Structural heterogeneity defines the properties of many functional polymers and it is often crucial for their performance and ability to withstand mechanical impact. Such heterogeneity, however, poses a tremendous challenge for characterization of these materials and limits our ability to design them rationally. Herein we present a practical methodology capable of resolving the complex mechanical behavior and tracking mechanical impact in discrete phases of segmented polyurethane—a typical example of a structurally complex polymer. Using direct optical imaging of photoluminescence produced by a small‐molecule organometallic mechano‐responsive sensor we observe in real time how polymer phases dissipate energy, restructure, and breakdown upon mechanical impact. Owing to its simplicity and robustness, this method has potential in describing the evolution of complex soft‐matter systems for which global characterization techniques fall short of providing molecular‐level insight. Understanding the mechanisms that define mechanical stability of polymers is crucial for designing robust materials resistant to wear and failure. Although conventional macroscopic techniques such as rheology or dynamic mechanical thermal analysis (DMTA) may be sufficient to characterize most polymers, the macroscopic picture provided by these techniques often lacks molecular‐level resolution. This limitation is particularly valid for functional polymers that are typically non‐uniform and heterogeneous on multiple length scales. To understand properties of materials composed of several distinct or mixed phases a conceptually different characterization approach would be required.A complex multi‐phase material at the core of this work, is a chain‐extended polyurethane (PU)—a copolymer composed of flexible polyether or polyester “soft” blocks and rigid “hard” blocks that form a hydrogen‐bonded crystalline phase Figure .1 The ubSeeking to overcome the limitations of global characterization techniques lacking molecular‐level resolution, several force‐responsive molecular probes have been developed over the last decade. With few exceptions utilizing nanoparticle moieties,Herein we address this challenge and develop a simple and straightforward methodology that allows the direct monitoring of the real‐lime evolution of discrete phases in a complex polymer system under mechanical stress. Instrumental to this work was the development of new robust organometallic mechanophores that combine high aerial stability and facile response with straightforward chemistry for their incorporation into distinct phases of polyurethanes. Relying on direct optical imaging as the primary analysis technique, the methodology holds great potential for general use in soft matter characterization.4CuN, Figure I halide complexes.The preparation of the new mechanophore and its selective incorporation in PUs was the first objective of our study. We based our mechanophore design on a new class of recently developed phosphorescent complexes that were shown to reversibly respond to mechanical stress . To identify potential mechanophores we evaluated a series of CuI complexes with different NHC ligands using DFT calculations (S6 of Supporting Information) targeting complexes with the isomerization mechanism identical of that of previously described Cu halides. Specifically, we analyzed how mechanophore isomerization is influenced by substitution of the halide ligand in the parent halide 4CuN by NHC ligands with different steric bulk. We identified one of the smallest known NHC ligands derived from N,N′‐dimethylimidazolium carboxylate . FTIR spectroscopy confirmed no impact of mechanophore incorporation on the hard phase of SP40 samples and indicated a marginal increase in the relative content of disordered carbonyl groups in HP40 further confirming that the labelling impacted specific synthetically targeted phases . Size exclusion chromatography indicated similar molecular weights of all samples ranging from 70 to 90 kDa with nearly identical polydispersity of 1.9–2.1. Finally, the polymer‐bound ligand L was complexed with CuI following the procedure developed for the synthesis of complex 1 to produce emissive polymers with 1H NMR spectra nearly identical to those of the reference complex 1 (See Section S3 of Supporting Information). The latter, importantly, confirms the identity of mechanophore embedded in the PU material and suggests no ligand exchange between mechanophore and polymer matrix taking effect.We further incorporated this phosphorescent motif into hard and soft phases of polyurethane samples. Inspired by the strategy reported by Braun and co‐workersL Figure  to presc1 in solution but not in the solid state. This confirms that mechanophore in the PU is well dispersed and not aggregated as the aggregates would produce emission similar to that of crystalline 1, which is significantly blue shifted to approximately 555 nm. Films of Cu‐HP samples incorporating the mechanophore in the hard domain exhibit nearly three‐fold longer PL lifetimes compared to their soft phase labelled counterparts Cu‐SP suggesting that Cu‐SP indeed contains mechanophore in the phase with significantly higher chain mobility as expected when the mechanophore is embedded in the soft phase of PU.Copper‐containing PU films fabricated by mold casting were photoluminescent and emitted at around 580 nm upon excitation with UV light. Importantly, the emission of PU films was identical to that of reference complex Once discrete phases of PU samples were labelled with mechanophore, we focused on tracking the differences in force‐induced behavior of those phases. To do that, PU films were subjected to uniaxial extension with simultaneous detection of the photoluminescence intensity produced by these films. For tensile tests the isotropic PU films were pre‐stretched to 250 % of their initial length to prevent necking and associated changes in sample geometry during the imaging experiment.0 s−1 to produce a fixed (7.5 MPa) increase in stress at each loading step followed by a 5 second hold allowing for stress relaxation to be monitored. Afterwards, the loading/relaxation was repeated until the sample failed. Typical curves produced in these tests are depicted in Figure To observe both deformation and stress relaxation kinetics we adopted the testing protocol Figure  similar Cu‐SP40 containing mechanophore in the soft phase produces an immediate increase of PL intensity that further decays when the sample is allowed to relax . IR spectroscopy, similarly indicated no detectible change in the overall hard phase composition in all Cu‐HP40 samples as evidenced by identical absorbance in the characteristic regions for urethane NH and C=O vibrations  indicating a major restructuring of the PU surface as the PU samples are subjected to mechanical impact.Aiming to identify the origin of a different PL response profile observed in the case of fast deformation we extended our structural studies to Cu‐HP40‐5/50 were revealed by the 2D‐SAXS analysis depicted in Figure Cu‐HP40‐50 produced by the fast loading are actually more ordered macroscopically —a value similar to that observed in imaging experiments. Although the H‐bond scission in PUs likely impacts a minor fraction of the hard phase, our methodology appears to have sufficient sensitivity to produce a readily detectible response to these transient physical events that otherwise remain undetected by techniques lacking temporal or spatial resolution.Although highly unusual to occur at room temperature, the hard phase crystallization is consistent with our experimental data—in PUs crystallization is a fast process with a time scale of multiple seconds at room temperature.In conclusion, our work provides a direct observation of diverse and distinctly different phase behavior patterns within a single polymer family and offers an unprecedented insight into the mechanically induced evolution of complex polymer system. The response of robust mechanophores has been used as a diagnostic tool to identify the deformation mechanisms leading to the development of distinct polymer morphologies that can hardly be predicted using global characterization techniques. Since the response of new mechanophores originates at the molecular level, it allows us to describe force‐induced phenomena locally. At the same time, the non‐destructive nature of mechanophore action allows these dynamic phenomena to be tracked in real time. This methodology is efficient in dealing with the case of structural complexity in polyurethanes. We, therefore, see no limitation for it to become a common tool assisting materials scientists in resolving soft matter complexity in the near future.1849321 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.Full synthesis and characterization data and testing methodology description can be found in Supporting. CCDC The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training.We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self‐generated data sets.Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs “healthy”, “old” vs “young”, and facilitates the identification of cell populations with potential biologic relevance to states of disease and health.We developed We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets. For example, this accounts for 80 informative populations for four markers, 728 for six markers, and 59 048 for 10 markers, thus providing a considerable amount of information. In contrast, conventional approaches focus on the identification and measurement of a few populations defined by the sequential combination of markers.Flow cytometry is a powerful tool enabling high‐throughput, multiparametric assessment of biological properties at the single cell level. However, conventional data analysis approaches have relied mostly on the retrieval of data from bivariate plots each combining two markers of interest to the question investigated, and subsequent gating on further subpopulations defined by additional markers. This allows targeted assessment of predefined cell populations, which in most cases are known to the investigator, but leaves the overwhelming majority of information obtainable from a given panel of markers unaccounted for. In fact, the information content that can be retrieved from any given panel increases exponentially with the number of markers used in the panel as each maker can assume at least two mutually exclusive conditions: positive or negative. Considering that each given marker in the panel may or may not be taken into account to depict a cell population, the number of cell populations that can be described by the panel equals ncp = 32The supplement section of this paper contains all the information necessary for the independent reproduction and application of this tool, including the source code of the program.2.1Leukocytes were purchased from the New York Blood Center  which routinely obtains informed consent from all subjects and approved all experimental protocols. All methods were carried out in accordance with relevant guidelines and regulations.2.2Peripheral blood mononuclear cells were obtained through centrifugation over Ficoll  and cryopreserved on the day they were received. Cells from all recruited donors were thawed, stained with the antibodies specified in Table S1 for 20 minutes at room temeparture, fixed in 4% paraformaldehyde and acquired on an LSRII the following day. The staining panel for this experiment was optimized in various single‐color titration and add‐in experiments as described.2.3We aimed to collect samples from female and male humans in two age groups: 8 to 35 years old, and 65 to 95 years old and requested leukocyte enriched blood from presumably healthy human donors with these age and sex specifications from a commercial provider. We received 32 samples  in response to this request over a period of 7 months. The commercial provider of the blood samples allows verification of subject‐related health data retrospectively. All samples were verified to have the requested age and sex specifications, except two samples which were outside the requested age range (HDC‐39 and HDC‐40). For one sample, the precise age could not be reliably verified (HDC‐38). These samples were excluded. As classification was not satisfactory when middle‐aged donors were included in the analysis, we decided to exclude all donors above the age of 30 to escalate the age difference . Included study subjects are enlisted in Table S2. Serological test results for all donors were provided as well. All donors tested negative for HBC, HCV, HTLV, Chagas disease, and Zika virus. Forty‐seven percent of the included donors were seropositive for CMV, consistent with the expected CMV seropositivity rate in presumably healthy human subjects in the general population.2.4M markers using coding elements (the lowest level gated populations) and intermediate gated populations as input.The algorithm calculates all subpopulations for The number of subpopulations can be calculated as follows for four markers as an example:1), and in general 2m populations are defined for m markers. Enumerating the subpopulations involves going through all populations defined by one marker, two markers, and so forth. The number of marker combinations when choosing m markers among 4 is C = 4!/(m!(4−m)!), with markers defining 2m populations.Each marker defines one positive and one negative population  21 + C 22 + C 23 + C 24 = 80, and in general C 21 + C 22 + … + C 2M = 3M−1 populations for M markers.Thus, the total number of populations is A2/4   × %(A)/100, where % is the percentage of the parent population.Populations are computed from coding elements and higher level gated populations. Thus for four markers, the population 2.5For data entry, nested gating is performed in FlowJo , and populations are entered into the “Create Table” module from higher to lower gating levels  (see the Supporting Information file “instructions for running the program” for more details).A table is created for all samples, gathering population values in the specified order. The resulting table is pasted into the Java‐created.csv input file (named “4markers.csv” in the section describing “Running the program”) .From the input file, the Java program generates an output.csv file containing a table of all population values for each sample (named “4markers_output.csv” in the section describing “Running the program”) .To demonstrate the impracticality of performing calculations for even small numbers of markers, we computed output values under the application of the above‐mentioned principles for four markers in excel spreadsheets. An example is attached in  that exemplifies the summation of coding elements into final output values.2.6n present/absent/undefined combinations of the markers, then computes the percentage value for each combination by recursively summing the present/absent subtrees for every marker that is undefined. The value for a combination where all markers are defined is computed directly based on partial sums of the input percentage values.The Java program recursively generates all 3Instructions to run the program are attached as a Supporting Information file “Instructions for Running the Program”. The source code is provided in two Supporting Information files MarkerCombosInputGeneratorFlowJo.java and MarkerCombosBatch.java. Two versions are included, depending on whether the input tables are comma‐separated files  or semicolon separated files .2.7To verify the accuracy of computed population values, they were directly compared with manually gated subpopulation statistics. As shown in Figures S1 and S2, there was concordance between calculated values and direct gating, with both the formula‐based spreadsheet  and the Java program .2.8http://biit.cs.ut.ee/clustvis/).For principle component analysis and hierarchical clustering, data were entered into the ClustVis online program (https://discover.nci.nih.gov/cimminer/oneMatrix.do).For some supplementary analyses, hierarchical clustering was performed based on Euclidian distance applying default specifications of the CIMMiner online tool at .2.9All necessary ethical approval for data presented in this study has been acquired.3We implemented an approach to compute data sets generated through a minimal number of bivariate gating steps to generate metadata describing accurately the entire set of cell populations that can be retrieved from a panel, when each marker used in the panel is defined as either positive or negative, thus extracting the full information content obtainable from each gating choice. This approach was pioneered and applied in a low throughput setting by Hofmann et al,combinations of markers and their respective bi‐modal qualities (positive/negative) are taken into account. This involves the summation of coding elements specific to the representation level of the markers in the hierarchy of the gating tree. It is to be noted that this calculation cannot be performed easily by the current version of FlowJo or any similar flow cytometry analysis software. To enable the combinatorial population generation process for analysis of larger panels, we coded the algorithm in Java. For easy reference, we called the program Flow Plex.We called the numeric values for these populations “coding elements” as further computation was based on their rearrangement and combination. These coding elements depict the highest level of resolution for the differential representation of cell populations in the panel. Extraction of coding elements can be conveniently done and exported using the popular flow cytometry analysis software FlowJo, or can be performed on any other analysis platform. In the next step, coding elements are sequentially paired to determine the representation of each mathematically possible cell population within the starting population when all possible To verify the accuracy of the obtained meta‐data sets using this approach, we generated and mined flow cytometry data. Percentage values for computable cell populations were obtained through manual gating. There was an excellent correlation between computed and manually gated populations .+ CD4+ cells had been stained for CD62L, CD27, and for secretion of interleukin‐22 (IL‐22) and IL‐17A. Nested gating allowed measurements of all 80 coding elements derived from these four markers . Using the freely available online tool ClustVis,Next, we asked whether this approach could help classify biological samples according to immune phenotypes. To this end, we first mined a data set generated from the peripheral blood of healthy humans and subjects with a uniform ocular pathophenotype (pan‐uveitis) caused by two different autoimmune diseases: Behçet's disease (BD) and sarcoidosis (SAR).We also tested the approach on another mined data set,lo CD25hi lymphocyte starting population which contains the bulk of human regulatory T cells . The input of gated coding elements into the algorithm yielded the expected 728 populations. Despite the relatively low number of samples and the expected subtle differences in immune parameters between young and elderly populations, hierarchical clustering allowed very good categorical separation of samples . We created a panel incorporating common, well‐known immune‐markers: CD3, CD4, CD8, CD45RA, CCR7, and CD38 and gated on a CD1274Flow Plex, a computational tool we have developed for this purpose. For instance, for an eight‐marker panel, only four gating decisions need to be made and batched: one at each level involving two markers in the gating hierarchy—yielding a total of 38−1 = 6560 different cell populations. The information obtained through this approach increases exponentially with the number of markers in the panel while the number of necessary gating decisions increases only linearly. This results in a vast increase in information over conventional gating. The latter—with increasing numbers of markers used—yields a constantly decreasing fraction of the information that can actually be obtained from the same panel. Flow Plex can function as a pipeline for the generation of high dimensional data by immunologists familiar with standard flow cytometry analysis software such as FlowJo, but without specialized computational training. These high‐volume data sets can then be analyzed employing straightforward routine approaches, such as conventional principal component analysis and hierarchical clustering techniques to group peripheral blood samples by states of health, disease or other variables/categories of interest, identify outliers and point to populations of potential interest. These tools are easily accessible online  and/or part of the standard repertoire of statistical support typically available to immunologists. Clustering of populations can help with the biological interpretation, which may be of interest for further discriminative and mechanistic investigations. Additional methods can be used to test the statistical significance of population proportion differences if desired. As an example, the uncertainty/robustness of clustering for the AML/ healthy data set (used as a training set for the program in the workflow) was measured by bootstrapping using the R package pvclustThe approach described herein allows for the full exploitation of information available from a flow cytometry panel with minimal gating effort and is facilitated by The quality and standardization of the flow cytometry data are crucial to draw valid conclusions from flow cytometry‐based analysis. All of the general rules for standardization of flow cytometry data acquisition apply here: special care should be taken during acquisition, especially in longitudinal studies. Bead‐based standardization is strongly recommended. It is to be noted that the program and subsequent analyses such as PCA and hierarchical clustering—given their unbiased character—can often very efficiently detect systemic biases.Flow Plex, the number of input values (obtained through batching) also rises in an exponential fashion, however, on a lower scale. To directly demonstrate the feasibility of the method with standard computational power, we plotted the execution times vs increasing numbers of markers for MarkerCombosBatch (execution time for MarkerCombosInputGeneratorFlowJo is negligible), demonstrating that analyses for up to 10 markers require less than a minute for subpopulation generation, even for large numbers of samples . Clustering methods may become infeasible with a high number of markers without prior feature selection. If manual gating is used, the method probably works best at an optimum in the 4 to 10 marker range (representing 80‐59 048 cell populations per sample), a number of markers often used in clinical studies. By using only a few markers, we could generate meta‐data that helped categorize clinical samples with a high degree of accuracy, even in samples where relatively subtle differences are expected . Also of importance is that many immunologically relevant markers do not follow clear bivariate partitioning, that is, they are expressed as a continuum instead of one positive population distinct from autofluorescence or other background signals. The strict application of FMO (n−1) controls helps to clearly differentiate positive from negative signals and is highly recommended when generating data sets to be analyzed with this approach. Considering the former, this method will not discriminate between low/dim vs high/bright vs expression levels of antigens, however, understanding these limitations will enhance its useful application as a discovery tool, stimulating, not replacing, hypothesis‐driven research.While there theoretically is no limitation to the number of markers that can be computed with The authors declare that there are no conflicts of interests.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Environ Toxicol Chem 2019;38:2764–2770. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETACEcological risk assessments are hampered by limited availability of ecotoxicity data. The present study aimed to explore the possibility of deriving species sensitivity distribution (SSD) parameters for nontested compounds, based on simple physicochemical characteristics, known SSDs for data‐rich compounds, and a quantitative structure–activity relationship (QSAR)‐type approach. The median toxicity of a data‐poor chemical for species assemblages significantly varies with values of the physicochemical descriptors, especially when based on high‐quality SSD data (from either acute median effect concentrations or chronic no‐observed‐effect concentrations). Beyond exploratory uses, we discuss how the precision of QSAR‐based SSDs can be improved to construct models that accurately predict the SSD parameters of data‐poor chemicals. The current models show that the concept of QSAR‐based SSDs supports screening‐level evaluations of the potential ecotoxicity of compounds for which data are lacking.  Extrapolation of data for species sensitivity distribution (SSD) parameters (mu and sigma) using a QSAR approach  may in the end result in filling in data gaps for ‐ in theory ‐ all chemicals. QSAR = quantitative structure–activity relationship; SSD = species sensitivity distribution. A recent publication presented species sensitivity distributions (SSDs) for 12 386 compounds, with potential utility for prevention, assessment and management of chemical pollution  model operating at the level of species assemblages. We investigated whether different combinations of physicochemical characteristics can be used to predict the SSD parameters (ecotoxicity) of nontested compounds by applying a QSAR‐type approach to the SSD parameters of data‐rich compounds.2) of toxicity endpoints such as the no‐observed‐effect concentration (NOEC) and the median effect concentration (EC50)—can be empirically related to different combinations of the chemicals’ physicochemical characteristics. With an eye on practical needs, easily obtainable parameters were selected for the latter, to judge the working hypothesis for compounds for which only very basic molecular data are available and to develop models for screening‐level assessments of chemical pollution. Limitations on ecotoxicity data are common when there are ecotoxicity concerns regarding chemicals of emerging concern.We made a preliminary investigation to assess whether and to what extent the variability of the 2 parameters of log‐normal SSDs across chemicals—the median (μ) and the variance (σThe aims of the present study were 1) to provide insight into whether and with what precision the 2 parameters of the log‐normal SSD model (the median of the toxicity endpoints [μ] and their variance [σ]) can be empirically described as a function of physicochemical characteristics, in the context of providing SSDs for, in principle, all compounds, and 2) to evaluate the potential utility of the resulting QSAR‐based SSDs. The exploration specifically considered freshwater ecotoxicity, given the array of curated ecotoxicity data for training the QSAR SSDs and in view of the European Union policy aims of reaching nontoxic surface water systems  selecting a database encompassing SSD parameters for a large number of compounds, 2) selecting physicochemical characteristics and adding them to the database, 3) assessing empirical relationships between ecotoxicity and one or more of these characteristics (deriving QSAR‐based SSDs), and 4) evaluating robustness and model performance of the derived models by means of 4‐fold cross validation.As a starting point, we selected the database on SSDs for 12 386 compounds. The SSDs in that database are summarized in the formats of EC50‐based SSDs (based on acute EC50 data) and NOEC‐based SSDs (based on chronic NOEC data) by applying a log‐normal model to a large data set of ecotoxicity data  to 0 and the corresponding standard deviation (si) to 1  as well as the adjusted R2. Afterward, the most influential predictors of ecotoxicity were identified by standardizing the regression coefficients using the Relaimpo package R statistics, Ver. 3.5.1.We derived 2 multiple regression models  that incorporated the full set of 6 physicochemical parameters  of each input variable  EC50‐based original SSDs and 37/246 SSD NOEC‐based original SSDs with complete data. These numbers of data were considered adequate for the exploratory purposes of the present study, given the relatively low number of predictors used (maximum 6).p < 0.001). This implies that the null hypothesis—that the model coefficients equal zero —should be rejected. The idea to obtain ecotoxicity information for nontested compounds by QSAR‐based SSDs has merit, be it that the relationships are statistically significant (apart from the estimate of σ from SSD NOECs) but at the same time not yet very precise for non‐screening‐level assessment (further discussion in the next section). The highest adjusted R2 values (and the lowest AICs) were found for models associated with NOECs rather than EC50s. However, models explaining the QSAR‐based SSD σ for both endpoints performed poorly (R2 < 0.4). The relative importance analysis reveals solubility (log S) as the most important parameter influencing ecotoxicity , accounting for 36.6 and 38.8% of all variance in μ for EC50s and NOECs, respectively. Solubility in water appeared to be positively associated with the variation in μ for both EC50 and NOEC modeling, implying that the species’ sensitivity toward a chemical decreases with increasing solubility. Of approximately equal importance (based on their z scores), the outcome shows a significant inverse relationship between primary biodegradability and the μ of EC50 modeling, implying that species sensitivity is higher for compounds that are less degradable . In the present study, adding molecular weight in the linear model did not increase the predictive power, in contrast to results from earlier work  to the available data.A comparison of the QSAR‐based SSD fits for μ between the NOEC‐ and the EC50‐based models showed that the former performed better than the latter  were mostly below 5, except for the nonsignificant SSD NOEC σ estimation . For the significant models, this indicates limited multicollinearity between input variables and a limited potential for interpretation error. Residuals of the QSAR‐based estimates of the QSAR‐based μ values were normally distributed, albeit heavy‐tailed, for EC50‐ as well as NOEC‐trained models . Averaged RMSEs calculated in the 4‐fold cross‐validation analysis  were lowest in the models for μ and σ associated with acute EC50 values as training set . Differences between model p < 0.001, linear multiple regression) for SSD μ (median sensitivity of species), indicating that the slopes of the regression lines for the chosen set of physicochemical characteristics are greater than zero: the idea of QSAR‐based SSDs has merit to predict ecotoxicity of nontested compounds. Although the models explained 56 and 72% of the medians of SSDs based on EC50s and NOECs, respectively, variation in the medians of the SSDs explained in the external data set by our model  was much lower for both SSDs based on EC50s and NOECs , implying that these models perform worse when applied to compounds for which less ecotoxicity data were available. Because the out‐of‐sample predictive power for both SSD NOECs and SSD EC50s was much lower, this may suggest overfitting of the model in the case of modeling SSD parameters. Regarding prediction precision, although the present study showed that mainly the variance in the SSD parameter μ can be explained by a nontested compound's common physicochemical characteristics, more research on models with additional descriptors is necessary to derive accurate user‐oriented models to estimate SSD parameters. So far, the utility of the models would be screening‐level lower‐tier assessments which can discriminate low and high relative toxicity rather than precise prediction of molecularly relative closely related compounds.The present study resulted in the derivation of models to predict the 2 parameters of log‐normal SSDs (μ and σ) from ecotoxicity data of data‐rich compounds combined with easily obtainable molecular characteristics. Their potential use asks for an evaluation, and possible improvement, of the significance of the relationships as well as the precision of the predicted toxicity when applied in practice for nontested compounds. The models showed high statistical significance  to the SSDs of the training set . However, the slopes may differ among toxic modes of action and chemical classes and may need further investigation to obtain appropriate average slopes per chemical class or mode of action, suitable for environmental risk assessment.In the present study no information is included on the exact endpoint tested per individual data record. Because all data entries of the data set used in construction of the model can refer to a variety of effects, it is uncertain to what extent the composition of tested endpoints is similar among the 2 training data sets for NOECs and EC50s. Because acute EC50s represent a set of similar adverse outcomes, whereas chronic NOECs may represent a wide variety of effects, we would expect NOECs to be less easy to predict than EC50s. The association for the shape parameter of the SSD  was much lower. This was to be expected because interspecies differences in ecotoxicity are thought to originate from differences in physiology and metabolism between species, rather than differences in molecular properties of chemicals. Because QSAR‐based SSD σs are highly related to interspecies and interindividual correlations, diversity in species and individuals plays a large role in the observed variation and thus the prediction of σ. Because some compounds have a very species‐specific toxic mode of action, the accuracy of predicted σ values strongly depends on the taxa represented in the training set. Although in the present study this was mitigated by truncating data based on taxonomical coverage Table , the exaKOW and log‐transformed toxicity endpoints, multiple studies suggest that the log KOW may be a stronger predictor for toxicity than solubility because this physicochemical characteristic may act as a proxy for Klipid membrane–water  and could, in addition, include certain traits of tested species in model derivation. The latter would yield QSAR‐based SSDs for separate taxonomic groups with an expectedly improved precision, given the fact that molecular characteristics embodying, for example, “insecticidal action” relate to “insect species traits.”To optimize their utility, QSAR‐based SSDs should be statistically significant and precise . A mere addition of trait‐specific predictors has, however, various consequences on the statistical power or on limitations such as on obtaining descriptor data for all chemicals in the study data set (loss of data in the “training set” for modeling). Conflicting  effects may occur, and conceptual improvements may trade off with statistical power and utility of results. In the present study, this bias was overcome by using the AIC scores. Further derivation of these models asks for a balance between the science‐based expansion of (molecular and/or species trait) descriptors and the opportunity to (easily) obtain descriptor data, to potentially use the net outcomes of this approach in the format of QSAR‐based SSDs in decision‐support processes for environmental protection, assessment, and management.NOEC) or exploratory toxic pressures  of observed environmental concentrations, there is a need for an improvement of the QSAR‐based approach to derive improved slope parameters (σ). Protection from chemical pollution, and the related assessment and management of these chemicals, hinges on approaches for their prioritization. Chemicals can be prioritized using protective benchmark concentrations based on chronic NOECs or similar endpoints. Protective benchmarks have been derived for some thousands of chemicals worldwide because of the importance of benchmark concentrations for achieving a level playing field for a jurisdiction such as the European Union to judge for allowing chemicals on the market or to initially judge environmental quality. Only a fraction of the 100 000+ compounds in commerce are currently evaluated regarding potential impacts so that the potential impact of mixtures of these chemicals cannot be evaluated, often resulting in neglect. Both QSAR modeling as well as SSD modeling are generally used toxicity extrapolation methods in environmental risk assessment. However, predicting ecotoxicity based on physicochemical characteristics or structural alerts is mainly done for only one species or one substance at a time, requiring extensive data on toxicity as well as on the predictive variables in the model, to provide a statistically sound model .The models derived in the present study could potentially serve as a screening‐level exploration into the potential of chemicals to pose harm to ecosystems via direct effects on species assemblages for those chemicals lacking in ecotoxicity data. For such chemicals, the QSAR‐based SSDs would enable translation of molecular characteristics into the μ and/or σ of associated SSD models. We note, however, that to be helpful to derive exploratory protective benchmarks (e.g., HC5 Dearden . The app Dearden  and Brac Dearden , who rep Dearden . Althoug Dearden , the var Dearden . The chaWe consider it highly relevant to assist focusing policy and management attention for a problem consisting of 100 000+ chemicals and their mixtures by providing a method—QSAR‐based SSD modeling—that can help in identifying the compounds for which it is highly unlikely that they pose harm by direct effects of species in species assemblages. Given our earlier findings (Posthuma et al. https://doi.org/10.1002/etc.4601.The Supplemental Data are available on the Wiley Online Library at DOI: This article includes online‐only Supplemental Data.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Post–infarction ventricular septal defect (VSD) is a fatal mechanical complication of myocardial infarction. Although the incidence has decreased to less than 1% after the extensive use of reperfusion strategies, post–infarction VSD still carries a high mortality risk. Management is controversial, whether to wait for surgery after a stabilisation period or to perform emergency surgery when diagnosed. We report on a case of post–infarction VSD that was detected with severe haemodynamic instability, beginning immediately after the patient’s Valsalva manoeuvre on the sixth day of a non–reperfused inferior myocardial infarction. In the early period, the post–infarction VSD was repaired via a trans–aneurysmal approach. The incidence of post–infarction ventricular septal defect (VSD) was 1–3% in the pre–thrombolytic era, but it has declined to nearly 0.2% in the era of thrombolysis.4We report on a case of post–infarction VSD in a patient who did not receive thrombolytic therapy because of late presentation. On the sixth day of the inferior MI, after a period of Valsalva manoeuvre, she developed sudden haemodynamic deterioration and symptoms and signs of acute heart failure. After the diagnosis of post–infarction VSD, we performed emergency surgery and repaired the defect by teflon strip with pladgeted prolene sutures via a trans–aneurysmal approach.A 62–year–old female patient was admitted to our hospital with typical enduring chest pain that had started two days earlier. Her past medical history was normal and she was a non–smoker. Her physical examination was normal. Surface electrocardiogram showed 2–mm ST elevation in leads D2, D3 and aVF with pathological Q waves and negative T waves, suggesting a sub–acute phase of inferior MI. Her biochemical markers of MI were higher than the upper limits of normal.She was admitted to the coronary care unit with a diagnosis of sub–acute inferior MI and started on isosorbite mononitrate 60 mg/day (p.o.), metoprolol succinate 100 mg/day (p.o.), acetylsalicylic acid 300 mg/day (p.o.), clopidogrel 75 mg/day (p.o.) and enoxaparine 1 mg/kg, bid, (s.c.). Transthoracic echocardiography was performed in the first hour after admission to the coronary care unit on day one and showed akinesia in the basal septum and mid–basal inferior wall, with a calculated ejection fraction of 45%.The patient was transferred to a room after an uncomplicated course of 36 hours. On the second day, at 08:00 her medical examination was normal. One hour later, after using the toilet and after several attempts at Valsalva manoeuvres, she felt sudden chest pain, dyspnoea and nausea. Her heart rate was 110/minute, blood pressure was 80/60 mmHg, and a new systolic murmur was recorded at the left sternal border with thrill and bilateral rales at the basal level of both lungs. Her surface electrocardiogram was unchanged. She was immediately transferred to the coronary care unit. This was the fourth day after admission to hospital.A second transthoracic echocardiography was performed and revealed a left ventricular infero–basal aneurysm, infero–mid and infero–septal akinesia and a ventricular septal defect in the basal mid–interventricular septum, creating a peak 80–mmHg gradient and mild pericardial effusion . IntraveThe patient underwent an operation two days after successful IABP insertion in the coronary care unit. The operation was performed with aorto–bicaval cannulation under extra–corporeal circulation (ECC). The heart was arrested by anterograde and retrograde blood cardioplegia. Exploration of the ventricular septum showed an inferior aneurysm in the right ventricular side approach . InspectThe ventricular septal defect that was near the apex of the septum was closed with the trans–aneurysmal approach. It was infixed to the left ventricular free wall to be repaired by teflon strip with pladgeted prolene sutures. The aneurysmotomy was closed in a buttress of strips of teflon felt and sutured with pladgeted prolene sutures . After aThere was no difficulty weaning the patient from the ECC with 2:1 IABP assistance in sinus rhythm. The postoperative course was uncomplicated. Early transthoracic echocardiography revealed no shunt. The patient was weaned from the IABP on the first postoperative day and inotropic drugs were reduced on the third postoperative day with stable haemodynamic parameters.Post–infarction VSD was first shown by Latham in an autopsy in 1845, and clinical diagnostic criteria were first introduced by Sager in 1934.7Post–infarction VSD is usually located in the anterior or apical portion of the ventricular septum (about 60% of cases) as a result of an anterior MI. Twenty to 40% of patients have a defect in the posterior portion of the ventricular septum as a result of an inferior MI.6Symptoms are mostly chest pain, dyspnoea, and symptoms due to low output and cardiogenic shock.1In patients who do not receive thrombolytic therapy, coagulation necrosis begins within three to five days of infarction. Neutrophils penetrate into the necrotic zone. By triggering apoptosis, released lytic enzymes cause the necrotic myocardium to rupture.Although the exact mechanism of rupture was not known, we presume that on the sixth day post MI, after our patient performed several forceful Valsalva manoeuvres, the thinned, necrotic septum was affected by the sudden volume and pressure changes created by the Valsalva manoeuvres. This may have caused the septum to rupture.It has been shown that an approximately 20–mmHg fall in the systemic blood pressure occurs during early phase 2, which is followed by an abrupt rise in phase 4 of the Valsalva manoeuvre (about 43 mmHg) in a patient who is sitting and performing it.8 An overshoot of arteriolar vasoconstriction during late phase 2 and a 42% transient increase of the cardiac output above resting state are believed to be the reason for the blood pressure rise during phase 4.9 ACC/AHA practice guidelines recommend daily stool softeners in the hospital management of ST–elevation MI and our opinion is that this recommendation should be closely followed.102), serious right ventricular dysfunction and low systolic blood pressure.4,11 If the right ventricle can handle the volume overload, the mean PAP and PCWP increases, as well as the survival rate.11Previously reported studies have shown that mortality is higher with: early VSD with surgery in the first 24 hours, inferior MI, the need for inotropic agents, cardiogenic shock, low mean pulmonary arterial pressure (PAP) and pulmonary capillary wedge pressure (PCWP), low cardiac index (CI < 1.75 ml/min/ mThe first repair by Cooley et al. in 1957, a case of acquired ventricular septal defect, was accomplished using an approach through the right ventricle with incision of the right ventricular outflow tract.12Our choice was to attach the apical–septal VSD to the left ventricular free wall and repair with teflon strips and pladgeted prolene sutures. The aneurysmotomy was closed and buttressed with strips of teflon felt and sutured with pladgeted prolene sutures after RCA bypass with a saphenous vein. The left internal mammary artery to the left anterior descending anastomosis was performed at the last stage.It was recently reported that a small or medium–sized postinfarction VSD could be treated successfully with a ventricular septal occluder. This intervention may be permanent or it may stabilise the patient and allow myocardial fibrosis, thus facilitating delayed subsequent surgical correction.13Post–infarction VSD is a serious mechanical complication of MI. Close monitoring of the non–reperfused patients is mandatory and a forceful Valsalva manoeuvre may facilitate the rupture of the myocardium, causing post–infarction VSD. Repair of postinfarction VSD can be accomplished with a trans–aneurysmal approach. The potential advantage of this technique is that an incision in the intact left ventricular myocardium is avoided.


Parasites are a major component of all animal populations. Males and females often differ in their levels of parasite prevalence, potentially leading to sex differences in the impact of parasitism on fitness, with important implications for the evolution of parasite and host traits including resistance, tolerance, and virulence. However, quantitative measures of the impact of parasitism under free‐living conditions are extremely rare, as they require detailed host demographic data with measures of parasite burden over time. Here, we use endoscopy for direct quantification of natural‐parasite burdens and relate these to reproductive success over 7 yr in a wild population of seabirds. Contrary to predictions, only female burdens were associated with negative impacts of parasitism on breeding success, despite males having significantly higher burdens. Female reproductive success declined by 30% across the range of natural parasite burdens. These effects persisted when accounting for interannual population differences in breeding success. Our results provide quantitative estimates of profound sub‐lethal effects of parasitism on the population. Importantly, they highlight how parasites act unpredictably to shape ecological and evolutionary processes in different components of the same population, with implications for demography and selection on host and parasite traits. Understanding the key drivers of demographic processes is crucial in predicting population growth and persistence , and relate this to their concurrent breeding success. Endoscopy more accurately reflects absolute parasite burdens than traditional indirect techniques of quantifying parasite load, such as fecal egg counts, which only capture the reproductively active subset of the parasite population  Does natural parasite load negatively impact individual breeding success? (2) Does parasite load and the effects of parasitism differ between the sexes? (3) Do environmental conditions alter the impact of parasitism on host breeding success?°11′N, 2°33′W), during chick rearing in the breeding seasons of 2011–2017. One hundred and one adult European shags were endoscoped to determine natural parasite load of gastrointestinal nematodes Contracaecum rudoliphii, a generalist parasite infecting a large number of seabirds. Seabirds can act as the definitive host to C. rudolphi, which is transmitted via fish prey , and were fitted using the To test for a difference in parasite loads between sexes (Q2) we compare linear mixed models describing parasite load (worm count) with and without the term sex using a likelihood‐ratio test, while accounting for the explanatory variables age, a quadratic effect of age and year . After carrying out a preliminary analysis on the relationship between parasite load and body mass, it was not included as an explanatory variable in these models; see r = 0.083, n = 34).In all other models, the response variable was the number of chicks fledged, relative to the maximum number of eggs laid, representing “fledging probability per egg,” which was assumed to have a binomial distribution (with logit link function). For these analyses, we fitted models for males and females separately, because of nonindependence of nest pairs, to quantify the impact of parasitism on the breeding success of both sexes directly. Preliminary analysis showed no correlation between male and female parasite load from the same nest in the same year  and led to predicted breeding success values that were 30% lower at the highest observed parasite loads than at the lowest observed loads, equivalent to a loss of 0.7 chicks per adult  and although there was evidence for a significant quadratic effect of age on the number of chicks fledged , but females did not. This occurs in a number of wild vertebrate populations and is well documented in seabirds (Daunt et al. Effects of parasitism are often context and host‐condition dependent, with effects expected to be more apparent in bad conditions (Bustnes et al. In conclusion, our results demonstrate that although parasite burden may be associated with differences in breeding success within the population, the direction or strength of this relationship can differ between sexes. They also highlight that although one sex may be more heavily parasitized than another, this does not translate directly into parasitism impacts. Differences between the sexes may have led to different selective pressures affecting the ability of individuals to tolerate a given level of infection (Duneau and Ebert  Click here for additional data file. Click here for additional data file.


HIV prevention offers promise for preventing new HIV infections, access to and uptake of these technologies remain unacceptably low in some settings. New models for delivery of HIV prevention are clearly needed. This commentary highlights the potential of person‐centred programming and research for increasing the cultural relevance, applicability and use of efficacious HIV prevention strategies. It calls for a shift in perspective within HIV prevention programmes and research, whereby people are recognized for their agency rather than assumed to be passive beneficiaries or research participants.While biomedical HIV prevention reorientates power dynamics so that individuals (rather than interventions) are at the centre of the response. Respecting personal choice and agency – and understanding how these are shaped by the context in which people exercise these choices – are critical dimensions of the person‐centred approach. Community‐based participatory research should be employed to inform and evaluate person‐centred HIV prevention. We argue that community‐based participatory research is an orientation rather than a method, meaning that it can be integrated within a range of research methods including randomized controlled trials. But embracing community‐based participatory approaches in HIV prevention research requires a systemic shift in how this type of research is reported in high impact journals and in how research impact is conceived. Community‐based organizations have a critical role to play in both person‐centred HIV prevention and research.Person‐centred HIV prevention is situated at the intersection of unprecedented opportunity and crisis. Person‐centred approaches to HIV prevention and research shift power dynamics, and have the potential to ensure a more sustainable response with each individual actively participating in their own care and meaningfully contributing to the production of knowledge on HIV prevention. This approach taps into the resourcefulness, resilience and knowledge of the person and their communities, to strengthen research and programmes, making them more relevant, appropriate and effective. It has been established that antiretroviral treatment (ART) is an efficacious HIV prevention tool But this decline is far from the prevention target that most governments pledged to achieve when they signed the 2011 Political Declaration on HIV and AIDS. The target was a 50% reduction in new infections acquired through sexual transmission or injecting drug use between 2010 and 2015 New models of delivery of HIV prevention are clearly needed to ensure that nobody is left behind. In this commentary we highlight the potential of person‐centred programming and research for increasing the cultural relevance, applicability, efficacy and uptake of HIV prevention strategies 22.1There is an increasing recognition that HIV prevention must be reorientated so that it places people (rather than interventions or disease) at the centre of our response At the core of person‐centred HIV prevention is the acknowledgement that people are best placed to decide which prevention methods are right for them While person‐centredness Evidence on person‐centred HIV prevention programming is scarce but emerging studies suggest it may help reach the most marginalized populations who may have intersecting vulnerabilities and are not being reached through public health systems. For example, Women Initiating New Goals of Safety (WINGS) is an individualized screening, brief intervention and referral to treatment model for addressing intimate partner violence and HIV risks among women who use drugs or engage in heavy drinking There is an urgent need for more evidence on which person‐centred approaches work for whom and in what contexts, and for evidence‐informed implementation guidance. The following sections of this paper highlight the need for person‐centred HIV prevention research to meaningfully engage with communities and call for a shift in how community participation in HIV prevention research is reported.2.22.2.1focus of enquiry: it is defined as research examining person‐centredness orientation for developing or evaluating HIV prevention interventions that aim to be person‐centred.Person‐centred research is determined based on the with the people whose life‐world and meaningful actions are under study” Community‐based participatory research involves planning, executing and disseminating research “person‐centred, then the focus of enquiry must expand from a disease (or vulnerability to the disease) to the whole person and their lived experience Building further from the aforementioned participatory practices, if a study is concerned with also being Person‐centred research is grounded in the belief that the evidence on HIV prevention must adequately respond to the broad needs and aspirations of people who take part in the research and who we hope to uptake the HIV prevention technologies and interventions. For example, a mixed‐methods longitudinal study of adolescents living with and affected by HIV in South Africa, has used a participatory approach to examine what might improve young people's uptake of health services. Through the “dream clinic” exercise 2.2.2Participatory research has often been categorized as a qualitative research method – portrayed in contrast to positivist quantitative science Embracing community‐based participatory approaches in HIV prevention research requires a systemic shift in how this type of research is reported in high‐impact journals. High impact peer‐reviewed publications featuring emerging evidence on HIV prevention, including this journal, require that authors adhere to gold standard reporting guidelines for effectiveness and epidemiology studies. But the relevant reporting guidelines for randomized controlled trials Further, for those of us providing HIV prevention services and strategies, the outcome of community‐based participatory research cannot be stand‐alone research outputs. Rather, the research process should be fully embedded in and intertwined with all other elements of HIV prevention. For us, HIV prevention research is a tool for optimizing service delivery. In order to inform person‐centred HIV prevention, the research must also be participatory, whereby people are not merely participants but rather essential technical advisors, partners in the research design and implementation, co‐owners of data and key stakeholders for dissemination Networks of key populations and people living with HIV, community groups, women's rights groups and community activists can play instrumental roles in posing difficult ethical questions, identifying relevant community partners and helping ensure that the research is conducted in a way that maintains accountability to communities. Community‐based participatory research in the context of HIV is challenging. Debates around these challenges are important and, in our view, reinforce the importance of engaging with community‐based organizations in HIV prevention research. For example, researchers have expressed tensions between the basic tenets of ethics to protect participants versus the basic principles of community‐based participatory research which recognizes people's autonomy and authority over their own lives 3HIV prevention is situated at an intersection of unprecedented opportunity and crisis, with prevention targets not being met for marginalized populations In order to achieve this, a reorientation of power dynamics in research is essential. We posit that community‐based participatory approaches to research are highly relevant to shaping person‐centred HIV prevention. Here, community‐based participatory research is employed as an orientation to scientific enquiry, which can be applied to both qualitative and quantitative research methods. Community‐based organizations have a critical role to play in strengthening community–academic partnerships and ensuring that research is done ethically in a way that is accountable to communities.Person‐centred approaches to HIV prevention services and research shift power dynamics, and have the potential to ensure a more sustainable response with each individual actively participating in their own care. This approach taps into the resourcefulness, resilience and knowledge of the person and their communities, to strengthen research and programmes, making them more relevant, appropriate and effective.The authors declare that they have no competing interests.CR, ER, MP and SS conceptualized the commentary. MP, CR and SS provided content on person‐centred HIV prevention programming. MP and ER provided content on implications for research. MP drafted the manuscript, and all authors contributed to revisions.


Inflammatory diseases are a major socio‐economic burden, with the incidence of such conditions on the rise, especially in western societies. For decades, the primary treatment paradigm for many of these conditions was to develop drugs that inhibit or antagonize the production and biological actions of molecules that were thought to be the culprits in propagating disease; these include cytokines and eicosanoids. This approach is effective in controlling disease propagation; however, long‐term exposure to these anti‐inflammatories is also associated with many side effects, some of which are severe, including immune‐suppression. The discovery that termination of self‐limited acute inflammation is an active process orchestrated by endogenous mediators, including the essential fatty acid‐derived resolvins, protectins and maresins, has provided novel opportunities for the design of therapeutics that control inflammation with a lower burden of side effects. This is because at variance to anti‐inflammatories, pro‐resolving mediators do not completely inhibit inflammatory responses; instead, these mediators reprogramme the immune response to accelerate the termination of inflammation, facilitating the regain of function. The scope of this review is to highlight the biological actions of these autacoids and their potential utility as lead compounds in developing resolution pharmacology‐based therapeutics.http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetocThis article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit  These specialized organs required protection from invading microbes as well as mechanisms to repair and regenerate damaged tissues and organs, thus paving the way to the evolution of the immune system  phases , the initiating enzyme in the lipoxin biosynthetic pathway , protectins and maresins and the eicosapentaenoic acid derived resolvins (RvE)   was converted in inflammatory exudates to novel mediators that potently and stereospecifically promoted the termination of inflammation and were thus coined as E‐series resolvins (RvE) , downstream genes, as well as microRNA (miR)‐21 and 155, resulting in a reduction in inflammatory signalling. In vitro, RvD1 up‐regulates P. aeruginosa phagocytosis by both neutrophils and macrophages  during ischaemia/reperfusion, it enhanced perfusion recovery and promoted arteriogenesis   by its cognate ligand    was not simply a precursor in the biosynthesis of DHA from EPA. This essential fatty acid is converted by leukocytes to several mediator families that are congeners to those produced when DHA is the substrate Figure  ‐hydroxyperoxy‐7Z,10Z,13R,14E,16Z,19Z‐docosapentaenoic acid (13‐HpDPA). This or its reduced form, namely, 13‐HDPA, are then donated to neutrophils for transcellular biosynthesis to produce RvT  inflammasome in murine and human macrophages, reducing the expression of caspase 1 and the production of IL‐1β as well as the release of LDH, a hallmark of pyroptosis   is part of the GPCR family  and operates in a ligand‐biased fashion. Furthermore, signalling pathways activated by these different dimerization states are distinct   receptorresolvin E1  leads to the activation of PI3K and ERK resulting in the phosphorylation of both Akt and the ribosomal protein S6  in human monocyte‐derived macrophages was recently shown to be regulated by LPS and IFN‐γ, where incubation of monocyte‐derived macrophages with these inflammatory signals activates the promoter of these receptors  is a receptor for RvD2. The binding of the agonist to its receptor leads to an increase in intracellular cAMP as well as CREB, ERK1/2 and STAT3 phosphorylation. Loss of this receptor in transgenic animals is associated with delayed resolution response to both sterile and infectious inflammatory insults ‐induced spatial memory impairment, actions that were CB1 receptor‐dependent . A later generation analogue of RvE1 displays potent protective actions in dry eye syndrome in humans. In a phase 2 trial, this RvE1 analogue significantly reduced disease incidence, controlling dry eye‐related inflammation, findings that are under further investigation in a phase 3 trial (Wire, From these efforts, several generations of analogues and mimetics are now developed (see Table al Wire, ..et al., et al., et al., et al., Additionally, recent studies have demonstrated the protective actions of analogues of RvD1 (Orr et al., et al., et al., et al., et al., et al., et al., The appreciation that resolution of inflammation is an active process and SPM are central in controlling both cellular trafficking and responses has opened up new and exciting horizons for the development of new therapeutics. Given that resolution pharmacology‐based medicines will harness endogenous reparative responses, it is anticipated that they will be burdened with lower side effects since they will not interfere with natural host immunity, potentially also increasing patient compliance. Furthermore, these findings shed new light on the roles of omega‐3 essential fatty acids in the control of acute inflammation. The protective actions observed in many clinical studies using these fatty acids are not simply due to the inhibition of inflammatory eicosanoid formation by competing for the biosynthetic enzymes. Indeed, these substrates are precursors to structurally unique molecules, the SPM, that carry potent tissue protective actions. Thus, these mediators may represent novel markers to determine the pharmacodynamics and pharmacokinetics of omega‐3 supplements and their ability to influence the host response in both healthy people as well as patients with inflammatory diseases. While the evidence in humans for the utility of this approach is still being investigated, results from animal and initial human experiments are encouraging (Dalli http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., et al., Key protein targets and ligands in this article are hyperlinked to corresponding entries in The authors declare no financial conflicts of interest.


The study was considered a success for ODM, and therefore should not be included in a meta‐analysis comparing ODM with conventional care.However, the study selection raises concerns that impact the result substantially. The included study by Brandstrup Furthermore, the most influential study regarding ODM by Calvo‐Vecino and co‐workerset al.Consequently, the meta‐analysis should be modified by exclusion of the study by Brandstrup and colleaguesThe authors declare no conflict of interest.


Task‐sharing in surgery is well established, with associate clinicians performing successful surgery in many countries. Little is known about the process of surgical skill acquisition by associate clinicians, or whether this differs from that of doctors.A blinded experimental study compared surgical skill acquisition by Sierra Leonean associate clinicians enrolled in an essential and emergency surgery training programme with that of a matched group of UK surgical trainees. After identical instruction, practice time and with identities disguised, trainees were videoed performing simulated surgery. Trainees were marked on 12 performance parameters and five behaviour characteristics using validated tools and qualitative comment.The Sierra Leonean group comprised 19 associate clinicians and one doctor; the UK group comprised 20 doctors in their first 5 years of training. The UK group had significantly more surgical and postgraduate experience than the Sierra Leonean group. The Sierra Leonean trainees outperformed the UK trainees on three of the 12 performance parameters and four of the five behaviour characteristics. UK trainees did not outperform Sierra Leonean trainees on any parameter or characteristic. Qualitative differences in learning style were observed.Sierra Leonean associate clinicians demonstrated equal or superior skill in all objective parameters tested, despite having less experience than the UK doctors. Like many countries in sub‐Saharan AfricaCommunity health officers in Sierra Leone are a cadre of associate clinicians who have undergone a 3‐year basic medical diploma and work in community health centres and hospitals providing general medical care. A 2‐year surgical task‐sharing programme commenced in Sierra Leone in 2011 to expand the practice of selected community health officers and non‐specialist doctors in providing essential and emergency surgery and obstetric care. For logistical reasons, the opportunity for doctors to enrol in the programme has been limited and, to date, 55 of 57 (96 per cent) of the trainees in this programme are health officers. The 5‐year outcome, productivity and safety of this surgical training programme have been described recentlyThe primary aim of this study was to evaluate whether, with identical training, those enrolled in this predominantly associate clinician training programme could achieve the same level of competence, in the same time, as matched UK trainees, all of whom were medically qualified. A secondary aim was to understand any qualitative difference in learning between the two groups of trainees.All participants were consenting volunteers, recruited between October 2015 and April 2016. The UK group involved medically qualified UK surgical trainees in their first 5 years of training. The Sierra Leonean group involved surgical trainees  enrolled in the surgical training programme.Sierra Leonean trainees were invited to volunteer when they attended the base hospital for training or other purposes. Consecutive trainees attending the base hospital were approached, and all agreed to take part. UK trainees were invited to volunteer when they attended regional in‐service training days. All those attending were invited; a few individuals also asked to take part, having heard about the study through colleagues. Consecutive volunteers were recruited with no selection process. Any trainee who had previously performed simulated or actual venous cut‐down was excluded.Skill acquisition was assessed using a medium‐fidelity simulated venous cut‐down model . The simulation requires the following surgical skills: incision, dissection, venotomy, knot‐tying and suturing. This construct was selected for ease of video assessment, speed of assessment and unfamiliarity to all trainees. It had the advantage of being an attractive skill for both groups, being part of life‐saving skills for obstetrics necessary in Sierra Leone and of Advanced Trauma Life Support training in the UK.Both groups were exposed to identical training materials over a 1‐h period, consisting of an instructional video, a paper fact sheet, and access to the simulation model to practise as often as they wished. All materials were in English. English is the official language and language of higher education in Sierra Leone, although the second or third language for participants in the Sierra Leonean group. To mitigate this possible bias, the operator in the training video was a Sierra Leonean surgeon speaking in English. There was additional instructional commentary on the training video by one of the authors familiar to both groups of trainees. After 1 h, trainees each performed the simulation with one assistant taken from the same study group. Each assistant was only used once. To mask ethnic identity, all participants wore identical surgical gowns and opaque gloves, and videos showed only the covered arms and hands. Work surfaces were covered with identical drapes. Procedures were video‐recorded. Participants were asked to talk about what they were doing during the video, and all speech was removed and dubbed back on by a single investigator.Videos were randomized and uploaded online. They were viewed in unedited format by two consultant surgeons independently. Both assessors participate in UK National Selection at Speciality Trainee  Year 3 level and were therefore familiar with the scoring methods used. Performance parameters were assessed using validated Objective Structured Assessment of Technical Skills (OSATS), and trainee behaviour characteristics were assessed using a 5‐point Likert scale. Each participant completed a 12‐question true/false multiple choice question (MCQ) test to ascertain factual knowledge about the procedure and its application.Ethical approval for the study was obtained from the University of Oxford in the UK; an ethical committee waiver was granted in Sierra Leone as no patients were involved in the study.2 test, parametric data with Student's t test, and non‐parametric data with two‐tailed Mann–Whitney U tests using a 95 per cent confidence level. Spearman's rank correlation test and a logistic regression model were used to identify independent factors that might predict candidates' performances using Microsoft Excel®  and SPSS® version 22 software .Assuming considerable intragroup variation, 20 participants in each group had a power of 0·8 to detect a 10 per cent difference between groups. The null hypothesis assumed no difference in performance and skills acquisition between the two groups. Categorical data were analysed with the χQualitative analysis was undertaken using the quasi‐quantitative methods described by Burnardversus 1·78 years respectively). The Sierra Leonean trainees were significantly older and less likely to have undertaken a basic surgical skills course than the UK trainees , older age (P = 0·011) and having more actual surgical experience (P = 0·004) all predicted a higher estimation of length of time in surgical training. Only being a Sierra Leonean trainee independently predicted a higher Global Impression Score (P = 0·006) , there were no significant differences between the two groups (P = 0·072). Positive critiques in both groups and in all domains were generic. Negative comments were more revealing as ‘dangerous technique’ and ‘risk of needlestick injury to the assistant’ would require direct intervention from trainers in a real‐life procedure. Sierra Leonean trainees showed a preference for verbal learning, talking through the steps of the procedure and their cognitive processes. They interacted more with their assistant and were bolder in their actions.Results and examples of the qualitative assessments for types of comment are shown in Despite evidence of safetyIt is unusual for medically qualified and non‐medically qualified surgical trainees to receive identical training, and therefore difficult to make comparisons regarding skill acquisition. The present study found that, with identical training, Sierra Leonean community health officers showed equal or superior skill acquisition, greater confidence and better communication than UK surgical trainees, despite UK trainees having more previous surgical experience.Categorization of behaviour patterns can help an understanding of how individuals learnPrevious years of surgical experience correlated with performance and the assessors' estimate of length of time in surgical training amongst the UK trainees. Previous experience and transferable skills might be expected to improve effectiveness when learning a new procedure. This was not seen amongst the Sierra Leonean trainees, who demonstrated equal poise to perform the procedure regardless of experience. It appeared they either had more trust in the training materials or paid more attention to them. This is consistent with the assessors' findings that the Sierra Leonean trainees demonstrated greater confidence.The demonstrator in the instructional video was Sierra Leonean, and his features were not masked. This may have led to a role‐modelling effect on the trainees' learning. Medical students have been shown to identify their ideal clinical role model along racial groundsDetractors could argue the Sierra Leonean trainees' performance simply reflects more didactic learning rather than being a true representation of cognitive process. Perhaps the Sierra Leonean trainees simply followed the instructions better. The observations suggest, however, that the Sierra Leonean trainees adopted a more conscious and stepwise approach to completing the task, with deliberation and verbalization of what they aimed to achieve for each step of the procedure, voicing potential pitfalls of the steps before they interacted with the simulation, recognizing and correcting errors if necessary. This transformed them from being technicians to deliberating practitioners with contextual awareness, a hallmark of deliberate practiceIn contrast the UK trainees were more silent, showed less preparation, and appeared less discriminatory in their choice of instruments and sutures. This could indicate lower cognitive engagement with the task, perhaps being accustomed to an environment where they are handed equipment without the need to consider its suitability. Perhaps UK trainees may simply have been less talkative with their cognitive processes occurring silently. This could make them appear more hesitant. The cognitive process seems more explicit in the more communicative Sierra Leonean trainees.This study aimed to see whether, given the same training, Sierra Leonean associate clinicians achieved the same level of competence as medically qualified UK trainees. Superiority had not been anticipated, raising a number of questions. It may reflect cultural background. Sierra Leoneans tend to be vocal. Perhaps this can contribute to their ability to learn surgical techniques. Training in low‐resource settings can be hard to obtain and is seen as a privilege; this could contribute to an eagerness to learn. In addition, the vocal commentary of the Sierra Leoneans often alluded to recognition of their crucial role as the only professionals able to perform this task to save lives in deprived localities. The poorer performance of the UK trainees, despite their greater experience and in many cases more formal training, could represent complacency among those who benefit from a sophisticated training structure, or a knowledge that they are exceptionally unlikely to be left alone with this task in the near future.There are limitations to this study, including assessments of more complex procedures, real‐life surgery and a closer examination of cultural differences in the cognitive process of learning. Nevertheless, the study provides some insight into the educational ability of associate clinicians performing surgery in a low‐resource setting. It is reassuring that they were not found lacking. The poorer performance of the UK trainees raises questions about whether we currently provide early‐years trainees with the best possible training environment. Perhaps they could learn from those facing much tougher working environments early in their careers.


To compare and analyze the clinical outcomes of the proximal humeral internal locking system (PHILOS) alone and the PHILOS combined with fibular allograft in the treatment of Neer three‐ and four‐part proximal humerus fractures (PHF) in the elderly.From January 2014 to January 2018, a total of 42 elderly patients with Neer three‐ or four‐part PHF admitted to our hospital were randomly divided into observation group and control group, with 21 patients in each group. The observation group was treated with the PHILOS combined with fibular allograft. The control group was treated with the PHILOS alone. Perioperative parameters and fracture classification were recorded in the two groups. Function results were assessed by Visual Analog Scale (VAS), Constant‐Murley score (CMS), American Shoulder and Elbow Surgeons (ASES) score, and the Disability of Arm‐Shoulder‐Hand (DASH) score. Radiological results were evaluated using the neck‐shaft angle (NSA) and humeral head height (HHH), and complications were also recorded in each group.P < 0.05). The CMS and ASES were higher in the observation group than the control group, and there was significant difference between the two groups (P < 0.05). The mean difference in the NSA and HHH were lower in the observation group than the control group, and there was a significant difference between the two groups (P < 0.05). There was one postoperative complication in the observation group, which was humeral head avascular necrosis (AVN). There were seven postoperative complications in the control group, including three cases of humeral head collapse and three cases of screw cutout and one case of humeral head AVN. The incidence of postoperative complications in the observation group was significantly lower than the control group (P < 0.05), there was a significant difference between the two groups.There were no significant differences between the two groups in terms of preoperative status, age, gender, cause of trauma, fracture site, and fracture classification. The average follow‐up time was 12 months. At the last follow‐up, the VAS and DASH observation groups were lower than the control group, and there was significant difference between the two groups (For Neer three‐ or four‐part PHF in the elderly patients, PHILOS fixation with fibular allograft shows satisfactory short‐term results with respect to humeral head support and maintenance of reduction, and may reduce the incidence of complications associated with fixation using a PHILOS alone. Proximal humeral fractures (PHF) account for 4%–5% of the whole body bone fracturesAlthough various surgical techniques have been described for the unstable PHF, proximal humeral internal locking systems (PHILOS) are increasingly popular for treating these fractures because they offer improved biomechanical properties by providing divergent and convergent fixed‐angle screws that improve fixation and pullout strength in osteoporotic boneThe aim of this study was to compare and analyze the clinical outcomes of the PHILOS alone and the PHILOS combined with fibular allograft in the treatment of Neer three‐ or four‐part PHF in the elderly. The hypothesis was that patients treated using the PHILOS combined with fibular allograft would have better clinical and radiological outcomes with lower complication rates than those treated using only the PHILOS alone.This research was a retrospective study and was approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University. Written informed consents were obtained from all enrolled patients.From January 2014 to January 2018, a total of 42 elderly patients with Neer three‐ or four‐part PHF admitted to Nanjing First Hospital were randomly divided into observation group and control group, with 21 patients in each group. The observation group was treated with the PHILOS combined with fibular allograft. The control group was treated with the PHILOS alone. Perioperative parameters and fracture classification were recorded in the two groups. The demographic characteristics of the patients in the two groups are shown in Table Inclusion criteria were as follows: (i) patients over 60 years of age; (ii) unilateral acute shift of Neer classificationExclusion criteria were as follows: (i) a history of shoulder surgery or chronic bone nonunion, there was severe soft tissue injury at the surgical site or systemic or local infection; (ii) pathological fracture or open fracture; (iii) complications of serious nervous or vascular injury; (iv) hypertensive patients with poor blood pressure control and diabetic patients with poor glycemic control; and (v) patients who refused to participate or failed to cooperate during the trial.A proximal humeral locking plate  was used in all patients. A fibular allograft  was used in the observation group.Step 1: All patients were placed in the beach chair position on a radiolucent operating table followed by cervical plexus anesthesia.Step 2: A deltopectoral approach was performed for all patients. An approximate 15 cm skin incision was made. The insertion of the deltoid muscle was one‐half detached posteriorly subperiosteally.Step 3: Non‐absorbable sutures were passed through the junction of the greater tuberosity and the rotator cuff to promote mobilization and reduce fragmented tuberosities. A 2.0 mm Kirschner‐wire (K‐wire) was inserted into the humeral head to control rotation, and a periosteal elevator was used as a joystick at the fracture site to obtain reduction. After reduction of the greater tuberosity and humeral head, one or two 1.5 mm K‐wires were used for temporary fixation. The plate was placed in a position that did not impinge on the acromion. Indirect reduction was performed by inserting a cortical screw into the humeral shaft following the shape of the plate. In the observation group, fibular allograft  and humeral head height (HHH) on anteroposterior views Fig. .The humeral neck‐shaft angle (NSA) was defined as the angle between a line that is perpendicular to the articular segment or atomic neck of the humerus and a line that bisects the humeral shaft by the measurement tools of the hospital's PACS system. The change in NSA was measured to assess whether the humerus had varus deformity.HHH was defined as the distance between the upper end of the plate and the upper end of the humeral head by the measurement tools of the hospital's PACS system. The change in HHH was measured to assess the collapse of the humeral head. Comparing the immediate postoperative radiological findings with those at the final follow‐up, loss of fixation was defined if the varus NSA change was >5° or if HHH change was >3 mmClinical evaluation was assessed at the final follow‐up using a Visual Analog Scale (VAS)The VAS was used to assess the pain of the shoulder joint of the subject. The VAS total score is 10 points, with zero points indicating no pain and 10 points indicating unbearable pain.The CMS score was 100 points, which consisted of pain (15 points), muscle strength (25 points), functional activity (20 points), and shoulder mobility (40 points). Higher scores indicate better functionality. Among them, objective evaluation indicators included shoulder mobility and muscle strength (65 points), and subjective evaluation indicators included pain and functional activities (35 points).The rating scale of the ASES, which includes pain (50%) and life function (50%), is 100 points. The higher the score, the better the shoulder function.The DASH scoring system consists of 30 items. Each of the five options corresponds to one to five points. When there are more than three missing items, the score is not calculated. The better the activity, the lower the score.In addition, complications, such as AVN, screw cutout, implant failure, plate impingement, head collapse, and infection, were also recorded.t test to detect the group differences. Qualitative data of groups was compared by the χ2 test. P value <0.05 was considered as significant difference.Statistical analysis was performed using SPSS v. 19.0 . Continuous variables, presented as the mean and standard deviation (SD), were compared by the Student P = 0.000).The radiological outcomes are shown in Table P = 0.000).The average HHH immediately postoperatively in the observation group was 12.3 mm (6.7 mm–15.7 mm), the average HHH at the last follow‐up was 10.2 mm (6.5 mm–13.5 mm), and the mean difference in the HHH was 2.14 ± 0.33 mm. The average HHH immediately postoperatively in the observation group was 12.2 mm (7.0 mm–18.5 mm), the average HHH at the last follow‐up was 9.0 mm (1 mm–15.1 mm), and the mean difference in the HHH was 4.54 ± 0.42 mm. The mean difference in the HHH between the values immediately postoperatively and those at the final follow‐up were significantly greater in the control group (P = 0.734) score was not significantly different between the two groups. The CMS (P = 0.020) score, ASES (P = 0.024) score, and DASH (P = 0.023) score were significantly different between the two groups at the final follow‐up.The clinical outcomes are shown in Table There were seven postoperative complications in the control group Fig. , includiPHF has been ranked as the third most frequent fracture and has a strong correlation with osteoporosisAlthough there are many surgical options available, PHILOS have consistently demonstrated biomechanical superiority over other forms of fixation in osteoporotic boneThe importance of reducing and maintaining the medial calcar to provide biomechanical support for a laterally placed plate has been recognizedUse of an endosteal strut allograft can re‐establish medial support, even in the comminuted osteoporotic bone commonly found in these patients. We considered that the fibula might be the most suitable donor bone for reconstructing the medial support in these types of fractures. Its length, geometrical shape, and mechanical strength might also be appropriate for these fractures. The cortical bone of the fibula provides immediate structural continuity and stability at the fracture site. When used as an intramedullary bone graft, it also has some osteogenic potential in addition to acting like a strut across the fracture site. Allograft is cancellous or corticocancellous chips or tricortical graft used as osteoconductive filler for metaphyseal defects. The fibular strut allograft may also minimize postoperative osteonecrosis by increasing the biomechanical strength of the construct and resisting a loss of reduction. Maintenance of reduction may permit revascularization of head pieces rendered ischemic at the time of injury. An important finding of this study is that the rate of varus malalignment and loss of HHH was significantly less when plate fixation was accompanied by a strut allograft.The similarly good clinical outcomes in our study might be related with some factors. Fibular allograft used as volumetric filling in the bone void formed after reduction of humeral neck‐shaft angle could push the humeral head resistance to the force from the scapular fossa along with the screws, preventing the screw penetrating into the articular surface. Besides, this medial strut could prevent the varus placement of the head to diminish humeral head varus collapse and reduce the incidence of malunion. Anatomical medial strut with allograft bone has more potential to prevent humeral head varus displacement compared with the isolated fibula allograft. Anatomical allograft should be modified into a specific shape to fill the bone void according to the intramedullary geometry of the proximal humerus through computer virtual design with Pro‐E software. This kind of structural allograft provides enough medial stability and allows the formation of osteogenic tissue across a fracture site along with the surface of the allograft followed by bone formation. In addition, faster fracture healing could minimize articular segment AVN or collapse. Anatomical allograft is a plane contacted with the humeral head, and the support position could be pushed to the inferior medial point. However, isolated fibula was just a point‐to‐point support of the humeral head, and the support point is just at the line of extension of the intramedullary canal direction.This study has several limitations. Firstly, it is not a randomized trial because the two techniques were performed at different times. Secondly, the study was limited by its retrospective design, which could introduce selection bias and the potential for confounding. Thirdly, although a large number of patients were included, this study had relatively low power to detect differences in forward elevation between the two groups. Subtle differences between the groups may not be identified in a single‐center study. A large‐scale, multicenter study would be required to investigate these clinical outcomes further.In conclusion, the present results showed that that patients treated by PHILOS combined with fibular allograft had a better functional outcome and a lower complication rate comparted to patients treated by PHILOS alone. Suitable void filler in the proximal humerus for supporting the head fragment, medial cortical bone and greater tuberosity might play a key role in reducing the incidence of complications in elderly patients, especially with osteoporosis.


TBRF) caused by the bacteria Borrelia, is poorly documented in veterinary medicine. Given the widespread presence of the soft tick vectors – Ornithodoros and the recently discovered hard tick vectors, as well as their close association with animal hosts, it is highly likely that infection occurs, but is rarely reported to be of veterinary importance. Sporadic reports of canine infection, some being fatal through to probable cause of abortion in horses have been published. Some of these pathogens exist in regions where there are limited diagnostic facilities, hence, they are likely to be missed and their impact on productivity may be unquantified. Here we review available literatures on cases of TBRF in domestic and wild animals in order to show their potential veterinary medical impact. Future efforts using field and laboratory surveys are needed to determine pathogenesis, vector competence and distribution in animals, their impact on animal health and productivity as well as to prevent further spill to the human population, where it is already a public health problem in some parts of the world.Tick‐borne relapsing fever ( Borrelia species are tick‐borne, Gram‐negative, spiral‐shaped bacteria that causes several diseases across the world, grouped into Lyme borreliosis (LB) and tick‐borne relapsing fever (TBRF)  species during feeding. These ticks are widely distributed in sub‐Saharan Africa, Asia, the Americas and some parts of Europe  group transmitted by Argas ticks and is not currently reported in man  Borreliae. New species of Borrelia (B. miyamotoi) transmitted by hard‐bodied (Ixodid) tick species that transmit the Lyme borreliosis have been recently reported to cause relapsing fever in some humans in North America, Asian region of Russia, Europe and Japan . Although, sequence obtained from the 16S rRNA gene in that study from Nigeria for Borrelia species identification was unsuccessful, it showed 99% nucleotide homology to B. burgdorferi sensu lato (the Lyme disease pathogen), thus may possibly belong to a presently unclassified Borrelia species  causing pathogenic illnesses in dogs in Africa, where there are endemic foci of infection, and are pathogenic to human. This may warrant specific studies to determine the presence or absence of the disease in dogs because of the close interaction of dog with human and the reports of tick vectors in human dwellings where dogs are also resident.Tick‐borne relapsing fever is a potentially fatal infection in pet animals such as dogs and cats   is the causative agent of bovine borreliosis. The disease has also been reported from cattle, sheep and horses from several countries in Africa, South America, Europe and Australia  for which the mare might have been an incidental host for an infected tick  with a very high prevalence of 58% , during human disease outbreak investigation of relapsing fever in United States  in the United Kingdom. A PCR‐based analysis targeting the flaB and glpQ gene fragments showed that the causative agent had close similarities with the relapsing fever Borrelia previously reported in Africa – a cluster containing B. recurrentis, B. duttonii and B. crocidurae  and glycerophosphodiester phosphodiesterase (GlpQ) antigen have also been carried out and have shown usefulness in differentiating RF Borrelia and Lyme disease group, although caution should be taken in interpreting results as current or past infection , and they should be considered as differentials in animals presenting with similar clinical signs  or carbamate (Kartzimet 20). Although Jarisch–Herxheimer reaction has been documented in humans following antibiotic treatment for RF borreliosis, so far there is no evidence of such reactions in animals (Baneth et al. et al. Drugs used in treatment of human TBRF have shown usefulness in treating canine TBRF. There are very few information on specific treatment of TBRF in other species of animals. Doxycycline administered orally at the dose of 7.5 mg/kg for 6 weeks (Piccione B. theileri in animals and world widely distributed B. anserina, majority of studies on other soft ticks TBRF in animals have are very few – summarized in Fig. Although a number of studies have been carried out on hTBRF such as None to declare.No ethical approval was required as this is a review article with no original research data.