductions in mortality associated with reduced bleaching are outweighed by increased mortality associated with severe low light periods and high levels of sediment deposition and impacts become synergistic (greater than what would occur independently). The findings suggest efforts to assess global cumulative impacts need to consider how pressures interact to impact ecosystems, and that the cumulative outcome may vary across the range of realised pressure fields. Multiple aspects of anthropogenic change threaten coral reefs. Here, the authors show that bleaching associated with thermal stress was low when local dredging released moderate amounts of suspended sediments, but high sediment loads coupled with high temperatures had synergistic negative effects on coral survival. With nearly the entire ocean affected by multiple stressors simultaneously2, an understanding of how they interact is critical to predicting their cumulative impacts. While most assessments of cumulative impact assume additive effects6, stressors may in fact interact such that their combined impacts are greater (synergistic) or even less (antagonistic) than might be expected in isolation7.The world’s coral reefs are under increasing pressure from a range of global and local threats9, potentially transforming coral reef assemblages11. In addition, elevated suspended sediment concentrations (SSCs), through runoff13, resuspension14 and dredging activities15 are an important local source of reef degradation in coastal waters. Suspended sediments can impact corals through three interrelated pressure pathways16, including: interfering directly with heterotrophic feeding18, attenuating light and reducing rates of algal symbiont photosynthesis19, and increased sediment deposition leading to smothering21.A key global pressure is climate change-induced increases in sea surface temperature, leading to recurrent mass coral bleaching22 associated with a week of unusually warm water temperatures in the region23. The dredging project included extensive water quality and coral health monitoring, before, during and after completion of the dredging activities, at locations covering a gradient of dredging-related exposure, from within a few hundred meters up to more than 30 km from the dredging activities. This monitoring resulted in one of the largest datasets ever collected on individual tagged coral colonies exposed to dredging activities, and included estimates of dredging-related pressure , thermal stress (water temperature), along with measurements of coral health  throughout an entire 1.5 year dredging campaign. The fact that the data set captures not only a complete gradient of dredging-related pressure, but also temporally spans a thermal stress event, provides a unique opportunity to look at the cumulative impacts of these two important stressors in situ, and examine evidence for synergistic or antagonistic cumulative effects.Using data from a large-scale dredging project undertaken on the reefs around Barrow Island in north Western Australia, we examine the relative importance of the three suspended sediment pressure pathways, along with thermal stress and associated coral bleaching, in predicting coral mortality. The 530 day Barrow Island dredging project coincided with a mass coral bleaching eventThe analyses show that, depending on the severity of their impact on benthic light and sedimentation, suspended sediments may have both negative and positive effects on corals during periods of thermal stress. Low-to-moderate reductions in available light from suspended sediments can reduce the incidence of coral bleaching, and may reduce overall coral mortality, particularly for branching corals. However, when sediment loads are high any reductions in bleaching incidence are outweighed by increased mortality associated with severe low light periods and high levels of sediment deposition. The outcome is that under low sediment loads the cumulative impact of suspended sediments and thermal stress may be less than expected (antagonistic), whereas at high sediment loads the overall impact is greater than when these stressors occur in isolation (synergistic). The results highlight that while pressures such as thermal stress associated with climate change can only be managed at a global scale, management of local pressures may, in some cases, have the capacity to modify their overall impact.Acropora spp. and Pocilloporidae) and massive (Poritidae) corals was observed as a result of both the combined impacts of dredging activity, and thermal bleaching  the incidence of partial mortality increases to values of around 30–40% at the highest temperatures (>31.5 °C), but has no negative impact on corals in the absence of thermal stress  at low temperature (22–28 °C), and as high as 60% at high temperature  exceeds any gain associated with reduced incidence of bleaching.Using a Monte–Carlo simulation approach based on Bayesian fits of the best models we predicted fortnightly partial mortality under the following three scenarios: (1) thermal pressure; (2) dredging pressure  is critical for effective management. Evidence for a shading effect associated with reduced light lends support to the idea that it may be possible to alleviate the coral loss associated with thermal stress through active mitigation efforts41, at least at local scales.Our results highlight that the outcome of concurrent pressures is not always additive, and in fact may change from antagonistic to synergistic across realised pressure fields, potentially complicating efforts to assess global cumulative impacts3 of predominantly unconsolidated, undisturbed carbonate sediments overlying limestone pavement42. It was predicted during the environmental impact assessment processes that the dredging campaign would have some effect on corals, and these impacts were permitted to occur under state and federal approval conditions. The project was carried out under a rigorous environmental management plan43.From 19 May 2010 to 31 Oct 2011 (530 days), a large-scale capital dredging project was undertaken on the reefs around Barrow Island located ~50 km offshore the Pilbara region of NW Western Australia .The project involved collection of extensive time series of both water quality and coral health monitoring across 25 sites covering a gradient from near ( < 1 km) to = far (>30 km) from the dredging activities46). For the purposes of this study, analysis of water quality and coral health was restricted to 17 sites to the south of the main dredging and 2 reference sites (AHC and REFN) located > 28 km to north of the study area , intermediate distances from dredging (4–15 km), and far from dredging (24–32 km), encompassing sites both to the north and south of the primary dredging site  monitoring sites ranging in depth from 4 to 11 m were installed from a few hundred metres to several tens of kilometres away from the future dredging activities  for each day separately, with predicted values used to determine the sum of the per second quantum flux measurements42 as the daily light integral (DLI) calculated as mol photons m-2 d-1. The instrument platform also included a sediment accumulation sensor48 which, while unable to provide absolute units of sedimentation, provides a relative sedimentation index49, and was scaled to between 0 and 1. Water temperature  was recorded by the in situ loggers. All data were visually screened for anomalies and evidence of logger failure using time series plots of the raw data, and any suspect data were removed, with all removed data recorded in a data screening log42.Water quality measurements were made at 10 min intervals for the pre-dredging and dredging phases using instruments attached to a seabed mounted steel frame. Turbidity was measured using sideways facing optical fibre backscatter (OBS) nephelometers while photosynthetically active radiation (PAR) was recorded using 2π quantum sensors. For all turbidity data, any values < 0 NTU were removed, and a smoothing filter was applied where any value > 3 NTU if the value was more than 2.5 × the mean of the preceding and following valuePorites lobata and Porites lutea, hereafter referred to as Porites spp.) colonies per site. Each colony was photographed in plan view (from above) using a digital camera, with a set of reference photographs taken at the start of the program and used to create a reference guide showing the original image of each colony to compare against for each additional survey, to ensure that the correct corals were photographed on each occasion, and that consecutive photographs were taken at the same orientation50. A metal frame was used to ensure all photographs were taken at the same distance from the substratum, and also provided a scale. The colonies were photographed every ~14 d for the 530 d duration of the dredging (until 11 November 2011) with 27–40 separate field surveys undertaken to photograph each individual tagged coral colony at each site over the duration of the project.For each of the water quality monitoring sites, 50 coral colonies considered representative of the local reef community were selected for sequential monitoring and tagged. Additional colonies were also tagged to ensure a minimum of 20 massive Poritidae .The growth form of each colony was recorded as either encrusting, foliose, corymbose, branching or massive. Consecutive photographs of each individual colony were scored for partial mortality, cover by sediment, and thermal bleaching, as a percentage of live tissue of the specific tagged colony, based on a relative assessment of the preceding photograph(s). Percentages were recorded on a categorical scale ranging from 1 to 7 where 1 = 0%, 2 = 1–5%, 3 = 6–33%, 4 = 34–65%, 5 = 66–95%, 6 = 96–99%, and 7 = 100% coverage, which were converted to their equivalent proportional cover midpoints  and used as a continuous response variable in subsequent summaries and analyses. Because of the photographic time sequence, it was possible to follow the fate of the coral tissue in time, making it possible to clearly observe mortality and/or bleaching of tissue, and in many cases identify the cause . Colonies often became partially covered in sediment and if sediments were ultimately washed off the coral surface (by waves or currents) revealing live tissue, the sediment covered tissue was classified as ‘live’ throughout the relevant period of the photograph sequence. If the sediments remained on the surface, or once washed off revealed a dead surface, the tissue was classified as ‘dead’ from when the sediment covering was first observed. The same principle was used in the case of mucous sheet formation in massive Where it was clear from the photographic time series that a colony had been moved or dislodged either by swells and waves associated with tropical storms  they occurred in sufficient numbers across the full design to be analysed as a single group . While other massive colonies were also observed  these were not generally well represented across sites, and their inclusion may bias results given that family level differences can be much stronger predictors of bleaching susceptibility than overall growth form52. Thus for the purposes of this study the term ‘Massive’ refers specifically to massive and sub-massive Poritidae, most likely a mix of Porites lobata and Porites lutea (see above). Compared to the massive Poritidae, observations of branching corals within any one family were scarce and it was necessary to aggregate the branching and corymbose Acropora spp. with the Pocilloporidae . The genus Acropora was the most abundant branching genera across the data set, with good spatial representation. The Pocilloporidae included representatives from Seriatopora spp., Stylophora spp. and Pocillopora spp., all of which have relatively similar morphologies and thermal stress sensitivity to the branching/corymbose Acropora spp.53, and were also well distributed across the study design. Studies including species from both the Acropora spp. and Pocilloporidae indicate similar sediment clearance capabilities30 and similar responses to light reduction19. The only other (non-Poritidae) branching/corymbose corals in the data set were a small number of Astreopora spp. and Hydnophora spp., both occurring only at two sites. These two additional branching groups were not included as they differ markedly to the branching Acropora spp. and Pocilloporidae in their sensitivity to bleaching38 and because of their poor spatial representation. While there are certainly some differences in the life history characteristics of our ‘Branching’ group (such as mode of reproduction) that might result in different rates of recovery from disturbances54, for the purpose of exploring the relative effects of thermal and sediment related stress on adult massive versus branching corals, members of the ‘Branching’ group can reasonably be considered to be functionally similar.While a broad range of coral families and genera were included across the original data set as being ‘representative of the community’, in most cases there was insufficient replication at each site for meaningful analyses of individual families. Instead, we focus on two key groups: ‘Massive’ and ‘Branching’, representing two contrasting growth forms that likely differ in their susceptibility to both sedimentation and light stress. Because massive 16) . We used two approaches to investigate which mortality pathways have most support based on this Barrow Island data set, for both the branching and massive coral groups. In the first approach we used path analysis implemented in a structural equation modelling framework, via the sem package57 in R. We used the logit of the proportional mortality of corals between each image (live cover at time t – live cover at time t + 1) as the response, for the branching and massive coral groups separately , averaged at the site and field survey level (n = 473 field survey x site means for ‘Branching’ and n = 478 field survey × site means for ‘Massive’). An a priori full model was developed for each coral group  as well as thermal stress indicators  in causing mortality of corals. The suspended sediment concentrations (SSC) could directly cause mortality, or could cause reductions in light availability and increased sedimentation. Reduced light availability could directly cause mortality. Sedimentation could cause partial mortality directly, or via sediment cover, which may subsequently result in mortality. As branching corals were never observed with sediment covering their surfaces, this link was not included in the branching coral partial mortality pathways. The total standardized effect of SSC, light and sedimentation was calculated by multiplying the standardized coefficients within a pathway then summing the coefficient values for all relevant pathways. The total standardized effect for thermal stress was the sum of the pathway coefficients via the effect of elevated temperature on bleaching, which in turn impacted the response, as well as the direct effect of elevated temperature  and AICc weight values (ω)59. The relative importance of each predictor variable was determined by summing the ω values for all models containing the variable, with higher summed values representing increased importance of that predictor to the response variable59. To avoid issues associated with collinearity between predictors, predictors were only included in a single candidate model when their absolute Pearson correlation coefficient was less than 0.4. To ensure models remained ecologically interpretable, only models with up to three predictors were included in any one candidate model . We also investigated interactions between factors (Bleached and Sediment Cover) and continuous predictors where these were deemed appropriate.In addition to the path analysis, we used a full-subsets modelling approachTo explicitly separate bleaching impacts from mortality, the probability (or incidence) of bleaching was examined as a function of light and temperature, with morphological group (branching or massive) included as a factor. Data were the number of colonies observed during a field survey showing a higher bleaching score than the previous survey  modelled as a function of the number of observed colonies . A site identifier was included as a random effect to accommodate repeated sampling of the same sites over time.As the observed partial mortality events across the time series were highly zero inflated for both morphological groups, we used a two-step hurdle approach to model mortality. Firstly, the probability of observing a partial mortality event  was modelled at the individual colony level, including colony ID as a random effect to accommodate repeated sampling of the same colonies over time . In the next step, only the data where partial mortality events were actually observed were included, and the amount of coral loss (live cover at time t – live cover at time t + 1) modelled as a function of the proportional live cover at time t. Colony ID was again included as a random effect. Sample size for the presence only model was obviously much lower than for the presence-absence model, as this was limited only to colonies showing some partial mortality during a field survey. There were 318 and 164 colony level observations across all sites and field surveys for the ‘Branching’ and ‘Massive’ group respectively.60, with the presence of mortality modelled as a binomial distribution. We used generalised additive mixed models rather than generalised linear mixed models to allow for potential non-linear relationships between the response variable and the various continuous environmental predictors. Smoothing terms were fit using a cubic regression spline61, with the ‘k’ argument limited to 5 . To further reduce overfitting, the maximum number of predictors allowed in any given model was restricted to three. A null model consisting of only an intercept and the random factors was also included in the model set to test if any of the included variables were indeed useful predictors of coral mortality events. While the data modelled here are time series, temporal variograms did not show strong consistent trends in temporal autocorrelation that could be modelled effectively using the correlation structures available in mgcv. Overall, temporal dependence was not strong because only a single data point from the daily water quality time series were used from the time step between observations of individual coral heads. Inclusion of colony level ID random effects effectively deals with the non-independence associated with some colonies being simply more robust or more sensitive to mortality and bleaching.Models were fitted using the gamm4 function in the gamm4 package62 in R. During initial Bayesian fits, we used three chains with 10,000 iterations and the rstanarm default warmup of (floor(iter/2) and default uninformative priors (see62). As there was good chain mixing final models were fit using only one chain to obtain posterior samples for each step in the mortality pathway to use in a Monte–Carlo simulation to explore the impacts of thermal and suspended sediment related stress individually and as cumulative impacts. We focused on light as the primary suspended sediment related stressor because light was overwhelmingly the most important variable for bleaching in both groups of corals, the best predictor of mortality incidence in branching corals, but was also an important predictor of the amount of lost tissue in massive corals  in a Bayesian framework using the function stan_gamm4 from the rstanarm package-2 d-1); dredging stressors alone ; and under cumulative thermal and dredging stress (high temperature [31 °C] across a range of light levels). A theoretical ‘additive’ curve was estimated as the sum of coral loss associated with thermal stress alone and that predicted for different levels of dredging stress at low temperature.For each iteration of the Monte–Carlo we predicted the incidence of bleaching, the incidence of mortality , and the overall coral loss given mortality . By multiplying the predicted probabilities for both the bleached and unbleached mortality pathways and then summing both pathways, we could calculate the total combined probability of coral loss under thermal pressure alone , our thermal stress scenario was based only on the data from the months of December, January and February. The total predicted fortnightly coral loss was modelled as a binomial function of starting coral cover  against log(distance), where distance was the minimum distance to the dredging footprints as per46. We modelled distance on a log scale as previous analyses had indicated that water quality conditions change as an exponential function with distance from dredging46. Models were fit using stan_gamm4 with default priors and warmup, with 3 chains and 10,000 iterations. Site identifier and fortnight number were included as random effects to accommodate non-independence in time and space. An estimated 10% ‘Effect Distance’ (ED50) was calculated as the distance at which the predicted curve drops below the 50% change from the maximum predicted value (at the closest observed distance of 0.19 km) to the minimum predicted value  and low (25 °C) thermal stress scenarios. The data used were the observed worst case fortnightly values for light and sedimentation (see Supplementary Table  °C and lFurther information on experiment design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer review file


In a recent study in older men (MrOS cohort), D3‐Cr muscle mass was associated with functional capacity and risk of injurious falls and disability, while assessments of LBM or appendicular lean mass by DXA were only weakly or not associated with these outcomes. Inaccurate measurements of muscle mass by DXA and other methods have led to inconsistent results and potentially erroneous conclusions about the importance of skeletal muscle mass in health and disease. The assessment of skeletal muscle mass using the D3‐Cr dilution method in prospective cohort studies may reveal sarcopenia as a powerful risk factor for late life disability and chronic disease.Sarcopenia has been described as the age‐associated decrease in skeletal muscle mass. However, virtually every study of sarcopenia has measured lean body mass (LBM) or fat free mass (FFM) rather than muscle mass, specifically. In a number of published sarcopenia studies, LBM or FFM is referred to as muscle mass, leading to an incorrect assumption that measuring LBM or FFM is an accurate measure of muscle mass. As a result, the data on the effects of changes in LBM or FFM in older populations on outcomes such as functional capacity, disability, and risk of injurious falls have been inconsistent resulting in the conclusion that muscle mass is only weakly related to these outcomes. We review and describe the assumptions for the most commonly used measurements of body composition. Dual‐energy X‐ray absorptiometry (DXA) has become an increasingly common tool for the assessment of LBM or FFM and appendicular lean mass as a surrogate, but inaccurate, measurement of muscle mass. Other previously used methods  also have significant limitations. D The assessment of body composition and its relation to health outcomes has been an integral component of medical research. While body mass index (BMI) is used as an index of overweight and obesity, the effects of the major individual components of body mass, including fat and skeletal muscle, on outcomes depends upon the accuracy of the method being used. Body composition may be measured using a number of different methods, each of which rely on assumptions that may not be true in all populations, ages, or clinical conditions.et al.In particular, the assessment of total body skeletal muscle mass has, until recently, been problematic. Skeletal muscle is a significant but not the only component of lean body mass (LBM) or fat free mass (FFM). In a large number of clinical trials,Body density may be accurately assessed using underwater weighing, or more recently, whole body plethysmography to determine fat and FFM.3H2O or 2H2O and sampling body water  after the dose has been completely mixed. TBW has been used as an indicator of FFM and, indirectly, of skeletal muscle mass. This estimate of FFM assumes a constant state of hydration and that skeletal muscle is a constant percentage of FFM. However, hydration status can be highly variable during illness, aging, exercise, and exposure to heat.Total body water (TBW) may be accurately measured by delivering a known amount of et al.r = 0.897 (P < 0.0001), the investigators concluded that these two methods are interchangeable for estimates of LBM at the population level; however, concordance between the two methods at the individual level is lacking, irrespective of BMI, and the same limitations for assessing muscle mass apply to TBW measured by BIA as for its measurement by isotope dilution (Table Bioelectric impedance (BIA) measures body conductivity or resistance to a small electrical current through the body or across a limb. The resistance is strongly related to TBW (electrolyte‐rich fluids) and is used to assess LBM and, by subtraction, total fat content.P < 0.000, the invFigure While CT and MRI have been used in clinical trials, each has limitations that reduce applicability for large cohort studies. CT and MRI provide a cross‐sectional measurement of anatomic composition and use of multiple CT or MRI slices can provide estimates of volume of muscle or fat tissues.Figure et al.et al.The most widely used method in current use to estimate lean mass is DXA. DXA machines are commonly used to measure bone mineral density for the diagnosis of osteoporosis or osteopenia and thus have the potential for widespread clinical use for other purposes, including body composition measures. DXA estimates body composition using a three compartment model; fat and bone mineral content are directly measured through the differential absorption of two photon energies, and then by subtraction, lean mass is determined.The use of many of these methods for whole body composition of lean and fat mass has provided essential data on effects of diet, disease, exercise, and aging, particularly with regard to total body fat mass and bone density. However, the measurement of one of the principal tissues of body composition, skeletal muscle mass, using these tools is not accurate, and has likely resulted in an erroneous estimate of the effects of muscle mass on outcomes such as risk of disability, falls, and all‐cause mortality.Nevertheless, a recent reviewA meta‐analysiset al.Indeed, this lack of a strong association between lean mass (as a surrogate measurement of muscle mass) and outcomes in elderly people has led to several consensus definitions of sarcopenia that include measures of muscle strength and/or physical performance in addition to measures of lean mass alone.et al.The use of creatinine (Crn) excretion provides a direct assessment of whole body creatine pool size and, as a result, of muscle mass. Heymsfield 3‐creatine (D3‐Cr) dilution method uses similar assumptions as the 24‐h Crn excretion assessment of muscle mass: specifically, that about 98% of the total body creatine pool is sequestered in skeletal muscle and that creatine is turned over in muscle through the conversion of creatine to creatinine. The additional assumption of this method is that an oral dose of a tracer quantity of deuterated creatine (D3‐Cr) is 100% bioavailable and once absorbed is transported across the sarcolemma and sequestered in sarcomeres. In some subjects, a small amount of the oral tracer dose is ‘spilled’ into urine and not transported into muscle. However, the amount of spillage can be estimated and corrected with an algorithm based on urine levels of creatine and creatinine.3‐enrichment in urine Crn provides an opportunity to ‘sample’ the intra‐myocellular enrichment of D3‐Cr and thus determines the dilution of the oral label in the whole body creatine pool in skeletal muscle. Importantly, the measurement does not require dietary control and relies on a single spot, fasted urine sample taken 48–96 h after dosing. The enrichment of urine D3‐Crn reaches isotopic steady state at about 48 h after dosing and remains stable for about 48 h.3‐Cr. Cross‐sectional3‐Cr muscle mass and lean mass measured by quantitative magnetic resonance  and muscle mass of the lower limbs (weighed after dissection) . A longitudinal validation study of muscle mass accrual of growing animals and loss of muscle mass due to dexamethasone treatment demonstrated a strong relationship between change in quantitative magnetic resonance lean mass and change in D3‐Cr muscle mass .The D3‐Cr muscle mass was strongly associated with whole‐body MRI of muscle mass  and DXA . The line of identity passed through the origin for whole body MRI vs. D3‐Cr muscle while whole body DXA overestimated muscle mass compared with MRI  associations with these outcomes. This is the first large cohort study with a side by side comparison of DXA estimates and D3‐Cr muscle mass measurements on outcomes related to functional capacity and disability. These powerful associations between D3‐Cr dilution estimate of muscle mass and functional capacity and disability provides evidence that D3‐Cr‐muscle mass is an assessment of functional muscle mass that is not available using DXA estimates of whole body lean mass or appendicular lean mass. In an accompanying editorial, SchaapNewly published data from the ongoing MrOS study3‐creatine dilution is that it can be assessed in large number of subjects with very little subject burden. A subject simply swallows a capsule and produces a fasting urine sample later.3‐creatine dilution method to be used in large subject and patient populations including many where CT or MRI cannot be used, such as paediatric or international studies in malnourished populations. For longitudinal trials, there is no limit to how many repeat measurements can be made as long as a urine sample is collected prior to subsequent doses of D3‐creatine to correct for baseline enrichment of urine D3‐creatinine.A particular value of Dstreetlight effect. It has led to inconsistent results and potentially erroneous conclusions about the importance of skeletal muscle mass in health and disease. In contrast, the D3‐Cr dilution method provides an accurate, non‐invasive, and easy‐to‐use assessment of functional muscle mass. Continued use of this method should be considered in longitudinal observation and randomized controlled trials to better understand the role of changes in muscle mass in the aetiology of age‐associated loss of functional capacity and development of chronic diseases. The potential of the continued use of this method in prospective cohort studies is that a single diagnostic criterion of sarcopenia as skeletal muscle mass corrected for body mass may emerge as a powerful risk factor for late life disability and chronic disease.The initial definition of sarcopenia3‐creatine dilution method; however, they do not derive any income related to commercial use of this method nor do they control the intellectual property. The authors certify that they comply with the ethical guidelines for authorship and publishing of the Journal of Cachexia, Sarcopenia and Muscle.No conflicts are reported for the authors. Drs Hellerstein and Evans are listed as co‐inventors on the filed patents for the D


Immune checkpoint inhibitors (ICIs) have shown promising efficacy in the treatment of non‐small cell lung cancer (NSCLC). Sex‐associated dimorphism in immune system response is acknowledged, but the effect of patients’ sex on efficacy of ICIs as treatment in NSCLC still remains controversial. The present study was conducted to investigate the difference in efficacy of NSCLC patients receiving immune checkpoint inhibitors according to the sex. A total of 9583 patients involved 6567 men and 3016 women with advanced lung cancer from 15 randomized controlled trials were included in this study. An overall survival (OS) benefit of immune checkpoint inhibitors was illustrated in both male  and female  patients, and a progression‐free survival (PFS) benefit was also found in both men  and women  in NSCLC. Both PD‐1/PD‐L1 inhibitors alone and PD‐1/PD‐L1 plus chemotherapy significantly improved the OS and PFS in male patients. Whereas in females, PD‐1 inhibitors or monotherapy significantly benefited the OS but not the PFS, PD‐L1 inhibitors or combination therapy significantly prolonged the PFS but not the OS. No survival benefit was found in both male and female patients from the CTLA‐4 inhibitors. The current study indicated that the magnitude of survival benefit is sex‐dependent and male patients seemed to obtain more consistent and favorable outcomes from ICIs than women patients in NSCLC. How sex associates with ICI efficacy is incompletely elucidated. To address these concerns, we perform the current study of 9583 patients to investigate the association of patient sex with the benefits from immune checkpoint inhibitors in NSCLC with recently accumulated evidence.22.1We searched the Embase, PubMed, and Cochrane Central Register of Controlled Trials (CENTRAL) for phase 2 and phase 3 randomized controlled trials (RCTs) from the inception to December 2018. The main search terms used in the search strategy included immune checkpoint inhibitors , specific drug names , randomized controlled trials, lung cancer . The search strategy is provided in the supplementary materials. We also performed a computerized search of the major international conference proceedings of ESMO , ASCO , AACR (American Association for Cancer Research), and WCLC (World Conference of Lung Cancer). Study selection was carried out in line with the PRISMA  guidelines.2.2To be eligible, randomized controlled trials had to assess the inhibitors of PD‐1, PD‐L1, CTLA‐4, or their combination comparing immunotherapy with other systemic treatment regimens including chemotherapy in patients with lung cancer. Studies that investigated immune checkpoint inhibitors plus chemotherapy compared with chemotherapy alone were also taken into account. Eligible studies had to have data available for the hazard ratio (HR) for overall survival (OS) or progression‐free survival (PFS) according to the sex of lung cancer. Trails that belonged to retrospective or prospective observational cohort studies or reported subgroup analysis for one sex only were excluded.2.3Two investigators independently reviewed and extracted the data including first author, publication year, study ID, trial phase, treatment, hazard ratio for overall survival or progression‐free survival stratified by patient sex with a standardized data collection form. Any discrepancies or disagreements were discussed and addressed with the consensus of all investigators. A risk of bias assessment was evaluated by using the tool recommended by the Cochrane Collaboration Handbook.2.4Q and I2 statistic. For the Q test, P‐value less than 0.10 implied significant heterogeneity; for the I2 statistic, I2 value more than 50% indicated significant heterogeneity. Random effects model by DerSimonian‐Laird method was applied; otherwise the fixed effects model by Mantel‐Haenszel method was performed. We derived the hazard ratio and 95% confidence intervals (CI) for OS or PFS in the intervention/treatment group and control group for men and women patients separately. We calculated the pooled HR of OS or PFS in male and female patients applying random effects model or fixed effects model. We also did subgroup analyses to investigate the variation of effect of sex immunotherapy efficacy. Egger’s test and Begg’s test were used to evaluate potential publication bias. All analysis was performed with Stata version 15.0 . All reported P values were two‐sided and P‐value less than 0.05 was used to indicate statistical significance.The heterogeneity was identified using Cochrane’s 33.1A total of 2784 potentially related articles were identified from online database by the initial search strategy. After eligibility screening the abstracts and reviewing the full texts, 15 randomized controlled trials (RCTs) involving 9583 patients were finally included in the present study Figure .16, 17, 3.2The main characteristics of the included 15 randomized controlled trials were summarized in Table Several studies may warrant further explanation due to the unique designs. The KEYNOTE 010 study tested two different doses of pembrolizumab (2 mg/kg and 10 mg/kg) vs docetaxel in advanced NSCLC patients. In this scenario, the pooled HR for OS and PFS was considered. CheckMate 227 trial was designed to evaluate different nivolumab‐based regimens  versus chemotherapy in distinct patient populations. The part of CheckMate 227 trial focusing on nivolumab plus ipilimumab versus chemotherapy among patients with NSCLC was identified due to available data.3.3P ＜ 0.001) and women  , but not in male patients . In subgroup analysis of male patients, there was an OS benefit in the anti‐PD‐1 treatment  and anti‐PD‐L1 treatment . Whereas in female patients, OS benefit was found in the anti‐PD‐1 treatment  but not in anti‐PD‐L1 treatment . Monotherapy  seems to have an OS benefit compared with combination therapy (ICI inhibitors combination or ICI plus chemotherapy) for both men and women. No survival benefit was found in both male and female patients from the CTLA‐4 inhibitors  had a significantly reduced risk of death for both men  and women  who received immune checkpoint inhibitors (compared with available standard therapies) Figure . Heterog3.5Results from the risk of bias of the included RCTs are shown in Table 4Sex‐based differences of the immunologic responses may reflect the different outcome in the efficacy of immune checkpoint inhibitors in male and female. The aim of the current study is to analyse differences in the response to immunotherapy in both sexes. We investigate OS data from 12 RCTs with 8023 patients and PFS data from 10 RCTs with 5622 patients. The result of the current study demonstrated that immune checkpoint inhibitors could improve overall survival and progression‐free survival for patients of both sexes in non‐small cell lung cancer. Interestingly, this study indicated a greater net value of immune checkpoint inhibitors for women  than for men  in terms of overall survival. One possible explanation is that the stronger immune responses from women might lower the risk of cancer mortality. This result seems consistent with Wallis's study  which suggested a greater immunotherapy advantage in women, but contrary to Conforti's study  that indicated a greater immunotherapy benefit in men in NSCLC.Five different meta‐analysis have been reported to investigate the efficacy of immune checkpoint inhibitors according to the sex of cancer patients.It still remains unknown which is better efficacy of PD‐1, PD‐L1, or CTLA‐4 inhibitors for men and women. A previous meta‐analysis emphasized that PD‐1 inhibitors for men would consequently have better OS benefit than women.Preclinical studies showed that conventional therapy including the radiotherapy and chemotherapy could kill tumor cells and induce PD‐L1 expression on tumor cells.To the best of our knowledge, we speculate on three possible reasons that could explain the difference between men and women. First, sex dimorphism in immunity indicates that the stronger immune responses occurring in women could lower the risk of cancer mortality and higher incidence of autoimmune diseases also could make them more susceptible to produce ICI‐related adverse events causing higher possibility of treatment discontinuation. Second, gender dimorphism in behaviors such as higher smoking frequency in men, which increases tumor mutational burden (TMB) greatly and such correlates with immune checkpoint inhibitor efficacy. Third, lower smoking prevalence with lower tumor immunogenic landscape and higher EGFR mutation might lead to these results among women patients with lung cancer, and those female patients with EGFR tumor mutations receive no survival benefit from the immune checkpoint inhibitors. We performed a systematic review and meta‐analysis to investigate the efficacy of immunotherapy in lung cancer according to sex and demonstrated that male patients could derive larger relative survival benefit from immune checkpoint inhibitors (compared with available standard therapies) then female patients.The present study had several limitations. First, this study relied on published randomized controlled trial subgroup HRs of OS and PFS rather than individual participant data. Second, several RCTs of immune checkpoint inhibitors excluded due to lack of available sex‐subgroup analysis data that might indicate sex differences if included in the analysis. Third, the OS and PFS data from all included RCTs were not mature enough and update studies will provide final survival data. Fourth, differences from results between both male and female patients might be attributed to other factors such as prevalence of autoimmune diseases, difference in the number of both men and women, and difference of life style. The above limitations may have a certain influence on the final outcome. Despite the limitations, the current study is the largest meta‐analysis incorporating outcomes from 15 RCTs involved over 9000 patients in NSCLC, providing the increased credibility of results.5In the current study, immune checkpoint inhibitors are significantly related with prolonged overall survival and prolonged progression‐free survival in both male and female patients in NSCLC. Both PD‐1/PD‐L1 inhibitors alone and PD‐1/PD‐L1 plus chemotherapy significantly improved the OS and PFS in male patients. Whereas in females, PD‐1 inhibitors or monotherapy significantly benefited the OS but not the PFS, PD‐L1 inhibitors or combination therapy significantly prolonged the PFS but not the OS. No survival benefit was found in both male and female patients from the CTLA‐4 inhibitors. The current study indicated that the magnitude of survival benefit is sex‐dependent and male patients seemed to obtain more consistent and favorable outcomes from ICIs than women patients in NSCLC. Prospective randomized controlled trials of greater inclusion of women stratified by sex and deeper understanding of molecular mechanisms of tumor immune response and response are needed to validate the difference of efficacy of immunotherapy in male and female patients in NSCLC.The authors declare no conflict of interests.Weimin Li and Dan Liu contributed to conceptualization and supervision. Chengdi Wang, Wenliang Qiao, Yuting Jiang, Pengwei Ren contributed to data acquisition, data interpretation and statistical analysis. Chengdi Wang, Min Zhu and Jun Shao wrote and reviewed the manuscript. All the authors approved the final version. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


RNAs in skin wounds reveals miR‐223 as a key regulator of inflammation during tissue repair. The findings suggest targeting of miR‐233 as an effective strategy to enhance neutrophil activation after bacterial infection and to improve healing of infected wounds.A novel approach for identification of functional mi Live imaging revealed increased recruitment of neutrophils in miR‐223Y/− mice as early as 12 h post‐injury. Increased neutrophil activation and production of reactive oxygen species were also observed. This led to increased macrophage infiltration and concomitant impairment of wound healing during normal conditions, including slower re‐epithelialization and an increase in early scar tissue formation. These findings are in line with a deleterious effect of an excessive inflammatory response on wound repair and promotion of scarring ‐6 mRNA. IL‐6 is expressed and secreted by activated neutrophils and is important for fighting infection. Importantly, human neutrophils expressed less miR‐223 upon recognizing Collectively, the authors demonstrate a key role for miR‐223 in limiting neutrophil activation. Silencing of miR‐223 effectively promoted the hyperactivation of neutrophils, leading to elevated IL‐6 secretion and improved bacterial defense. These findings highlight the therapeutic potential of targeting specific miRNAs during chronic wound infection and suggest miRNA therapy as a promising alternative to already existing strategies. Future work in this area will certainly be of great interest, as the need for better approaches for treating chronic wounds remains a major priority for health care providers.


Surgical resection and adjuvant chemotherapy are the only treatment modalities for localized colorectal cancer that can obtain a “cure.” The goal in surgically treating primary colorectal cancer is complete tumor removal along with dissection of systematic D3 lymph nodes. Adjuvant treatment controls recurrence and improves the prognosis of patients after they undergo R0 resection. Various clinical studies have promoted the gradual spread and clinical use of new surgical approaches such as laparoscopic surgery, robotic surgery, and transanal total mesorectal excision (TaTME). Additionally, the significance of adjuvant chemotherapy has been established and it is now recommended in the JSCCR (the Japanese Society for Cancer of the Colon and Rectum) guideline as a standard treatment. Herein, we review and summarize current surgical treatment and adjuvant chemotherapy for localized colorectal cancer and discuss recent advances in personalized medicine related to adjuvant chemotherapy. Patients with colorectal cancer considered to be curative now have several options for surgical approaches, adjuvant chemotherapy regimens, and duration of therapy. Although the current situation makes it difficult for oncologists and patients to make clinical decisions, future developments are expected to lead to more effective personalized medicines and treatment. However, the same principles of resection with lymph node dissection applicable to open surgery are also applicable to the laparoscopic approach.Although the concept of complete mesocolic excision (CME) has begun to emerge in recent years,Additionally, the Japanese specific lymph node dissection is pelvic lateral lymph node dissection, which is considered a distant metastasis in Western countries. Lateral lymph node dissection is indicated when the lower border of the tumor is located distal to the peritoneal reflection and the tumor has invaded beyond the muscularis propria. Prophylactic lateral lymph node dissection has a weak recommendation in the JSCCR guideline.3The combination of advances in diagnostic methods and the use of many newly developed treatment methods has steadily improved the results of treatment. However, treatment methods vary among the medical institutions in Japan that treat patients with colorectal cancer, and this can lead to differences in treatment results.Therefore, the JSCCR guidelines 2019 for the treatment of colorectal cancer,4Laparoscopic surgery, which is the standard treatment for colon cancer in the USA and Europe, has several benefits over open surgery in terms of short‐term outcomes such as decreased pain, improved postoperative pulmonary function, reduced postoperative ileus, lower incidence of wound infection, faster recovery, and shorter hospital stay. Further, as shown by the results of several randomized controlled trials,Several meta‐analyses of randomized trials also showed faster recovery and no detrimental impact on recurrence or survival rates for laparoscopic vs open colectomy for the treatment of colon cancer.As another possible benefit of laparoscopic surgery, adjuvant chemotherapy is more likely to be started in patients who undergo laparoscopic colectomy for node‐positive cancer. Among 12 849 patients who underwent colectomy for stage III colon cancer, those undergoing the laparoscopic approach were more frequently administered adjuvant chemotherapy than those undergoing open colectomy (72% vs 67%).5Rectal cancer surgery is performed via the open, laparoscopic, and robotic approaches and is chosen based on surgeon and patient preference. No approach has been shown to be superior to another. Medical insurance coverage for the robotic approach began in Japan in 2019.Traditionally, curative resection of rectal carcinoma was obtained by the open surgical techniques of low anterior resection or abdominoperineal resection. Laparoscopic rectal cancer surgery was compared with open surgery in four randomized trials that reported conflicting results. Therefore, until further data become available, the best surgical approach should be determined individually based on tumor and patient characteristics and surgeon experience. Among these trials, the European Colorectal cancer Laparoscopic or Open Resection (COLOR II) multicenter trial randomly assigned 1044 patients with a solitary rectal adenocarcinoma located within 15 cm of the anal verge to undergo laparoscopic or open surgery.A systematic review and meta‐analysis of randomized trials that including the four above‐mentioned studies concluded that, for rectal cancer, a higher rate of “noncomplete” (composite of incomplete and near‐complete) total mesorectal excision was achieved with laparoscopic surgery than with open surgery (13.2% vs 10.4%). However, the rates of circumferential and distal margin involvement, mean numbers of lymph nodes retrieved, and mean distances to radial and distal margins were similar between the two techniques.6Robot‐assisted surgery is an emerging technology combining the advantages of the laparoscopic approach (e.g. faster recovery with less postoperative pain) with those of open surgery .Short‐term (6‐month) outcomes of 466 patients who underwent low anterior resection or abdominoperineal resection for rectal cancer were compared in the RObotic vs LAparoscopic Resection of Rectal Cancer Trial (ROLARR).The ROLARR trial showed that operative time was longer and cost was higher when robotic‐assisted laparoscopic rectal surgery was performed by surgeons with varying degrees of experience with robotic surgery, and it offered no incremental benefit over conventional laparoscopic surgery. Of note, the issue of oncologic equivalency of robotic‐assisted laparoscopic surgery to conventional or open rectal surgery was not addressed in the published data from the ROLARR trial.A systematic review and meta‐analysis performed in 2018 of five trials, including ROLARR, comparing robotic‐assisted resection for rectal cancer with that of conventional laparoscopy found similar perioperative outcomes regarding mortality, rate of circumferential margin involvement, and number of lymph nodes harvested.7In centers with experienced surgeons, TME has also been attempted transanally for distal rectal tumors, particularly in obese men with a narrow pelvis.Other studies following up patients for up to 29 months showed comparable rates of local recurrence and survival between TaTME and standard TME.8Among patients who have undergone a potentially curative resection, disease recurrence is thought to arise from clinically occult micrometastases present at surgery. The goal with postoperative (adjuvant) therapy is eradication of these micrometastases to increase the cure rate. The benefits of adjuvant chemotherapy have been most clearly demonstrated in stage III disease. The JSCCR guideline recommends several agents and regimens described below.An oxaliplatin‐based regimen, rather than a fluoropyrimidine‐based regimen alone, is recommended for patients <70 years old with resected stage III disease who are expected to tolerate oxaliplatin. Multiple randomized trials have shown a survival benefit by adding oxaliplatin to adjuvant fluoropyrimidines in patients with resected stage III colon cancer, and the benefit appears across diverse practice settings and patient subgroups. However, the benefits of an oxaliplatin‐ over a non‐oxaliplatin‐containing regimen are less clear in patients ≥70 years old. In contrast, orally active fluoropyrimidines such as capecitabine and UFT are more convenient and may offer an improved therapeutic ratio.More recent trials suggest that the benefit achievable with either capecitabine or UFT is at least equivalent to that of FU/LV administered by intravenous bolus. Two randomized trials of oral capecitabine compared with intravenous fluoropyrimidines showed equivalent rates of 6‐month DFS.Six months of UFT plus LV is a standard approach for adjuvant chemotherapy of stage III colon cancer in Japan.9The optimal duration of adjuvant oxaliplatin chemotherapy for patients with stage III colon cancer is still evolving. An analysis of the International Duration Evaluation of Adjuvant Chemotherapy (IDEA) collaboration  revealed that in the overall population of patients with stage III colon cancer receiving adjuvant therapy with FOLFOX or CAPOX, non‐inferiority of 3 vs 6 months of therapy was not confirmed. For the patients treated with CAPOX, and particularly those in the lower‐risk subgroup, 3 months was as effective as 6 months of therapy.In a combined analysis of 12 834 patients with resected stage III colon cancer enrolled in all six trials, 3 months of oxaliplatin‐based therapy did not meet the prespecified criteria for non‐inferiority, as the 3‐year rate of DFS of patients with 3 months of therapy was 74.6% vs 75.5% for 6 months of therapy .The 2019 Clinical Practice Guideline from the American Society of Clinical Oncology (ASCO) states that patients with stage III colon cancer who are at a higher risk of recurrence (T4 and/or N2) should undergo 6 months of adjuvant oxaliplatin‐based chemotherapy, whereas lower‐risk patients can undergo either 3 or 6 months of therapy.Six months of oxaliplatin‐based chemotherapy has been the standard of care for adjuvant treatment of stage III colon cancer. However, due to the cumulative and dose‐limiting peripheral neuropathy caused by oxaliplatin (grade 3 neuropathy in 13% of patients receiving 6 months of FOLFOX in the MOSAIC trialIn Japan, the JSCCR has developed a nomogram to calculate risk of recurrence after curative resection that includes data on age, gender, tumor location, stage, and preoperative tumor markers from 19 institutions. This is expected to be an important information tool for use in personalized medicine. Additionally, a report based on data from NSABP C‐07 suggested that use of the Oncotype DX colon cancer assay for genomic profiling might improve risk prognostication in stage III disease and better clarify the absolute benefit that might be achieved by adding oxaliplatin to the therapy of these patients.10Clinical outcomes of patients with colorectal cancer have improved as a result of the variety of surgical approaches and agents developed in this past decade. Patients with colorectal cancer considered to be curative now have several options for surgical approaches, adjuvant chemotherapy regimens, and duration of therapy. Although the current situation makes it difficult for oncologists and patients to make clinical decisions, future developments are expected to lead to more effective personalized medicines and treatment.Conflicts of Interest: Authors declare no conflict of interest for this article.


NAI). Currently in the United Kingdom there are insufficient pathologists with experience in histological ageing of fractures to meet the medicolegal need for this examination. This study provides a practical tool that will allow those skilled paediatric and forensic pathologists currently involved in assessing infants for evidence of accidental or non‐accidental injury a basis for extending their assessment into this area of unmet need.This study is the first to systematically document histological features of fractures of known age in infants (≦12 months). It has been used to develop a tabulated database specifically to guide histopathologists to age fractures in children considered to have suffered accidental or non‐accidental injury (MSB) were used to identify specific histological features and to relate them to fracture age. In 1999 the data were entered into a tabulated database for fractures accumulated between from 1985 to 1998 inclusive. Thereafter cases were added, and at 2‐yearly intervals the accumulated data were audited against the previous database and adjustments made.One hundred and sixty‐nine fractures of known age at death were obtained from 52 anonymised infants over a period of 32 years (1985–2016 inclusive). Sections stained using haematoxylin and eosin (H&E) and Martius scarlet blue (This paper describes the final data set from the 2017 audit. The study was terminated at the end of 2016, as there had been no material changes in the data set for three consecutive audits. Recognition of fractures in life and at autopsy is a key element in the assessment of infants believed to have suffered accidental or non‐accidental injury.Radiological skeletal survey (SS) is the current method of choice in detecting fractures in life.decide if bone injury has occurred;age fractures relative to the time of death; anddetermine whether there is an abnormality of the bone  that might make bones more easily fractured.Part of the full investigation of fractures detected/suspected at autopsy is histological examination of the bone in order to:It is accepted that post‐mortem histological examination of infant bones can detect fractures not seen radiologically.Ageing fractures is complementary to their detection. In the absence of underlying bone disease, fractures of multiple ages strongly correlate with NAI. Distinguishing ante‐ from post‐mortem fractures and placing fractures within a temporal landscape can assist in testing witness statements and contribute to understanding the chronology of events prior to death. However, while there are time‐lines for the processes of fracture healing in experimental animals and experiential evidence of similar timelines in humans,Here we describe the results of a 32‐year study to generate a practical tabular database allowing recognition of histological characteristics of fracture healing for assessing fracture age.The starting point was a data set of eligible fractures from cases received in the University of Manchester's Osteoarticular Pathology laboratory (UMOAP) from January 1985 to December 1998, analysed in 1999. Thereafter cases were added and the accumulated data analysed every 2 years. This paper describes the final data set from the 2017 audit. The study was terminated at the end of 2016, as there had been no material changes in the data set for three consecutive audits (i.e. since 2011).Fractures from infants (≦12 months) were sent to UMOAP by forensic and paediatric pathologists. In every case employed in this study the age of the fracture  was documented. This took the form of a precise history and fractures confirmed radiologically. It is very uncommon to acquire such evidence in cases of NAI, but less so in real or claimed accidental injury. All cases were documented anonymised of all patient details except age and fracture site (bone and position within that bone) by one specialist osteoarticular pathologist (A.F.). They included single and multiple fractures from the same individual, including individuals with fractures of different known ages.The data set analysed in 1999 consisted of 99 fractures from 27 infants. During the subsequent 18 years a further 70 fractures were added from 25 infants. The rarity of having timed fracturing events in cases of NAI, and accidental injuries being uncommon in this age group, is reflected in there being only 169 fractures in this study from post‐mortem examination of ~3200 fractures of infants in UMOAP during the same period. In some cases, in any one infant, evidenced timing was available for a proportion of the fractures only and in some for more than one episode of fracturing.All tissue was decalcified in 10% formic acid under radiological control, and resultant tissue blocks were sectioned and stained with H&E and MSB.Underlying bone disease that might affect the rate of healing (e.g. osteoporosis/vitamin D deficiency) was excluded histologically.Haemorrhage in and around the fracture lineVisible strands of fibrin in/adjacent to the fracture linePolymorph infiltrate between the bone ends and within haemorrhageMacrophage infiltrate in the fracture line/adjacent marrowGranulation tissue formationOsteocyte lossOsteoclasts removing cortical/trabecular bone at fracture sitesCondensation of mesenchymeEarly woven bone formationDistinct bone trabeculae and cartilage nodulesCalcificationFracture unionOrganisation of primary callus and deposition of lamellar boneNormal bone structure restoredFrom 1985, all eligible fractures were documented for the presence of histological featuresData were tabulated to show the time prior to death at which each feature was seen and the proportion of fractures from each time‐point showing the feature. The time‐points were largely self‐selecting by the number of fractures, each time‐point having to have at least four fractures from at least two infants.After 1998, all data (including new cases added in period) were summated every 2 years and an audit performed comparing the ‘new database’ with that of 2 years earlier. While this led to changes in consecutive databases, most of the changes were caused by the increasing number of samples resulting in increased numbers of time‐points. However, from the 2011 audit (cases to December 2010), no changes at all were made to the database despite adding further cases. As per the original protocol, the study was terminated after three successive unchanged 2‐yearly audits at the end of 2016.The age of the infants did not conform to a conventional Gaussian distribution, two‐thirds being less than 6 months old. The median was 4.1 months and the range 3 weeks to 11.9 months (M:F = 28:24).The full 2017 data set recording the appearance, presence and disappearance of each histological feature and the number of cases showing that feature tabulated against time from death in Figure de‐novo (in fibrous tissue) or endochondral ; osteoclastic/osteoblastic bone remodelling with lamellar bone formation; and return of a more normal structure. Features of progression varied between cases, presumably through biological variation, but were remarkably consistent in different fractures from the same individual.The data indicate a reproducible and universal sequence of histologically defined events as healing progresses, including haemorrhage; clot formation; inflammatory cell infiltration; granulation tissue formation; stimulation of mesenchymal stem cells; production of matrix: appositional (on pre‐existing bone surfaces), The presence of RBC within the fracture line Figure A is usedIn keeping with the literature,Distinct strands  are universally seen at 12 h, increasing appreciably over 24 h  Figure I,J. It wAll cases showed bridging periosteal bony/cartilaginous fracture callus by 22–28 days.Deposition of lamellar bone on pre‐existing woven bone or cartilage nodules was taken as the first sign of remodelling towards a normal bone microstructure. Best identified in polarising light Figure K1, lamelReturn towards the structure of ‘normal’ bone was clearly established by 92 days after fracture, after which differential histological ageing became difficult.Mobile fractures, particularly rib fractures, often showed healing reactions of different ages. We hypothesise that this is due to recurrent trauma at the fracture site consequent upon motion. In this setting, we found that the periosteal healing reaction away from the fracture line most accurately reflected the fracture's age.Refractures are recognised by a fracture line passing completely through existing fracture callus Figure L. While fractures through the growth plate cartilage do not show features of repair and cannot be aged; andmost metaphyseal fractures propagated through bone at their periphery. Here the healing process was identical to bone elsewhere Figure M,N,O.Many fractures were metaphyseal, typically of anterior and posterior ribs and the long limb bones. The metaphysis contains a cartilage/bone interface, a site of weakness. Forceful pulling and twisting can cause fractures along the interface, which are well described in the literature.The periosteum was frequently damaged during fracturing, and healing responses appeared to have exactly the same histological progression to those in the medulla. A periosteal reaction was sometimes seen in the absence of fracturing.We present here a 32‐year study of histological findings and their relative time of occurrence in 169 fractures of known age from 52 infants. An initial tabulated data set based on 99 fractures from 26 infants was drawn up in 1999 and a rolling audit performed for a further 18 years.This study was conducted to provide histopathologists examining fractures from cases of accidental or non‐accidental injury with a practical tool to age traumatic injuries of bones. While others have given valuable data on ageing fractures based on clinical observation,The tabulated data can be considered an algorithm which, if one or more of the features described is recognised and then singly or in combination mapped onto the table, can be used to define an age range within which the fracture occurred. Similarly, if a feature is not present, the fracture has either not reached, or has passed, the age at which that specific feature becomes universally seen or disappears.Data presented here indicate that all fractures appear to go through similar linear patterns of histological changes, with biological variation influencing differences in the rate of healing between individuals. Because it is not possible to know where within the biological spectrum an individual might lie fractures cannot be aged exactly, but our data indicate that a time range, during which the fracture occurred, can be given with a high degree of certainty. Age ranges for fractures of different ages can overlap; however, from examination of fractures of different known ages from the same individual, histology allows fractures to be clearly recognised as being of different ages.loss of osteocytes from bone at the periphery of very early fractures (1–2 h). All our observational data suggest that this cell death is energy‐requiring, conforming with current understanding of bone cell biology where osteocyte apoptosis is a necessary precursor of osteoclast‐driven bone remodelling;while metaphyseal fractures are difficult to age, if they involve bone the bone‐healing process matches that of fractures in other sites. Fractures involving only cartilage or non‐vascularised primary spongiosa cannot be aged with any certainty; andperiosteum can be injured without fracturing and heals with the same processes over the same time‐frame as periosteal reactions associated with fractures.In addition to producing a novel dating algorithm for infant fractures we report other new findings. We have shown:None. Professor Freemont has presented evidence in writing and in court based on these data.


The transition from recreational drug use to compulsive drug‐seeking habits, the hallmark of addiction, has been shown to depend on a shift in the locus of control over behaviour from the ventral to the dorsolateral striatum. This process has hitherto been considered to depend on the aberrant engagement of dopamine‐dependent plasticity processes within neuronal networks. However, exposure to drugs of abuse also triggers cellular and molecular adaptations in astrocytes within the striatum which could potentially contribute to the intrastriatal transitions observed during the development of drug addiction. Pharmacological interventions aiming to restore the astrocytic mechanisms responsible for maintaining homeostatic glutamate concentrations in the nucleus accumbens, that are altered by chronic exposure to addictive drugs, abolish the propensity to relapse in both preclinical and, to a lesser extent, clinical studies. Exposure to drugs of abuse also alters the function of astrocytes in the dorsolateral striatum, wherein dopaminergic mechanisms control drug‐seeking habits, associated compulsivity and relapse. This suggests that drug‐induced alterations in the glutamatergic homeostasis maintained by astrocytes throughout the entire striatum may interact with dopaminergic mechanisms to promote aberrant plasticity processes that contribute to the maintenance of maladaptive drug‐seeking habits. Capitalising on growing evidence that astrocytes play a fundamental regulatory role in glutamate and dopamine transmission in the striatum, we present an innovative model of a quadripartite synaptic microenvironment within which astrocytes channel functional interactions between the dopaminergic and glutamatergic systems that may represent the primary striatal functional unit that undergoes drug‐induced adaptations eventually leading to addiction. Astrocytes may channel functional interactions between the dopaminergic and glutamatergic systems within a quadripartite synaptic microenvironment which may represent the primary striatal functional unit that undergoes drug‐induced adaptations that eventually lead to addiction. A1adenosine 1 receptorA2Aadenosine 2A receptorAcbnucleus accumbensAcbCnucleus accumbens coreAcbSnucleus accumbens shellAdenoadenosineaDLSanterior dorsolateral striatumA‐Oaction‐outcomeAstroastrocyteATPadenosine triphosphateB‐endbeta‐endorphin2+CacalciumCB1cannabinoid receptor 1CeNcentral nucleus amygdalaCNScentral nervous systemCOMTcatechol‐o‐methyltransferaseCSconditioned stimulusD1dopamine receptor D1D2dopamine receptor D2DAdopamineDATdopamine active transporterDLSdorsolateral striatumDORδ‐opioid receptorDRdopamine receptorDyndynorphineCBsendocannabinoidsEnkenkephalinGABAgamma‐aminobutyric acidGLASTglutamate aspartate transporterGLT‐1glutamate transporter 1GlutglutamateKORκ‐opioid receptorLgAlong accessLTDlong‐term depressionLTPlong‐term potentiationMAOmonoamine oxydasemGluRmetabotropic glutamate receptorMORμ‐opioid receptorMSN(s)medium spiny neuron(s)NACN‐acetylcysteinepDMSposterior dorsomedial striatumPFCprefrontal cortexPLprelimbic cortexShAshort accessVTAventral tegmental areaxCTcystine‐glutamate exchanger1Drugs of abuse induce a broad spectrum of central and systemic effects that vary depending on their mechanisms of action Nestler, . DespiteThe striatum is the main entry point of the basal ganglia, receiving glutamatergic inputs from the cortex following a topographical organisation in parallel loops Haber, . BroadlyThis cytoarchitectural landscape is highly contrasted between the ventral and dorsal territories of the striatum, which have different cortical and limbic inputs and functionally distinct roles , the most ventral territory of the striatum and long considered a “limbic‐motor interface”  that reliably predicts the outcome, the firing of these neurons shifts to the CS. Consequently, the activation of mesolimbic dopamine neurons has been suggested to convey a prediction error signal about the value of future outcomes Schultz, . Within These complementary roles of the striatum have been formalised into a putative model, wherein the Acb is considered the “Critic,” whereas the dorsal striatum is considered the “Actor,” mapping relationships between states and action propensities  dopamine‐dependent control over behaviour  via antecedent glutamatergic mechanisms in the AcbC  homeostasis  and reinstatement Kalivas,  which haThus, at the level of the synapse, upon an action potential reaching the presynaptic terminal, a sudden increase in calcium concentration triggers the release of glutamate into the synaptic cleft wherein glutamate binds to its postsynaptic ionotropic or metabotropic (mGluR1/5/6) receptors, thereby triggering depolarisation of the postsynaptic neuron and regulation of the function of that glutamatergic synapse respectively , the activation of which suppresses glutamate release by presynaptic terminals, thereby contributing to downregulate glutamatergic transmission   under continuous reinforcement and are subsequently subjected to several consecutive daily instrumental extinction sessions (often 12 days). Instrumental responding is then reinstated either by an injection of the drug (drug‐induced reinstatement) , NAC was shown to be mostly ineffective, except in a subset of individuals who had volitionally initiated abstinence at the onset of the trial  and orbitofrontal cortex  environment, including dopamine  expressed on astrocytes by adenosine released by neurons triggers astrocytic glutamate release (Li, Nomura, Aihara, & Nishizaki, in vitro and in vivo studies that astrocytes contribute to the function of dopaminergic synapses (Jennings & Rusakov, The pivotal role of astrocytes in glutamate homeostasis has been relatively well described. However, the contribution of these cells to dopamine homeostasis and its impact on glutamatergic transmission remains poorly understood, despite increasing evidence from both Apart from their role in dopamine clearance, cultured astrocytes have been shown to respond to dopamine, in that direct application of dopamine modulates cytosolic calcium signalling in astrocytes. These effects occur both in a receptor dependent manner, through D1/D2 receptors (Jennings et al., Collectively, these data suggest an important role of the DAT, the gateway for dopamine into astrocytes, in the coupling between dopaminergic synaptic activity and calcium signalling.Thus, investigation of the role of astrocytes in the regulation of striatal synaptic physiology should go beyond the prototypic tripartite glutamatergic synapse and should consider the striatal dopaminergic synapse in a quadripartite synaptic environment. A more complete understanding of this quadripartite system is paramount for our understanding of drug addiction. The contribution of dopamine signalling to the homeostasis and function of the striatal quadripartite synaptic microenvironment remains to be elucidated but could represent the cellular mechanism by which drugs of abuse, which aberrantly increase dopamine levels in the striatum, trigger long‐lasting alterations of glutamate homeostasis that span the entire striatum.Furthermore, endogenous systems, namely endocannabinoids, endogenous opiates and the adenosinergic system, have been shown to influence glutamatergic and dopaminergic homeostasis as well as the dynamic function of astrocytes in the striatum (for review see Fouyssac, Everitt, & Belin, 4The purpose of this review was to summarise experimental evidence in support of the involvement of astrocytes in the dysregulation of the glutamate homeostasis induced by exposure to drugs of abuse across the functional domains of the striatum and offer a broader mechanistic view of the striatal quadripartite synaptic microenvironment. The disruption of the homeostasis and function of this striatal microenvironment may contribute to the emergence of maladaptive drug‐seeking habits and ultimately compulsive behaviour, the hallmark of addiction. A better understanding of the neural, behavioural and psychological consequences of the restoration of glutamate levels by NAC both in preclinical and clinical models has helped shed a new light on the cellular mechanisms in the striatum that contribute to several facets of addiction. However, the drug‐induced alteration of glutamate homeostasis characterised by the downregulation of astrocytic xCT and GLT‐1 may only be the tip of the iceberg of the many adaptations these cells may undergo in response to drug exposure, including those associated with dopaminergic transmission. Further research is warranted better to understand the physiology of the quadripartite synaptic microenvironment which, we hypothesise, represents the functional unit of the striatum, and the extent to which interactions between dopaminergic and glutamatergic systems are channelled by astrocytes. The molecular landscape of the quadripartite synaptic microenvironment and its associated physiology might differ between individuals and such difference may represent molecular and cellular vulnerability factors which could account for the inter‐individual propensity to develop maladaptive drug seeking habits and eventually drug addiction (Anthony, Warner, & Kessler, The authors have no financial or potential conflicts of interest to declare.MF and DB have contributed equally to the writing of the manuscript. Click here for additional data file.


IAC) is essential in vestibular schwannoma removal, cochlear implantation (CI) surgery, vestibular nerve section, and decompression procedures. Here, we analyzed the acoustic‐facial cistern of the human IAC, including nerves and anastomoses using synchrotron phase contrast imaging (SR‐PCI). A total of 26 fresh human temporal bones underwent SR‐PCI. Data were processed using volume‐rendering software to create three‐dimensional (3D) reconstructions allowing soft tissue analyses, orthogonal sectioning, and cropping. A scalar opacity mapping tool was used to enhance tissue surface borders, and anatomical structures were color‐labeled for improved 3D comprehension of the soft tissues. SR‐PCI reproduced, for the first time, the variable 3D anatomy of the human IAC, including cranial nerve complexes, anastomoses, and arachnoid membrane invagination  in unprocessed, un‐decalcified specimens. An unrecognized system of arachnoid pillars and trabeculae was found to extend between the arachnoid and cranial nerves. We confirmed earlier findings that intra‐meatal vestibular schwannoma may grow unseparated from adjacent nerves without duplication of the arachnoid layers. The arachnoid pillars may support and stabilize cranial nerves in the IAC and could also play a role in local fluid hydrodynamics.A thorough knowledge of the gross and micro‐anatomy of the human internal acoustic canal ( Vestibular nerve schwannoma removal and cochlear implantation (CI), as well as other surgical interventions such as facial nerve (FN) decompression and vestibular nerve section, require a detailed comprehension of the complex anatomy of the internal acoustic canal (IAC). Proper identification of various nerve components is essential in patients with congenital malformations  analysis of the fundus of the human IAC, using micro‐computed tomography , compared with a conventional radiogram, which does not. To obtain SR‐PCI images, each sample was scanned using the BMIT 05ID‐2 beamline at Canadian Light Source Inc. in Saskatoon, SK, Canada. This device provides an SR beam produced by a superconducting wiggler source ‐stained celloidin sections were scanned, and digitized Poschl reformats were collected.Dissections of the temporal bone occasionally injured the soft tissue in the IAC. Three cadaver bones showed comprehensive preservation of the entire IAC, which prevented more extensive quantitative evaluation of the anatomical structures. Pseudo‐colorization and adjustment of scalar opacity mapping were used to enhance surface borders and improve contrast separation and 3D conception.The 3D imaging allowed visualization of the cochleo‐vestibulo‐facial nerve complex but not the vestibular ganglia located in the nerves (ganglion Scarpa). The cochleo‐vestibular nerve (CVN) divided into the cochlear and vestibular branches near the mid‐portion of the IAC. The last part to separate was that between the inferior vestibular nerve (IVN) and the high‐frequency acoustic nerve fibers. These connections could be demonstrated using orthogonal sectioning from various angles.SR‐PCI reproduced the arachnoid and the acoustic‐facial cistern extending to the fundus of the IAC. It consisted of a thin, highly variable sheet surrounding the cochleo‐vestibulo‐facial nerve complex. The arachnoid was partly tagged onto the dura wall of the IAC, which was verified on the individual X‐ray section Fig. , inset. Figure At times, the arachnoid membrane was hardly visible at 3D, which was thought to be due to a limited optic resolution Fig. . HoweverConnections could be seen between the IVN (saccular nerve) and CN fibers directed to the base of the cochlea Fig. . The SVNInner ear blood vessels running in the IAC were generally difficult to discern. Both the CVA and blood vessels along the vestibular nerves could be identified near the fundus in a few bones. The CVA perforated the curtain of tissue embracing the high‐frequency cochlear fibers Fig. . One artRecently, we analyzed the fundus region of the human IAC using micro‐CT  innervating the posterior ampule entered the bony canal at variant distances from the fundus. This nerve can be used by the surgeon for anatomic guidance and nerve identification. After its separation, the IVN continues as the SN. Invariably, two SiNs entered separate channels Fig. , verifyiA notable finding was the arachnoid pillars or villi radiating from the surfaces of the cranial nerves. They ran between cranial nerves, and between nerves and the IAC wall. They resembled the pillars and veil‐like structures or plates that form networks to provide mechanical support to neurovascular structures between the pia mater and arachnoid in the brain  is between the saccular and CNs at the floor of the IAC. The nerve fibers direct to the basal turn of the cochlea Oort, , which, The main nerve supply to the saccule is from the IVN, which runs beneath the TC Retzius, . Voit 1 describeAnastomoses between the facial and vestibular nerves are known to exist in the IAC Paturet, , as wellIn brief, SR‐PCI identified the extension of the cerebellum‐pontine cistern into the fundus of the human IAC. The arachnoid wall of the ‘IAC cistern’ partly adhered to the cranial nerves at fundus. SR‐PCI verified results of earlier anatomic studies but added new 3D data on nerve anastomoses and blood supply Fig. . The tecSR‐PCI with 3D reconstructions was performed to analyze the complex anatomy of the cranial nerves and vessels in the acoustic‐facial cistern in the human IAC. Nerve anastomoses were verified. A system of arachnoid pillars and trabeculae were identified between the cranial nerves and arachnoid that were substantiated histologically. We conjecture that these structures support and functionally separate the cranial nerves, and may play a role in local fluid hydrodynamics. The present findings and descriptions may further assist in the surgical and radiological identification of anatomical structures in the human IAC. Novel techniques to image clinically the fine details of the inner ear with even higher precision are emerging.


We report a rare case where the inferior vena cava connected to the azygos vein, a diagnosis triggered by observation of an enlarged azygos vein traversing an azygos fissure. A 16‐year‐old male patient presented with an abnormal shadow on chest X‐ray. Chest computed tomography showed an enlarged azygos vein connecting to the inferior vena cava, with no other connection to the atrium. There were no associated malformations. The patient remains alive and has been asymptomatic for the past two years. If the flow through the connection was to be interrupted during the course of thoracic or abdominal surgery, this would invariably prove to be fatal. In addition, this abnormality prevents direct access to the atrium on attempting interventional radiology via the inferior vena cava, for example, during ablation. When an azygos lobe is identified on a chest X‐ray, a prominent solid structure traversing it may represent an engorged azygos vein with an anomalous course. We report a rare case of the inferior vena cava connecting to the azygos, in a 16‐year‐old male patient who presented with an abnormal shadow on chest X‐ray. The azygos vein was enlarged and ran through an azygos fissure, inviting the diagnosis. An azygos connection of the inferior vena cava is a rare condition, formed during embryonic development when the renal segment of the inferior vena cava does not connect to the hepatic segment We report a rare case of the inferior vena cava connecting to the azygos, in which the azygos vein was enlarged and ran through an azygos fissure, inviting the diagnosis.A healthy 16‐year‐old male patient of mixed Japanese and Filipino descent presented with an abnormal shadow on a chest X‐ray taken during an annual check‐up. His family history was not remarkable. The patient had no past history of note. He was a non‐smoker. The chest X‐ray showed cardiomegaly and a falciform shadow in the right upper lobe angled towards the mediastinum, accompanied by an azygos lobe with a prominent fissure Fig. A. Blood The inferior vena cava is composed of a hepatic segment, a suprarenal segment, a renal segment, and an infrarenal segment. If the renal segment does not connect with the hepatic segment during the embryonic period Fig. B, arrow.The azygos lobe is formed when migration of the azygos vein over the apex of the right lung towards the mediastinum is delayed during the embryonic period; the right upper lobe then becomes divided. Considered a normal variant, the azygos vein that runs through the azygos fissure is usually thin and not remarkable. The presence of an enlarged azygos vein in the azygos fissure indicates high flow through this vein. When an azygos lobe is identified on chest X‐ray, a prominent solid structure passing through it may represent an engorged azygos vein with an anomalous course.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Gerris gracilicornis (Hemiptera: Gerridae), we tested whether the level of female resistance affected the relationship between the sexual size ratio and latency to pair. We found that optimally sized males mated sooner than other males when females resisted a male's mating attempts. When females did not resist, an effect of sexual size ratio on latency to pair was not found. Our results thus imply that increased female resistance to male mating attempts may strengthen the pattern of size‐assortative mating. We provide clear empirical evidence that female resistance to mating influences the effect of mechanical constraints on size‐assortative mating under sexual conflict. This result further suggests that patterns of size‐assortative mating can be altered by a variety of ecological circumstances that change female resistance to mating in many other animal species under sexual conflict.Size‐assortative mating, defined as a positive linear association of body size between members of mating pairs, can arise from mechanical constraints on pairing efficiency, particularly when mating success is affected by males’ mate‐grasping force. In this context, female resistance is predicted to have an important role in changing the threshold force necessary for males to hold females, thereby contributing to the effect of mechanical constraints. Thus, increased female resistance is expected to increase the paring success of an optimally sized male relative to the female body size , which leads to positive size‐assortative mating. However, very little is known about the extent to which female resistance affects mechanical constraints on mate grasping. Here, using the water strider  Mate‐grasping mechanics leading to size‐assortative mating of G. gracilicornis has been supported by a theoretical model and empirical evidence . However, when the male fails to grasp the female firmly and copulation is delayed, other single males nearby may interfere with the first male's attempts to grasp the female, and an interfering male can sometimes take over the female  for different sexual size ratios  in mating pairs when males attempted to copulate with females. Then, we assessed whether female resistance altered the relationship between sexual size ratio and a male's latency to pair. We predicted that the degree of deviation from the optimal sexual size ratio of a pair (M/F = 0.86) determined a male's latency to pair more strongly when females resisted male mating attempts.The Korean water strider 22.1G. gracilicornis is divided into several steps  every 2 days. Pieces of floating Styrofoam were used as rest sites for the water striders.Individual Under laboratory conditions, we closely observed 110 pairs to measure the effect of a pair's sexual size ratio on males’ latency to pair. For the tests, we put a male and a female, which were randomly selected and had been isolated from the opposite sex for 3 days, in a transparent experimental basin  and observed their behavior. We measured (a) the latency to pair (duration (in seconds) from the first grasp to the male aligning his body parallel to the female's body), (b) whether the female resisted during the period, and (c) the sexual size ratio  of the pair. The body size of water striders was measured with the ImageJ program  after taking digital photographs of the ventral surface of each individual.The latency to pair is highly dependent on the mechanical constraints related to mate grasping  standardized latency to pair were fitted as the response variable and where the z‐transformed sexual size ratio (M/F) of a each pair (covariate), its quadratic term, female resistance to mating (two‐level factor: absence versus presence), and their interactions were fitted as fixed factors. In addition, to measure the effect of the sexual size ratio on the level of female resistance, we used a generalized linear model, where the female resistance was fitted as the response variable and where the z‐transformed sexual size ratio was fitted as a covariate. All analyses were performed using the Statistica 10 (StatSoft Inc.).32 × resistance, Table df = 1, p = 0.45).The effect of sexual size ratio on the latency to pair strongly depended on the level of female resistance to mating . This inference by other single males sometimes leads to a takeover and the female mates with interfering male. Courtship interference by other males is particularly common in water strider species that produce courtship ripple signals , males hold and guard females during the precopulatory period (Borowsky, Gammarus males may depend on the level of female resistance to mating. Therefore, given that mate grasping with forelegs, midlegs, or even antennae is widespread in animals (reviewed in Eberhard, A relationship between female resistance and patterns of size‐assortative mating is also predicted to be present in other animal species that utilize coercive mate‐grasping mechanisms in the precopulation period. For example, in crustacean Our results imply that ecological factors can affect patterns of size‐assortative mating. For example, in the case of water striders, females are generally more reluctant to mate in an environment with a female‐biased sex ratio to avoid superfluous mating Rowe, . In addiFurthermore, ecological conditions that change the level of female resistance and the strength of size‐assortative mating may also explain variations in sexual size dimorphism across populations in water striders. Strongly positive size‐assortative mating can exert stabilizing selection on male and female size distributions and can contribute to the evolution and maintenance of sexual dimorphism (Han et al., Our study suggests that a change in female resistance to mating affects the degree of size‐assortative mating in a species where male premating success depends on mechanical constraints on the efficiency of mate grasping. We further suggest that it would be fruitful to test whether the strength of size‐assortative mating is influenced by a variety of ecological circumstances in other animal species that experience strong sexual conflict during the precopulatory period.The authors declare that they have no competing interests.CSH and PGJ conceived the study. CSH collected the data and carried out statistical analyses. All authors substantially contributed to drafting the manuscript and gave final approval for the publication. Click here for additional data file.