Muntiacus vuquangensis). Given the importance of scent marking in individual recognition among ungulates, this behavior may serve to communicate the fitness cost of antagonistic interactions among rival males and could serve as a mechanism for mate assessment among females.We present evidence of scent marking in the large‐antlered muntjac ( Muntiacus vuquangensis), a little‐known and threatened deer endemic to the Annamites ecoregion of Indochina. We discuss the function of this behavior within a wider behavioral and evolutionary ecology framework for mammals living in tropical forests.We document the first recorded evidence of scent marking in the large‐antlered muntjac ( Indirect forms of communication play an important role in the exchange of information among mammals. Scent marking, in particular, appears to be widespread among a range of species , a solitary and elusive deer found in the tropical forests of Laos, Viet Nam, and Cambodia  based on antler configuration in males, forehead coloration in females, and length of the tail in both sexes  documented 11 independent encounters (independence threshold = 60 min) of large‐antlered muntjac in closed‐canopy tropical forest habitat. Large‐antlered muntjac were distinguished from the morphologically similar and sympatric northern red muntjac ; formal analysis (lead); methodology (lead); writing – original draft (lead); writing – review and editing . Pablo Sinovas: Conceptualization (supporting); funding acquisition (lead); methodology (supporting); project administration (lead); writing – original draft (supporting); writing – review and editing .PS would like to thank the Ratanakiri Provincial Department of Environment, and in particular Thon Soukhon, for facilitating access to Virachey National Park, and Jeremy Holden for assisting with the deployment of camera traps. AT would like to thank Kathi Kasper and Thanh Nguyen for their help in providing second opinions on individual identification in the camera‐trap photos.https://doi.org/10.5061/dryad.j3tx95xk3.This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at the following link:


Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet‐based microfluidics to encapsulate a fuel‐driven cycle that drives phase separation into coacervate‐based droplets to overcome this challenge. This approach enables the analysis of every coacervate‐based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate‐based droplets and their properties. We observed that coacervate‐based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate‐based droplets enables future selection‐ or evolution‐based studies. Droplet‐based microfluidics were used to encapsulate a fuel‐driven cycle that drives phase separation into coacervate‐based droplets. This approach enables the analysis of every coacervate‐based droplet in the reaction container throughout its lifetime. The nucleation, growth, and dissolution of active coacervate‐based droplets were investigated with this setup. Liquid–liquid phase separation (LLPS) as an underlying mechanism for forming membraneless organelles is attracting increasing attention due to its role in regulating intracellular processes. It is involved in the promotionHowever, microscopy analysis of active coacervate‐based droplets has been limited. This limitation is partly because only a small fraction of the total reaction solution can be imaged. On the one hand, this leads to a bias depending on which part of the solution is imaged, e.g., imaging close or far away from the top or bottom coverslip can change the average size of the observed coacervate‐based droplets. On the other hand, droplets cannot be tracked over their entire lifetime. Instead, a snapshot of some of the droplets is obtained. Finally, the time of mixing and imaging is relatively slow, as droplets can already form and grow in the first tens of seconds after mixing. These limitations are challenging for further developing active droplets and studying their behavior within a population.Microfluidic technology is emerging as an essential tool in the analysis of LLPS systems,Therefore, this work aims to introduce a method that allows us to analyze the behavior of every fuel‐driven active droplet formed throughout its lifetime in an experiment. Thus, we use a microfluidic setup to analyze a fuel‐driven LLPS system in a confined volume. This allows simultaneous analysis of every droplet in the reaction volume immediately after the start of the reaction cycle and tracking and analysis of the behavior of individual droplets from their nucleation to dissolution.3D‐OH and either the polyanion polystyrene sulfonate (pSS) or polyuridylic acid (pU). The aspartic acid moiety of the peptide precursor can be activated to its corresponding anhydride through the reaction with 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) as the fuel . The microfluidic droplet was captured less than 5 seconds after its creation and analyzed by confocal microscopy through sequential Z‐stack imaging (17.4 seconds per stack). Similar to experiments in bulk, in the first minutes, the droplets grew predominantly through fusion . We also observed that in these cases, almost all of the coacervate‐based droplets were lying at the bottom of the microfluidic droplet . These observations can be explained by gravitationally induced fusion which has been reported previously in confined volumes,k1 and the deactivation constant k4 (Supporting Information). Indeed, when we correlate the nucleation times with the anhydride concentrations predicted by the kinetic model, we find that nucleation occurred at an anhydride concentration of 0.61 mM, independent of the amount of fuel added .There are different methods to determine the total volume of the separated phase, like centrifugation3N‐NH2 as peptide . We observe the same behavior for the dye sulforhodamine B . Those experiments confirmed that diffusivity decreases by increasing the peptide to precursor ratio until no more liquid droplets are formed. Instead, a more solid‐like precipitate is observed. We hypothesize that from around 25 mM EDC, no further polyanion is recruited into the coacervate‐based droplets upon further increasing the fuel concentration. Further increasing the fuel, and thus the amount of anhydride leads to replacing precursor with anhydride molecules in the coacervate‐based droplets. The higher affinity of anhydride for pSS leads to a denser packing of the coacervate‐based droplets and hence a stagnation in the total volume increase. For verification, we conducted fluorescence partitioning experiments with non‐dynamic droplets using Ac‐F(RG)3N‐NH2 as a model peptide for the anhydride. For non‐dynamic coacervate‐based droplets, we observed that the pSS recruitment into the droplets only increased up to 2 mM of the model anhydride and leveled off afterward .Next, we used the active droplets and tested how the fuel, the peptide precursor, and the pSS concentration influenced the maximum total volume of the coacervate‐based phase. The amount of fuel added determined the total volume of coacervate‐based droplets Figure a. HoweveIn contrast, the precursor recruited into the coacervate‐based droplets decreased with increasing model anhydride concentrations . We observed similar total peptide amounts recruiting into the coacervate‐based droplets for the fuel‐driven system for 20 mM and 30 mM EDC . These experiments conclude that the anhydride to precursor ratio in the droplets increased. Thus, more fuel leads to more droplet material, but it also leads to a denser packing of the droplet material.A significant advantage of our experimental setup is that each coacervate‐based droplet can be followed throughout its lifetime. In perspective, droplets prepared in larger containers are impossible to track due to Brownian motion in and out of the imaging area. Thus, we tracked the emergence, motion, fusion, and decay of an entire population of coacervate‐based droplets for the first time. We used automated particle‐trackingThe droplets’ tracking data allowed us to follow the “life” of every individual droplet, which offered insights into the behaviors of a droplet on an individual level. For example, the individual volume of every coacervate is tracked Figure c. In thak4). The calculated volume decay slightly overestimates the measured volume decay, most likely due to a residual activation that is still happening in the system . The correlation between the anhydride hydrolysis and the loss of droplet material was strong even at high EDC concentrations, where droplets become more viscous . We explain this observation because the reduced diffusivity at high EDC concentrations is not constant throughout the reaction cycle but tends to increase again at the end of the cycle. Therefore, coacervate‐based droplets get more liquid towards the end of the reaction cycle when the amount of activated species is reduced, and no diffusion limitation is observed for the dissolution of the coacervate‐based droplets. The decrease in the viscosity and, therefore, the increase in the diffusivity throughout the cycle can be observed by a reduction in the fusion time from 1.5 min at the start of the reaction cycle to less than 0.3 min right before the dissolution of the coacervate‐based droplets occurs . The close resemblance of volume decay and anhydride decay emphasizes that our coacervate‐based droplets are dynamic. The identical shrinkage rate constant also leads to a longer lifetime the bigger the coacervate‐based droplets are, as they can maintain a sufficient size for a more extended period.The primary mechanism of growth in this timeframe is fusion. Coalescence has been reported as the primary mechanism for droplet growth.We can also analyze the average likelihood of fusion for coacervate‐based droplets depending on the EDC concentration . Here, we observe that the fusion rate scales with the EDC concentration in the first minutes. We explain this trend by faster growth in the average volume of the coacervate‐based droplets and the resulting higher likelihood of fusion. For fuel concentrations above 20 mM EDC the fusion rate declines significantly after 5 minutes. In comparison, experiments with fuel concentrations below 20 mM EDC show a more homogeneous fusion rate throughout the reaction cycle. For high EDC concentrations, the rapid decrease in the number of coacervate‐based droplets and their decreased mobility due to their bigger average size leads to a decreased likelihood of droplet fusion.When we followed the life of a single droplet, we found that it underwent three major fusion events between 3 and 12 minutes Figure d. Each oWe successfully imaged the entire life‐cycle of fuel‐driven active droplets enabled by a microfluidic system for droplet formation and analysis. With the method, we found four new behaviors in our active droplets. The droplets nucleate rapidly, but the kinetics of the reaction cycle can tune the nucleation time, i.e., the nucleation time directly follows the rate of anhydride formation. The droplets grow because of fusion, they fuse frequently, and their frequency of fusion at the beginning of the reaction cycle scales with the fuel concentration. However, the droplet's density increases with an increasing amount of fuel added, making the fusion process slower. The droplets decay at roughly the same timepoint, the shrinkage rate constant is independent of their volume and is equal to the hydrolysis rate constant of the activated species.The advantages of studying our droplets in microfluidics combined with a dropspot chamber can be used with other active systems. We anticipate that the method will be applied to study other chemically fueled systems in more detail. We also anticipate that this setup will be handy for evolution‐based experiments where droplets will compete for scarce resources and the future observation of self‐dividing or self‐reproducing droplets systems.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Fucus vesiculosus  are among the best known and well‐studied biogeographic patterns in ecology Wallace . The myrsus Fig. , Capistrsus Fig.  explore sus Fig. . This unPrevious work examining LDG and Rapoport's Rule in seaweeds (not their microbiomes) on European (Atlantic) and the temperate Pacific coasts also revealed mixed results: Atlantic populations displayed increasing species richness toward the tropics, whereas Pacific populations displayed the opposite trend  have shown that rare taxa account for the “core” microbiome of plastic particles collected in different parts of the Atlantic Ocean, and the Baltic and Mediterranean Seas (Scales et al. Whereas a justified aspect of this study was the choice of sample collection in the summer for all sites to account for effects of photoperiod, from a temporal perspective, given that alpha diversity in microbial communities peaks in the temperate areas in the winter (Ladau et al. Fucus vesiculosus. The influence of bacteria on the morphogenesis of macroalgae is a significant aspect to consider in this respect and makes for a fascinating dimension lacking in studies that consider only abiotic substrates. To this end, a striking finding from this research was the discovery of tissue‐specific bacteria. For example, Ilumatobacter_t6062 and Alphaproteobacteria_or_fa_ge_t3536 occurred in holdfasts over all sampling locations, and to a lesser extent, Granulosicoccus_t3260 occurred in all vegetative tips and receptacles but was absent in some of the sampled holdfasts. Although the search for a “core microbiome” using the ASV‐approach was not definitive, these tissue‐specific ASVs suggest a possible symbiotic role for bacteria in host morphology and physiology.Unlike the afore‐mentioned microplastics and other abiotic substrates that float, however, the Capistrant‐Fossa et al. study focused on microbiomes of a hard substrate‐attached biotic host: the model benthic macroalga, Fucusvesiculosus. Whereas the genus Granulosicoccus was a quasi‐core member of the   microbiome of this seaweed, its presence correlated with cooler sea surface temperatures, based on environmental modeling done in the study. This finding has important implications for the geographic range of the host and the yet‐to‐be‐discovered role this quasi‐core microbiome member plays in host health, given future climate warming scenarios.More pronounced in this study, however, were abiotic factors shaping the microbiome of Lastly, the study leveraged unique historical range data for the host to determine that North‐South patterns in host ranges mirrored those of ASVs in the NW Atlantic. Parallel latitudinal microbial community structure and presence of common ASV's across the Atlantic sampling sites highlighted the non‐random nature of the study findings. Untangling host phylogeographic signal from biotic and abiotic influences remains an undertaking for future pursuit.


Rising ocean temperatures are the primary driver of coral reef declines throughout the tropics. Such declines include reductions in coral cover that facilitate the monopolization of the benthos by other taxa such as macroalgae, resulting in reduced habitat complexity and biodiversity. Long‐term monitoring projects present rare opportunities to assess how sea surface temperature anomalies (SSTAs) influence changes in the benthic composition of coral reefs across distinct locations. Here, using extensively monitored coral reef sites from Honduras (in the Caribbean Sea), and from the Wakatobi National Park located in the center of the coral triangle of Indonesia, we assess the impact of global warming on coral reef benthic compositions over the period 2012–2019. Bayesian generalized linear mixed effect models revealed increases in the sponge, and hard coral coverage through time, while rubble coverage decreased at the Indonesia location. Conversely, the effect of SSTAs did not predict any changes in benthic coverage. At the Honduras location, algae and soft coral coverage increased through time, while hard coral and rock coverage were decreasing. The effects of SSTA at the Honduras location included increased rock coverage, but reduced sponge coverage, indicating disparate responses between both systems under SSTAs. However, redundancy analyses showed intralocation site variability explained the majority of variance in benthic composition over the course of the study period. Our findings show that SSTAs have differentially influenced the benthic composition between the Honduras and the Indonesian coral reefs surveyed in this study. However, the large intralocation variance that explains the benthic composition at both locations indicates that localized processes have a predominant role in explaining benthic composition over the last decade. The sustained monitoring effort is critical for understanding how these reefs will change in their composition as global temperatures continue to rise through the Anthropocene. Global warming continues to transform coral reef assemblages throughout the tropics, impacting biodiversity and ecosystem function. Here we identify disparate disruptions between an Indoneisan coral reef and a Honduran coral reef system under climate change over the last 10 years. " cd_value_code="text In the Honduran reef systems, coral cover has been stable between sites  in the center of the Coral Triangle, harboring over 390 species of hard coral, and 590 fish species across the 50 k hectares of coral reefs , the reef flat (~2–3 m), and the reef slope (~12–15 m). Using a variety of depths at the Honduras sites, a variety of reef zones at the Indonesia sites, which encompasses a wide depth range of shallow reefs (2–15 m), allows us to generalize the shallow reef benthic compositions at these sites. Categories for the benthic classifications are identified in Table 2.3Heat stress was quantified as SSTAs measured in °C over the last 52 weeks preceding surveying at a 5‐km resolution, extracted from Coral Reef Watch (CRW) v3.1 5‐km product suite  for each year from 2012 to 2019. Benthic surveys where SSTA data were not available were excluded from the analysis, leaving a total of 1088 surveys over the 8‐year time period. While there are potential issues  with a quasi‐Poisson distribution was used to determine whether the average SSTA was increasing through time at each location, as data were Poisson‐distributed and over‐dispersed.2.4.1get_prior” function in the package “brms,” which specifies priors for the beta coefficients, intercept, and random of effects of each model  we used Redundancy analysis (RDA) from the “vegan” package  from the “vegan” package  and the Honduras location .The average SSTAs over the last decade were highly divergent between the Honduras and Indonesia locations, with peak SSTAs preceding the survey years of 2016 and 2017 for Honduras  and SSTA at Indonesia 5.9%) and Honduras (4.7%) location. However, when adding site in the RDA model  explained the largest proportion of variance for the benthic composition at both the Indonesia and Honduras locations, indicating fine‐scale variability as a key factor in explaining the benthic composition of these coral reefs.4.1Elevated sea surface temperatures appear to predict coverage of benthic components at the Honduras location only, and not the Indonesia location, indicating that the Honduran reefs surveyed in this study are more susceptible to compositional change under marine heatwaves. This can be seen with the increase in bare rock coverage at the Honduras location and a decrease in sponge coverage in association with SSTAs Figure  but noteIn contrast to the effects of SSTAs, temporal patterns of variation predicting the benthic composition of reefs at both the Indonesia and Honduras location are prominent. These temporal patterns, which predict compositional changes at the Indonesia location have been previously recorded for sponges, which showed the strongest temporal increase of all the benthic components. This stark increase is most strongly related to the Sampela site where high sedimentation has driven sponge dominance ; data curation ; formal analysis ; investigation ; methodology ; project administration ; resources ; software ; validation ; visualization ; writing – original draft ; writing – review and editing . Dan A. Exton: Conceptualization ; methodology ; supervision ; writing – review and editing . Jaimie T. A. Dick: Supervision ; writing – review and editing . Joseph Oakley: Visualization ; writing – review and editing . Jamaluddin Jompa: Data curation ; project administration ; writing – review and editing . Daniel Pincheira‐Donoso: Conceptualization ; investigation ; resources ; supervision ; validation ; writing – original draft ; writing – review and editing .All authors contributed to the manuscript writing and revisions and declare no conflict of interest.Figures S1‐S14Click here for additional data file.


TTC21B encodes the protein IFT139, a critical component of the retrograde transport system within the primary cilium. Biallelic, pathogenic TTC21B variants are associated with classic ciliopathy syndromes, including nephronophthisis, Jeune asphyxiating thoracic dystrophy, and Joubert Syndrome, with ciliopathy‐spectrum traits such as biliary dysgenesis, primary ciliary dyskinesia, and situs inversus, and also with focal segmental glomerulosclerosis. We report a 9‐year‐old male with focal segmental glomerulosclerosis requiring kidney transplant, primary ciliary dyskinesia, and biliary dysgenesis, found by research‐based exome sequencing to have biallelic pathogenic TTC21B variants. A sibling with isolated heterotaxy was found to harbor the same variants. This case highlights the phenotypic spectrum and unpredictable manifestations of TTC21B‐related disease, and also reports the first association between TTC21B and heterotaxy, nominating TTC21B as an important new heterotaxy gene. Patient's 17‐month‐old sister was subsequently evaluated clinically and was confirmed to carry the same pathogenic variants. Comprehensive metabolic panel and abdominal ultrasound were performed and were normal. There are no respiratory symptoms at this time. DNA could not be obtained for genetic testing for the deceased brother with heterotaxy‐spectrum disease nor from the sibling with anencephaly. All living siblings were tested for both familial variants and found to carry only the splice variant (unaffected brother and sister) or neither familial variant (brother with Wilms tumor).Patient was enrolled in a gene discovery research study through the Center for Applied Genomics (CAG) at The Children's Hospital of Philadelphia (IRB protocol # 16‐013278). Exome sequencing was performed, and revealed biallelic variants in 2TTC21B encodes the protein IFT139, a critical component of the retrograde ciliary transport system required for proper formation and function of the primary cilium. Its deficiency is associated with ciliopathy‐spectrum disease as well as FSGS  in European and North African populations (Davis et al., in trans with a nonsense or frameshift null allele is sufficient to cause a syndromic ciliopathy, and that the combination of nonsense and splice variants invariably produces severe disease such as Jeune asphyxiating thoracic dystrophy or early nephronophthisis with prominent extrarenal manifestations (Davis et al., TTC21B variants and increasing disease severity: siRNA‐mediated TTC21B knockdown in mIMCD3 cells results in severe structural ciliary differences, and morpholino‐mediated ttc21b knockdown in zebrafish causes gastrulation defects (Davis et al., in trans with a splice‐altering variant likely explains their disease severity and prominent extrarenal manifestations.More than 50 disease‐causing TTC21B‐related disease, heterotaxy‐spectrum disease is a new association, and TTC21B is not included in commercial heterotaxy gene panels. Interestingly, heterozygous copy number variations involving TTC21B have been identified in a cohort of heterotaxy and congenital heart disease patients, supporting a role for this gene in the pathogenesis of heterotaxy (Cowan, TTC21B is a novel component of the nodal cilium that facilitates left–right patterning during embryogenesis.Though situs inversus has been reported in y Cowan, . It is tTTC21B variants, as TTC21B is associated with neural tube defects. Additionally, the infant who died at 5‐weeks of age with complex congenital heart disease, midline liver, and polysplenia also likely carried both familial variants. Our proband has a structurally normal heart, but has kidney, liver, and lung disease, whereas his sister has no evidence of liver, kidney, or lung disease but has severe cardiac malformations. It is possible that she will go on to develop these extracardiac manifestations, and that her heart disease is related to additional variants in other ciliary genes exacerbating her phenotype, which is a well‐described feature of ciliopathy syndromes (Shaheen et al., The etiology for the phenotypic diversity in this family is unclear. Although not able to be genetically confirmed, it is likely that the fetus affected by anencephaly carried the familial biallelic TTC21B variants and heterotaxy‐spectrum disease as well as an example of extremely divergent ciliopathy phenotypes within a single family ranging from anencephaly to heterotaxy to multiorgan syndromic ciliopathy, and suggest that TTC21B be considered on the differential for children presenting with heterotaxy‐spectrum disease and situs abnormalities.Here we present a novel association between biallelic The authors declare no conflicts of interest.Table S1 Ciliopathy genes specifically interrogated in proband on exome sequencingClick here for additional data file.


Severe atherosclerosis of the ascending aorta (SAA) in patients undergoing surgical revascularization by coronary artery bypass grafting (CABG) is becoming an increasing problem as more elderly patients are diagnosed with coronary artery disease. Strokes and other neurologic insults are common complications in this group, with devastating impacts on outcomes and prognoses.Early detection of the atherosclerotic aorta and the application of a stroke prevention protocol will reduce the risk of stroke in patients with SAA.In 2012, we adopted a protocol devised to preemptively detect and manage patients suspected of having SAA. From the time of the application of the protocol, we compared the immediate and late outcomes of CABG in SAA in the 8 years preceding the protocol in a “control” group (30 patients) and in the 8 years following the protocol in a “brain” group (69 patients).More patients with SAA were detected after the initiation of the protocol. They had significantly more history of stroke, renal dysfunction, and left main coronary disease. The percutaneous coronary intervention was utilized more after the protocol (26% vs. 7%) and there was far less utilization of replacement of the ascending aorta (12% vs. 37%). Postoperative stroke rates were significantly less after the protocol (2% vs. 18%), with an almost twofold reduction in stroke associated with SAA even after risk adjustment. The composite endpoints of cardiac death, nonfatal myocardial infarction, and stroke were significantly reduced after initiating the protocol at a median of 2.3 years from the time of revascularization.Early detection of SAA and individualized therapeutic strategies for revascularization is effective in reducing athero‐embolic brain injury and are associated with better prognosis. The revascularization strategy and planned surgical techniques will need to be custom‐tailored to the individual. This includes opting for percutaneous coronary intervention (PCI) rather than CABG, hybrid revascularization (PCI + CABG), performing CABG without cardiopulmonary bypass (CPB) support (off‐pump coronary artery bypass [OPCAB]), performing CABG under CPB support and fibrillatory arrest with intermittent reductions or interruptions in CPB flow to construct proximal anastomoses (fibrillatory arrest coronary artery bypass [FACAB]), and performing CABG after graft replacement of the atherosclerotic aorta (aorta replacement coronary artery bypass [ARCAB]). The purpose of this study was to conduct a retrospective cohort review of the immediate and late outcomes of isolated CABG in patients with SAA before and after the implementation of the preemptive diagnosis and management measures for SAA.2In an effort to reduce the incidence of stroke post‐CABG in patients with SAA, our local heart team initiated a stroke prevention protocol starting in July 2012. This was conveniently labeled the “brain‐before‐heart” revascularization strategy. The strategy is predicated on increasing awareness of ascending aortic atherosclerosis to prevent the devastating complication of perioperative stroke. This was conducted through a stepwise process involving an initial risk stratification, preoperative noninvasive imaging, individualized revascularization planning, intraoperative assessment of the ascending aorta, and the use of alternative surgical techniques guided by the site and burden of atheroma  scan of the chest to detect signs of calcification in the thoracic aorta. Carotid doppler ultrasound (US) is done routinely before CABG.The extent and burden of atheroma in the ascending aorta are subjected to careful assessment and preoperative planning by the heart team to decide whether to pursue surgical revascularization, PCI, or a hybrid approach. All patients undergoing CABG are subjected intraoperatively to epiaortic US (EAU) scanning and careful digital palpation of the ascending aorta to choose a soft spot for cannulation. Depending on the site and burden of atheroma in the ascending aorta, the surgical team can tailor an individualized surgical strategy involving any of the following surgical techniques: routine CABG with cross‐clamp, OPCAB, FACAB, or ARCAB.Patient‐specific information was censored and individual consent was waived using the time of the implementation of the brain‐before‐heart stroke prevention protocol as a pivotal set‐point (July 2012). The selected cohort of patients with SAA was divided into a control group C, comprising patients referred for CABG from January 2004 to June 2012, and a study group labeled the brain group B, comprising patients referred for CABG from July 2012 to December 2019. All surgical revascularization was performed via a median sternotomy with bypass conduits selected according to the preference of the operating team. These included at least one left internal mammary artery (LIMA) and/or a combination of saphenous vein grafts (SVG), right internal mammary artery (RIMA), and radial artery (RA).EAU scanning of the ascending aorta was adopted only after the implementation of the stroke prevention protocol. Before this, the assessment of atheroma in the ascending aorta was done by digital palpation. Our protocol is as follows: Depending on the severity of ascending aorta disease and the site of the atheroma, a decision is made to choose between using OPCAP (with or without “no touch”) or utilizing CPB via the heart–lung machine. Aortic “no touch” revascularization strategy was mainly focused on grafting the LIMA to the left anterior descending and harvesting the RIMA as a free graft to construct a proximal Y anastomosis to graft the next largest target on the left system. A left RA or SVG was used to supplement the left system or the right coronary targets as an end‐to‐side graft from the mammary. For CPB, the cannulation sites can be in the distal ascending aorta, aortic arch, femoral artery, or axillary artery. If a safe disease‐free spot for the aortic cross‐clamp is available, then the ascending aorta is cross‐clamped and routine CABG is done with cold antegrade cardioplegia for myocardial protection. If the ascending aorta cannot be safely cross‐clamped, the surgical team is left to decide between doing FACAB or ARCAB. FACAB is done by cooling the systemic temperature to 28°C, fibrillating the heart by applying a shock using a fibrillatory device, then constructing all the distal anastomoses, and then performing the proximal anastomoses with the head down under low or short bouts of no flow in the aortic canula. If the ascending aorta has a large anterior atheroma that prevents proximal construction, then the systemic temperature is cooled down to 18°C and the ascending aorta is replaced under deep hypothermic circulatory arrest with a Dacron tube graft. The tube graft is then cross‐clamped and routine CABG is performed.Perioperative outcomes were assessed by a trained database manager who was unaware of the goals of this study. Death was defined as death at any time during the index hospitalization. Myocardial infarction (MI) was defined as a fatal or nonfatal MI as evidenced by new Q waves on the electrocardiogram or by a peak in creatine kinase isoenzyme MB level greater than 50 IU/L that represented more than 7% of the total creatine kinase. Echocardiographic corroboration of a new MI was routinely sought but was not necessary for the diagnosis. For the purpose of this manuscript, stroke was defined as signs and symptoms of neurological deficit due to cerebrovascular causes that persist beyond 24 h or are interrupted by death within 24 h. Neurological deficits that resolved completely within 24 h were labeled as transient ischemic attacks (TIAs). Strokes were confirmed by a staff neurologist and CT scans. Renal insufficiency was defined as having a decrease in the calculated GFR to less than 60 ml/min. Completeness of revascularization was determined by successfully grafting all diseased coronary distribution territories. Renal failure was defined as the requirement for dialysis. Smoking was defined as a current history of smoking, or cessation within the 3 months preceding surgery. Hypertension, dyslipidemia, diabetes mellitus, and peripheral vascular disease are referred to medically or surgically treated conditions. As of the first quarter of 2020, the follow‐up was 98% complete. Crude survival was determined by matching the unique national identification number for each patient in the series with the national death registry. The cause of death was determined by reviewing the death certificate for those who matched in the registry. Patients who were still alive were directly contacted after obtaining permission at the time of the inquiry. Primary cardiac death was determined if the cause of death was acute coronary syndrome, cardiogenic shock, congestive heart failure, valvular heart disease, death during a cardiac procedure, or sudden cardiac death. History of stroke, TIA, and nonfatal MI were determined at the time of follow‐up inquiry by the study team.n tables and the two‐group comparisons were made using the chi‐square test or Fisher exact test for categorical data. Continuous variables were tested using the Student t‐test or Mann–Whitney U‐test. The crude odds ratios (ORs) with 95% confidence intervals (CIs) were estimated using univariate logistic regression, and adjusted ORs with 95% CIs were estimated using multiple logistic regression analysis. Crude survival and event‐free survival curve comparisons were done using the log‐rank test. The results were expressed as hazard ratios (HRs), 95% CIs, and p‐values. A two‐tailed p‐value < 0.05 was considered significant for all statistical tests.Statistical analysis was performed using SPSS 21.0 software (SPSS Inc.). Categorical data were summarized as absolute numbers and percentages. Numeric data were summarized as the mean and standard deviation (SD) or median and interquartile range. The tested variables were grouped in 2 × 3A total of 2100 consecutive patients were referred for isolated CABG within the study time frame (2004–2019). Of those CABG patients, 902 (45%) had CABG before the implementation of the protocol, and 1198 (57%) patients had CABG after implementation. A significantly larger number of patients had a screening with chest CT before surgery and EAU intraoperatively. This resulted in a significantly higher rate of detection of ascending aorta calcification and atheroma after the implementation of the protocol  who were labeled as having SAA mandating re‐evaluation of the revascularization strategy. This cohort was divided based on the time of surgery into 30 patients in group C (30.3%) and 69 patients in group B (69.7%).p = 0.037). The B group also had more patients with chronic renal insufficiency  and more patients who presented with left main coronary artery stenosis . The surgical risk scores (EuroSCORE II) of the two groups were similar: 2.91% versus 2.34% in B and C, respectively (p = 0.670).Demographic analysis Table  revealedp = 0.027). Also, the use of ARCAB in the B group declined significantly with the adoption of the protocol  . Perioperative stroke and TIA were more pronounced in the C group when compared with the B group . With the exception of three patients in the C group who required dialysis for acute kidney injury, all other immediate surgical outcomes were comparable (Table p = 0.034). This association remained significant after adjusting for age, history of stroke, carotid stenosis, peripheral vascular disease, low ejection fraction (ejeciton fraction < 40%), and left main coronary disease .The patients who had surgical revascularization (20 patients were excluded from this outcome as they had PCI) in the B group had more multiple arterial grafts (38% vs. 13%), and more complete revascularization    from the time of revascularization was not statistically significantly different between groups B and C ; significantly more PCIs were done in the B group; more hybrid revascularization was done in the B group; and surgeons became more comfortable performing CABG with cross‐clamping after mapping the ascending aorta with EAU. The use of FCAB remained low for both groups.One big advantage of the preemptive approach in SAA is the ability to reduce the element of undesirable surprises during surgery. As with all our B group subjects, Park et al.All the surgical techniques used in this study have scientific merit in mitigating the effects of ischemic stroke and poor survival in SAA, but it is the application of our new protocol and algorithm that we tested against the operative outcomes and event‐free survival. We know of no study conducted on patients with SAA that is comparable to the current study. Our operative results clearly show that, since the application of the new protocol, we observed better use of arterial grafts (2 instead of 1), higher complete revascularization (96% vs. 79%), and more importantly, significantly fewer perioperative strokes/TIAs (2% vs. 18%), even after adjusting for other risk factors associated with stroke and cerebrovascular disease. The clinical endpoints of cardiac death, nonfatal MI, and stroke/TIA for the median follow‐up of 2.3 years clearly favor the outcomes in the patients who had revascularization under the new protocol in our cohort. In a long‐term follow‐up study on 1957 patients over 50 who underwent cardiac surgery, Davila‐Roman et al. found that over the period of 7 years after surgery, the patients who had “severe” atherosclerosis of the ascending aorta based on EAU criteria had a greater than 3‐fold increase in the incidence of both neurologic events and mortality compared with those with less severe atherosclerotic disease.This study is unique in the way it analyzed the immediate and mid‐term impacts of applying a stroke prevention protocol for detecting and managing patients with SAA. The revascularization strategy was individualized and based on an algorithm that uses pre‐ and intra‐operative imaging modalities. Despite this, our findings are limited by a number of factors. The study groups were managed and treated in different time periods. Although the time gap is not big, nonmeasurable subtleties such as an improved learning curve, better equipment, and access to more sensitive imaging technologies can account for better surgical results and outcomes. No distinction was made between the “soft” and calcific atheromas in the ascending aorta as the earlier can only be detected by EAU and has a higher propensity to cause stroke. The study's retrospective nature and single institution experience are inherent limitations. Determining the cause of death solely from death certificates can be inaccurate at times. Reporting nonfatal events based on verbal communication is subjected to recall bias and inaccurate reporting. The late outcomes of PCI as an alternative to surgical revascularization in SAA were not specifically analyzed in this study, due to the small sample size.In conclusion, this study is an attempt to evaluate practical measures for coping with SAA in the increasing number of patients who need surgical revascularization. Early detection and individualized therapeutic strategies play an important role in reducing the devastating neurologic insults that can result from manipulating a diseased ascending aorta during surgery.Rakan I. Nazer conceived the study, helped plan the methodology, helped collect the data, and wrote the manuscript. Ali M. Albarrati helped plan the methodology, helped collect the data, did the analysis, and reviewed the manuscript.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


An Austrian organic dairy sheep farm experienced cases of recumbency and sudden deaths in 3‐ to 4‐week‐old lambs. Two animals were subjected to thorough clinical and pathological investigations. Pathohistological analysis identified severe nonsuppurative myelitis and mild nonsuppurative encephalitis. A reverse‐transcription quantitative PCR (RT‐qPCR) assay for the recently discovered ovine picornavirus causing comparable lesions scored negative. By next‐generation sequencing‐based metagenomics, a nearly complete genome of a novel enterovirus could be detected and assembled. In situ hybridization using a specifically designed probe revealed robust signals in affected motoneurons of the spinal cord suggesting a causative role of the novel virus. Sample preparation was performed as described previously  or polyprotein amino acid sequences derived thereof were aligned using MAFFT  in combination with the Agencourt RNAdvance Tissue Kit (Beckman Coulter) and a KingFisher Flex instrument (Thermo Fisher Scientific). From the initial draft sequence, a reverse‐transcription quantitative PCR (RT‐qPCR) assay was designed. As a positive control, a gBlock oligo (Integrated DNA Technologies) was used. As RT‐qPCR was performed with a SensiFAST Probe No‐ROX Kit (Meridian) according to the manufacturer's instructions. The PCR was run as a duplex assay containing an internal control assay as described  was performed according to a previously published protocol  did not show any signals.The detection of viral signals in the affected motoneurons confirms the presence of specific viral RNA in the respective lesions and indicates that the newly discovered enterovirus is most likely the causative agent of the associated clinical signs.By RT‐qPCR, viral RNA was found in all examined spinal cord samples and in caudal brain areas, which corresponds well to the ISH results. In the majority of extraneural tissues, no viral RNA was identified; a low amount of viral RNA was detected in a liver, muscle, and intestinal sample of one animal, each Table . In bothIn the last decade, particularly NGS‐based metagenomics contributed to significant progress in the elucidation of previously elusive causes of nonsuppurative encephalomyelitides in many animal species. The most outstanding achievements are the identification of astroviruses as causative agents in numerous species  are very similar. The viruses are widespread within their host species and the resulting herd immunity prevents the viruses from spreading from the intestine to the nervous system. This is the reason why enterovirus‐associated cases of myelitis are quite rare. Nervous system manifestation occurs in individuals with immunity gaps, either due to failure of colostrum uptake or at the time of weaning, when maternal immunity wanes and active immunity is not yet fully developed. Also, in the cases described here, a lack of colostrum uptake was evident in at least one lamb. Still, it cannot be ruled out that further lambs received insufficient colostrum since the lambs usually stay with their mothers for 5–10 days, and feed uptake is solely visually monitored and not further verified.Taken together, this report describes a novel enterovirus which may be common in sheep and seems to be able to cause myelitis with severe motoneuron damage in colostrum‐deprived lambs. Thus, sheep farmers should be instructed that colostrum uptake has to be secured in order to provide adequate immunity to these potentially deleterious viral agents.The authors declare no conflict of interest.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The authors confirm that no ethical approval was required as this work was carried out according to the rules pertaining to the clinical and diagnostic services of Vetmeduni Vienna.


Bacteriophage (phage) therapy is re‐emerging a century after it began.Activity against antibiotic‐resistant pathogens and a lack of serious side effects make phage therapy an attractive treatment option in refractory bacterial infections.Phages are highly specific for their bacterial targets, but the relationship between in vitro activity and in vivo efficacy remains to be rigorously evaluated.Pharmacokinetic and pharmacodynamic principles of phage therapy are generally based on the classic predator–prey relationship, but numerous other factors contribute to phage clearance and optimal dosing strategies remain unclear.Combinations of fully characterised, exclusively lytic phages prepared under good manufacturing practice are limited in their availability.Safety has been demonstrated but randomised controlled trials are needed to evaluate efficacy. Shigella enteritis (bacillary dysentery) in 1919, in the first recorded description of phage therapy. In the years that followed, phages were used with varying success by d'Herelle and others for a variety of serious infections, including staphylococcal bacteraemia, typhoid fever and osteomyelitis,Caudovirales)  . Phages Box 1A) and electron micrograph of bacteriophage (Myoviridae) with 1% uranyl acetate negative staining, with size marker (B). Bacteriophage preparations were dialysed against 0.1 M ammonium acetate in dialysis cassettes with a 10 000 membrane molecular weight cut‐off (Pierce Biotechnology), negatively stained with 2% uranyl acetate, and visualised using transmission electron microscopy (TEM). TEM was conducted at the Westmead Scientific Platforms  on a Philips CM120 BioTWIN (Thermo Fisher Scientific) transmission electron microscope at 100 kV. Images were recorded with a SIS Morada digital camera using iTEM software (Olympus Soft Imaging Solutions).Schematic  in the 1970s of high dose phage for the treatment of cholera found tetracyclines to be more effective.Escherichia coli infection, and only half of these were phage‐susceptible in vitro. More recently, a phase 1 double‐blind RCT of a topical bacteriophage cocktail in chronic venous leg ulcers found no safety concerns,Pseudomonas with standard care with silver sulfadiazine, found that significant reductions in bacterial counts took longer in the phage group and that the silver sulfadiazine group had higher treatment success than the phage group. The PhagoBurn trial suffered from instability of the phage preparation, but showed that phage‐susceptibility of Pseudomonas isolates in vitro is crucial to eventual clinical success, with susceptibility rates of 89% in successful cases compared with 24% in clinical failures in the phage therapy arm  experience in humans is less impressive . The larrapy arm .Box 2Phages usually exhibit a high degree of host specificity and are most easily sourced from the habitat of their usual bacterial hosts. Phages specific for clinically relevant bacteria may be readily sourced from human and animal sources, hospital wastewater, and environmental soil and water. Their reproduction is dependent on the bacterial host, either integrated into the bacterial genome as a prophage or acting in a purely parasitic manner , the latBox 3A), the phage DNA (purple) is classically either reproduced and packaged as new virions at the expense of the cell  or reproduces with the host DNA .After infection  susceptible isolate — productive infection and formation of individual plaques detectable at serially diluted phage lysate — and (B) resistant isolate — single plaques absent and early bacterial lysis present only at high concertation of phages. Liquid culture‐based system: (C) across a gradient of multiplicity of infections (MOIs) against eight bacteria on a single 96‐well plate. There are seven MOIs tested from highest to lowest (left to right). Column eight and nine are phage‐only and bacteria‐only controls. (D) Bacterial growth kinetics in presence of phage at differing MOIs.Phage‐susceptibility testing of bacterial isolates with the standard double‐layer method:The plaque assay is time‐consuming and operator‐dependent, with automated methods still in their infancy. Liquid culture‐based systems that continuously monitor bacterial growth in the presence of bacteriophage in a standard 96‐well plate format , C may bAcinetobacter infection when antibiotics were failing.The development of in vitro bacterial resistance was a prominent feature of phage therapy for a high burden multidrug‐resistant input)A good understanding of the non‐linear kinetics of bacteriophage distribution in blood and tissues is necessary to maximise efficacy of future therapeutic protocols.1–105  colony‐forming units per mL of blood in severe sepsis,9 plaque‐forming units (PFU) into the human blood volume (~ 5 L) is expected to yield an MOIinput over 200.Numerous methods of bacteriophage delivery have been explored, including topical, inhalational, oral and injectable . When administering phages orally, concern has been raised regarding recombination between modular phage genomes in the gut, although there has been little evidence from trials to date to resolve this one way or the other.The paradoxical persistence of a narrow host range for phages is not fully understood, given the presumed ecological advantagesE. coli have added additional barriers (curli fibres) as a collective protection.Urine concentration of bacteriophage may be dose‐dependant,Optimised preparation of phage combinations (cocktails) for use in humans requires well characterised bacteriophages, a good understanding of the bacterial target population, and highly effective purification protocols to avoid inflammatory responses to contaminating residual bacterial endotoxin and protein.9–1010 PFU/mL) with unique but overlapping host ranges to guarantee lysis of the bacterial target and clinically relevant variants.General recommendations regarding the standardisation of key components of phage therapy have been published,Phage therapy may be presented as a discrete quality‐assured formulation either as several phages admixed to provide a broad spectrum of activity in a single medicine for a specific infection  clinical trials in humans means that phage therapy still has the status of an old technology based on anecdotal evidence, not much different to traditional medicines. Nevertheless, there are some successful phase 1 and 2 studies, including in Australia, which suggest the potential of phage therapy as an alternative or adjunct to antibiotics.S. aureus sepsis and infective endocarditis were treated twice daily for 2 weeks with intravenous AB‐SA01  — a GMP‐quality cocktail product with three bacteriophages — as adjunct to standard care.Recent studies in Australian centres of both intranasal instillationNatural bacteriophages are defined as investigational drugs by the Therapeutic Goods Administration in Australia. The regulatory side of bacteriophage therapy is beyond the scope of this review, but most medical communities are both sceptical and curious.To progress, we need at least to guarantee the availability of efficient phage susceptibility testing and of preparations that are safe for intravenous administration, define optimal dosing, and effectively monitor phage–bacteria–human host interactions and any important collateral effects on other members of the microflora.Realistically, application of therapeutic phages alone may never completely replace chemical antimicrobials as the standard of care. For now, it is reasonable to define phage therapy as a promising rescue therapy that has been associated with some spectacular resultsNo relevant disclosures.Not commissioned; externally peer reviewed.


Melatonin, the major secretion of the pineal gland, has beneficial effects on the cardiovascular system and might advantage heart failure with reduced ejection fraction (HFrEF) by attenuating the effects of the renin–angiotensin–aldosterone and sympathetic system on the heart besides its antioxidant and anti‐inflammatory effects.We hypothesized that oral melatonin might improve echocardiographic parameters, serum biomarkers, and a composite clinical outcome  in patients with HFrEF.A placebo‐controlled double‐blinded randomized clinical trial was conducted on patients with stable HFrEF. The intervention was 10 mg melatonin or placebo tablets administered every night for 24 weeks. Echocardiography and measurements of N‐terminal pro‐B‐type natriuretic peptide (NT‐Pro BNP), high‐sensitivity C‐reactive protein, lipid profile, and psychological parameters were done at baseline and after 24 weeks.p = .044). Moreover, the melatonin group had a significantly better clinical outcome , quality of life , and New York Heart Association class  at the end of the trial. Other studied outcomes were not significantly different between groups.Overall, 92 patients were recruited, and 85 completed the study . Serum NT‐Pro BNP decreased significantly in the melatonin compared with the placebo group (estimated marginal means for difference [95% confidence interval]: 111.0 [6.2–215.7], Oral melatonin decreased NT‐Pro BNP and improved the quality of life in patients with HFrEF. Thus it might be a beneficial supplement in HFrEF. Several effective medications have been developed which reduce symptoms and increase the survival of these patients.Melatonin is mainly secreted by the pineal gland with the primary role of coordinating the circadian rhythm. Recently, it has been shown that melatonin has beneficial effects on the cardiovascular system by its cytoprotective and antioxidant properties and by ameliorating mitochondrial dysfunction and regulating the endocrine system, such as SNS and RAAS.22.1ClinicalTrials.gov (NCT03894683).This report adheres to the Consolidated Standards of Reporting Trials (CONSORT).The MeHR trial was a double‐blinded randomized placebo‐controlled clinical trial with two parallel arms, conducted from January 2019 to August 2020 in outpatient Chamran Cardiology Clinics. The design and methods of the study are fully described elsewhere.2.2Participants were selected from patients with a definite diagnosis of HFrEF (left ventricular ejection fraction [LVEF] < 40%) who were symptomatic (New York Heart Association [NYHA] class II or III) and regularly attended a specialist in mentioned clinics. In addition, they had to be clinically stable for at least 3 months and on essential drugs for HFrEF according to the 2016 ESC guidelines, that is, receiving maximum tolerated doses of an angiotensin‐converting enzyme inhibitor/angiotensin‐receptor blocker, a beta‐blocker, and spironolactone/eplerenone unless contraindicated, and diuretics if needed. The medications and other indicated treatments were prescribed by the specialists who referred the patients for participating in the study. Patients suspected to need device therapy (pacemaker or implantable cardioverter‐defibrillator) within the next 6 months were not enrolled.Details of eligibility criteria have already been declared.2.3The intervention was 10 mg melatonin or placebo tablets, identical in shape and physical properties made by Sepid Teb (Sepid Teb Co.) and prescribed for at least 24 weeks, one tablet at bedtime. The drug was delivered to the patients in identical boxes containing 100 tablets and they were requested to return the unconsumed pills in the scheduled follow‐ups. Patients' adherence and the adverse effects were assessed regularly by telephone calls, and patients attended the study center after 12 and 24 weeks of intervention for outcome evaluation and pill count to objectively evaluate adherence to the intervention.2.4The primary outcomes of the study were variations in LVEF and left ventricular end‐diastolic diameter (LVEDD), measured by echocardiography at Week 24 relative to the baseline, and changes in serum levels of NT‐Pro BNP at Week 24. Also, a compound clinical score was calculated at Week 24 for each patient, composed from all‐cause mortality, any hospitalization for HF exacerbation, and changes in quality of life measured by the Minnesota Living with Heart Failure Questionnaire (MLHFQ).Clinical events were collected at baseline and during the study. Hospitalization was recorded from patients' medical documents, and mortality was recognized by verbal autopsy. An expert blinded committee of three cardiologists confirmed the events by evaluating the patient's medical records.Other outcomes analyzed in this report were serum levels of high‐sensitivity C‐reactive protein (hs‐CRP), lipid profile, and liver and renal function tests measured at baseline and end of the intervention (Week 24), as well as results of questionnaires measuring sleep quality , anxiety (the trait part of Spielberger Anxiety Inventory), and depression (Beck Depression Inventory‐II).Transthoracic 2D‐color echocardiography was done by a single specialist, blinded to the study groups, via the Simpson biplane method with concomitant electrocardiography monitoring . The intraobserver variability was determined by reassessing the stored images of 30 random baseline echocardiography measurements at least 1 month later. The intra‐class correlation coefficient of .97, .98, and .95 was obtained for LVEDD, left ventricular end‐systolic diameter (LVESD), and LVEF, respectively.A 5 ml fasting blood sample was drawn from each participant at baseline and Week 24. Routine blood tests  were performed by an autoanalyzer (Hitachi 902), and serum aliquots were stored at −80 for further analysis. Serum NT‐pro BNP was measured by a commercial enzyme‐linked immunosorbent assay (ELISA Kit) (Bioassay Technology Laboratory) according to the kit instruction. Serum hs‐CRP levels were determined by immunoturbidimetry (Pars Azmoon Inc).from best to worst).Validated Persian versions of relevant questionnaires were used in this study. The MLHFQ is a widely used health‐related quality of life questionnaire for patients with HF with acceptable validity.2.5T‐test/Mann–Whitney U test or χ2 test were used to compare baseline variables and the rate of adverse events between groups. The primary and secondary outcomes were analyzed using analysis of covariance, all adjusted for baseline values and other covariates as appropriate for the examined outcome. The missing data met the assumptions of missing completely at random. An intention‐to‐treat analysis approach was employed by including all cases with at least one measurement. A statistically significant level of less than 0.05 was acceptable for two‐sided tests.The sample size of 90 was calculated to find a 5% difference in LVEF between groups at the significance level of 0.05 (two‐tailed) and the power of 90%, based on a study by Garakyaraghi et al.33.1Figure Pill count showed a high adherence to the intervention in participants who completed the study, ranging from 80% to 100% (97.8% in the melatonin and 98.7% in the placebo group).3.2p = .044). Serum levels of hs‐CRP were not different between groups at week 24 . Table The echocardiographic parameters of LVEF, LVEDD, and LVESD were not affected by the intervention after adjustment for the age and HF type (ischemic vs. nonischemic). The estimated marginal means (EMMs) of serum NT‐Pro BNP at Week 24 was significantly lower in the melatonin group relative to the placebo group after adjustment for predefined parameters of age, sex, body mass index, and estimated glomerular filtration rate .During the study, 12 hospitalizations occurred among patients who completed the study, of which 3 were because of HF exacerbation (one from five in melatonin and two from six in the placebo group). Quality of life measured by MLHFQ improved at least five scores in 39 patients (22 [52.4%] in the melatonin and 17 [39.5%] in the placebo group) and worsened at least five scores in 13 persons (6 [14.3%] in the melatonin and 7 [16.3%] in the placebo group) after 24 weeks. The composite clinical outcome was significantly better in the melatonin than in the placebo group  and treatment and time interaction (p < .001) (EMM [95% CI] for difference: 5.8 [0.9–12.5]) in favor of the melatonin group. The NYHA class of the patients measured before and after 24 weeks of intervention did not change in 75 patients, improved in 5 patients (four in the melatonin group), and worsened in 5 patients (four in the control group). All patients were categorized in NYHA classes II and III after the intervention. The logistic regression adjusted for baseline values of NYHA class, age, and sex showed a significant effect for intervention on the NYHA class of the patients in the melatonin group .The MLHFQ was measured at Week 12, too; the generalized estimating equation model with three time‐points as within‐subject variable and age and sex as covariates showed a significant effect for treatment , however, they did not reach statistically significant difference. In addition, the renal function tests were not significantly affected by the treatment during the study. The aspartate transaminase and alanine aminotransferase levels were somewhat improved in the melatonin group, but they did not reach a statistically significant difference (Table 3.6p = .144). One patient in each group left the study because of skin eruptions after consumption of the drug. No serious adverse effect was seen during the study.From 92 allocations, three did not receive the intervention (two in the melatonin group and one in the placebo group). Throughout the study, 13 of 89 patients reported at least one type of drug‐related adverse effect Table . However4This study showed that oral melatonin supplementation lowered the serum levels of NT‐Pro BNP in patients with HFrEF and improved their disease‐specific quality of life. Moreover, patients who received melatonin had a better composite clinical outcome, and their overall NYHA class improved after 24 weeks of intervention. However, melatonin did not affect the echocardiographic parameters of the patients.In a study, Garakyaraghi et al.Melatonin decreased serum NT‐Pro BNP compared to placebo, but it did not affect hs‐CRP as an inflammatory marker. The positive effect of melatonin on NT‐Pro BNP level might be due to the synergistic effect of melatonin with angiotensin‐converting enzyme inhibitor/angiotensin‐receptor blocker and beta‐blockers in the modulation of RAAS and SNS.Melatonin's ability to improve mitochondrial dysfunction might alleviate myocardial function and decrease overload. In experimental studies, melatonin administration inhibited the opening of mitochondrial permeability transition pores in myocytes of the aging ischemic hearts and reversed the functional and biochemical changes in ischemic HF models.The beneficial effect of melatonin was also detectable on the composite clinical outcome, including mortality, hospitalization due to HF decompensation, and quality of life. However, we had no mortality and few hospitalizations during the study. Indeed, most of the clinical benefit of melatonin was its effect on the quality of life.Melatonin did not affect sleep quality and quantity, anxiety, and depression levels during this investigation. There are discrepancies among previous studies and recent systematic reviews regarding the effect of melatonin on sleep patterns; overall, it seems that the melatonin effect is dependent on several factors such as dose, duration of use, pharmacokinetic of the melatonin formulations, and the background characteristics of the studied population.Furthermore, the effect of melatonin on mental health is inconsistent. Jafari‐Vayghan et al.Melatonin is supposed to improve serum lipid profile in various target populations, possibly by directly regulating lipid metabolism and reducing the detrimental effect of oxidizing lipoproteins on the cardiovascular system.We performed liver and renal function tests for the primary purpose of detecting any drug‐related adverse effects in our patients. On the other hand, several studies have shown melatonin to ameliorate nonalcoholic fatty liver disease and decrease liver transaminases in concordance with moderately lower levels of these enzymes in our melatonin group.We found an overall 35% reduction in NT‐Pro BNP concentration in melatonin compared with the placebo group relative to baseline values; while some clinical trials defined a reduction of 30% in NT‐Pro BNP level during treatment as a primary endpoint,The low sample size was also a limitation of the study, which might have caused some insignificant results. This low sample size might also have affected the clinical outcomes such as death and hospitalization despite a respectively long follow‐up and made any conclusion about these outcomes unreliable. However, this sample size seems enough to evaluate the primary outcomes. Also, the high number of missing in the first follow‐up was another limitation imposed by the urgent COVID‐19 epidemics, although none of the primary outcomes had been planned to be evaluated at the first follow‐up.Extended follow‐up time relative to other clinical trials using melatonin in cardiovascular diseases is a strength of the MeHR trial. Also, up to our knowledge, this is the first study that includes biological biomarkers, structural parameters, and patient‐oriented outcomes to evaluate the melatonin effect in HFrEF.Overall, melatonin might lower serum NT‐Pro BNP and improve disease‐specific health‐related quality of life in patients with HFrEF. Thus it could be a valuable supplement for these patients. Further studies in subgroups of patients with HF, such as diabetic or hypertensive cardiomyopathic patients, and sensitive evaluation methods for cardiac function might provide new information in this regard.The authors declare that there are no conflict of interests.Supporting information.Click here for additional data file.


While reforestation is gaining momentum to moderate climate change via carbon sequestration, there is also an opportunity to use tree planting to confront declining global biodiversity. Where tree species vary in support of diversity, selecting appropriate species for planting could increase conservation effectiveness. We used a common garden experiment in Borneo using 24 native tree species to examine how variation among tree species in their support of beetle diversity is predicted by plant traits associated with “acquisitive” and “conservative” resource acquisition strategies. We evaluate three hypotheses: (1) beetle communities show fidelity to host identity as indicated by variation in abundance and diversity among tree species, (2) the leaf economic spectrum partially explains this variation as shown by beetle preferences for plant species that are predicted by plant traits, and (3) a small number of selected tree species can capture higher beetle species richness than a random tree species community. We found high variation among tree species in supporting three highly intercorrelated metrics of beetle communities: abundance, richness, and Shannon diversity. Variation in support of beetle communities was predicted by plant traits and varied by plant functional groups; within the dipterocarp family, high beetle diversity was predicted by conservative traits such as high wood density and slow growth, and in non‐dipterocarps by the acquisitive traits of high foliar K and rapid growth. Using species accumulation curves and extrapolation to twice the original sample size, we show that 48 tree species were not enough to reach asymptote levels of beetle richness. Nevertheless, species accumulation curves of the six tree species with the highest richness had steeper slopes and supported 33% higher richness than a random community of tree species. Reforestation projects concerned about conservation can benefit by identifying tree species with a disproportional capacity to support biodiversity based on plant traits. The functional relationship between plant diversity and diversity of organisms in higher trophic levels is a fundamental ecological question of what is important for understanding the contribution of restoration of plant communities for conservation. We show that tropical tree species vary in the level of diversity that they support and that this variation can be predicted by plant traits. We further illustrate how such variation can be utilized to increase the conservation value of plant diversity in restoration. While the United Nations has proclaimed the current decade (2021–2030) as the decade of ecological restoration, there is still a fundamental lack of knowledge on how different tree species used in reforestation could be selected to facilitate a broader set of ecosystem functions such as promoting biodiversity. Incorporating the rebuilding of biodiversity as a cornerstone to the global reforestation movement could help battle global biodiversity loss  were specifically chosen in this study due to their high diversity in the canopy of tropical forests , non‐dipterocarps, and fruits tree. The included species were selected to represent species that are commonly used in reforestation efforts in the mixed dipterocarp forests of Borneo, the INIKEA project area, and throughout the state of Sabah. The experimental common garden was established in the interior of the INIKEA project area, at an elevation of approximately 300 m. In the garden, 20 replicated trees of each of the 34 species, for a total of 680 seedlings, were randomly planted along lines separated by 10 m, and along lines, trees were planted at 3 m intervals. The plant material used to establish the garden came from seeds collected within the INIKEA project forest, germinated in the project nursery, and were of approximately equal age and height when out‐planted. All competing vegetation was removed in the lines prior to planting, for example, ground vegetation, bushes, small pioneer trees, and climbers. We performed weed maintenance to keep the lines free from competing vegetation when necessary, resulting in 1–3 rounds of weeding per year corresponding to the INIKEA standard procedure for restoration planting. More details on the experimental design can be found in Gustafsson et al. .2.3Sindora irpicina and Shorea parvifolia represented by four replicate trees, and Walsura pinnata was represented by three replicate trees. We sampled beetles using canopy fogging with a pyrethrum‐based insecticide. To account for seasonal variation in beetle occurrence and assess the overall contribution to biodiversity, we repeated sampling every 3 months for a total of four sampling periods between May 2016 and April 2017. For each selected tree and sampling period, a white canvas sheet (2 × 2 m) was laid on the ground below the tree canopy. We sprayed the insecticide for approximately 30 seconds with the fogging zone including the entire canopy of each tree . For taller trees, we used an orchard ladder to reach the upper part of the canopy. After fogging, we waited 30 s for the insecticide to take effect and then vigorously shook the tree from the tree bole for 15 s to help detach arthropods from the canopy. Arthropods falling on the canvas were collected using forceps for 15 person minutes following fogging and immediately placed in vials containing an aqueous solution of 70% ethanol. The same procedure was repeated on all trees across sampling dates. We identified beetles to the species level where possible; all species were sorted to at least the family level which allowed us to categorize beetles into functional groups for some of the families. Richness estimates were based on both species identification and by morpho‐species classification. The use of morpho‐species generally provides a conservative estimate of richness as certain groups of beetles may sometimes be morpho‐typed as the same species. However, morpho‐typing is necessary for ecological studies as identifying specimens to species level where there are thousands of species is logistically difficult and beyond the scope of our research funding. Also, there are few taxonomic experts in the region and many insects in Borneo have not yet been described, which is a common problem throughout the hyper‐diverse tropics , nitrogen (N), and potassium (K) concentrations (mass‐based), specific leaf area (SLA), and wood density (WD). Plants with high nutrient concentrations, high SLA, and low‐density wood tend to be correlated with fast resource acquisition and rapid growth  with tree species as predictor to test for significant differences among tree species in abundance, richness, and Shannon index of diversity of beetles. As both abundance and richness are expressed in counts, we fitted the model with Poisson distribution. Shannon index of diversity conformed to assumptions of normality and was hence fitted with normal distribution.richness, and Shannon index of diversity for each tree. Shannon index of diversity (H) was calculated using the formula:To test whether variation in support of biodiversity was based by a general beetle preference for plant traits, we first conducted principal component analyses to explore relationships among plant traits  and growth rate, and established a loading matrix for the two principal components describing most of the variation among tree species. Then, we tested if there was a relationship between either one of the two principal components and our beetle biodiversity metrics  using correlation analyses. In cases where the relationship between a principle component and biodiversity was significant, we then used the three traits with the highest loading on that component in further correlation analyses. As other studies have shown that trait to function correlations may vary between different functional groups of plants  followed by saprophagous , predators , taxa with mixed feeding , xylophagous, , and fungivores . Most of the beetles we collected were from the Chrysomelidae family with 38% (540) of all individuals collected and 31% (127) of all the species encountered . Shannon index of diversity also varied among tree species . Tree species also varied 6.0‐fold in the total richness of beetles that they supported and total richness roughly resembled patterns of mean richness  and in non‐dipterocarps (p < .002). We also found that the six species supporting the highest richness; Hopea ferruginea, Diospyros sp., Shorea ovalis, Shorea leptoderma, Pentace laxiflora, and Dipterocarpus conformis, together supported 62% of the total beetle biodiversity (253 of 405 species).Agreeing with our first hypothesis, we found large and statistically significant variation among tree species in support of beetles in the mean abundance, mean richness, and Shannon index of diversity. Abundance varied 6.7‐fold and richness 5.9‐fold among different tree species Figure  and GLM 3.2r = .5328, p = .0074) and to some extent also beetle richness . The traits explaining the association between PC2 and beetle diversity were not consistent across the full set of 24 tree species but differed between dipterocarps and non‐dipterocarps. In the dipterocarps, we found that a high Shannon index of diversity was correlated with slow growth  and high wood density . High wood density was to some extent also associated with beetle richness  and beetle abundance . In the non‐dipterocarps, we found that foliar K was positively correlated with beetle diversity as expressed as Shannon index of diversity  and richness  but not beetle abundance . Beetle diversity was also close to significantly (p < .1) and positively correlated with growth rate in the non‐dipterocarps; richness , abundance , and Shannon index of diversity .Using a set of six plant characteristics  that together are known to distribute plants along the acquisitive–conservative continuum, we found that 61.0% of the variation in traits among species could be described by two principal components Figure . Princip3.3We used species accumulation curves to assess the functional relationship between plant species richness and beetle richness to estimate the level of plant diversity needed to rebuild canopy insect diversity in degraded secondary tropical forests. We found that a random community of 24 tree species was far from enough to reach asymptotic levels of beetle richness Figure  by estimWe found that the six tree species supporting the most rich communities of beetles had a greater potential to support beetle diversity at lower plant species richness compared to a plant community of random species. This is shown by accumulation curves of the six selected species having a much steeper trajectory than the random tree species community Figure . We foun4The aim of this study was to establish a framework for evaluating if non‐random selection of tree species could be used as a starting point for restoring communities associated with trees in tropical forest restoration. If tree species vary in their importance for supporting biodiversity of associated communities and this variation can be predicted from their traits, then screening diverse forests can be accomplished without the need to intensively study biodiversity which is a more expensive and time‐consuming process than measuring traits. We found compelling evidence that tree species in tropical forests vary in their support of canopy beetles suggesting that by selecting tree species that harbor high diversity we can increase diversity and abundance over selecting random tree species. Because beetles make up large fraction of the arthropod biodiversity in tropical forests and include large functional variation, they are potential drivers of insect diversity and also higher trophic levels communities such as insectivorous birds and mammals ) ; Data curation (supporting); Formal analysis (lead); Funding acquisition ; Investigation ; Methodology ; Supervision ; Validation ; Visualization (lead); Writing – original draft (lead); Writing – review & editing . Jane Vanessa Abin: Data curation (lead); Investigation ; Validation ; Writing – review & editing . Maria Lourdes T Lardizabal: Data curation ; Funding acquisition ; Investigation ; Project administration ; Resources ; Supervision (lead); Writing – review & editing . Ulrik Ilstedt: Funding acquisition ; Project administration ; Resources ; Supervision (supporting); Writing – review & editing . Kevin C. Grady: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Supervision ; Writing – original draft (supporting); Writing – review & editing .


Wound healing is a complex process that achieves the impressive goal of sealing the breach in the tissue and reinstating its integrity via the activation of a multifaceted biological response involving hemostasis, inflammation, cell migration, cell proliferation and tissue remodelling.A fundamental step in tissue repair is the activation of a collective migration program that ensures rapid closure of the breach by the cells at the edge of the wound before cell proliferation can restore the initial architecture. The mechanics of collective migration in epithelial wound healing have been dissected by a number of studies, many of which make use of simplified in vitro models of cultured epithelial monolayers injured by removal of a strip of cells.The collective migration that enables epithelial repair is often initiated by a few specialized cells at the leading edge of migrating cell sheets that become migratory and guide cell migration. For this reason, these are called ‘leader cells’. Leader cells acquire a characteristic flattened morphology and activate migratory signaling cascades (such as elevation of integrin β1 and phosphoinositide 3‐kinase and increased activity of Rac1—Ras‐related C3 botulinum toxin substrate 1), which endow them with the capacity to drive migration of their neighbours.We found that p53 levels are elevated in leader cells and, remarkably, that induction of p53 was able to instruct leader behaviour.The discovery of the role of p53 in leader cell specification moves the question one step further: What induces p53 elevation in epithelial injury, triggering the commitment to leader fate?Using a fluorescent reporter of p53 activity, we showed that this is increased in cells at the edge of a scratched epithelium, compared to their undamaged neighbours.We then asked: What happens to leaders when the epithelium is repaired? Tracking leader cells as the epithelial gap closed, we could observe their elimination via cell competition,The novel involvement of the p53‐p21‐CDK signaling axis in epithelial healing raises the question of whether such mechanism is relevant for tissue repair in vivo. Previous studies seem to support this idea. For example, the presence of a non‐proliferative migrating leading edge has been observed in wounds in vivo,The presence of cell‐cycle arrested cells at the front of migrating cell populations is not limited to the context of tissue repair. During placenta formation, cytotrophoblasts penetrate the uterine wall to form the floating villi that mediate mother‐embryo exchanges.Given the established role of p53 in preventing neoplastic transformation and the importance of collective migration in tumour invasion and metastasis, it is tempting to question if p53‐mediated leader specification plays a role in cancer dissemination. Most human cancers carry mutations that abolish the normal function of p53. However, the tissue surrounding the tumour expresses functional p53, susceptible to be elevated by radiation and chemotherapy. It is thus possible that asymmetric expression of p53 or CDK activity in adjoining tumour–non‐tumour cell populations could result in leader‐follower behaviour, promoting tumour cell migration. This effect would explain the observation that chemotherapy‐induced senescent cells in tumour‐adjacent tissue promote metastasisThe discovery of p53 function in leader cell specification, if confirmed by in vivo studies, could therefore have broad implications in physiological and pathological activation of collective migration and, potentially, inform future therapeutic approaches. Figure The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


Rana [Lithobates] pipiens), American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum). Animals were exposed for 30 days  or until metamorphosis (toads) to 10, 100, or 1000 μg/L of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), or 6:2 fluorotelomer sulfonate (6:2 FTS). We determined that chronic exposure to common PFAS can negatively affect amphibian body condition and development at concentrations as low as 10 µg/L. These effects were highly species dependent, with species having prolonged larval development  being more sensitive to PFAS than more rapidly developing species (toads). Our results demonstrate that some species could experience sublethal effects at sites with surface waters highly affected by PFAS. Our results also indicate that evaluating PFAS toxicity using a single species may not be sufficient for accurate amphibian risk assessment. Future studies are needed to determine whether these differences in susceptibility can be predicted from species' life histories and whether more commonly occurring environmental levels of PFAS could affect amphibians. Environ Toxicol Chem 2022;41:1407–1415. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Per‐ and polyfluoroalkyl substances (PFAS) are contaminants of concern due to their widespread occurrence in the environment, persistence, and potential to elicit a range of negative health effects. Per‐ and polyfluoroalkyl substances are regularly detected in surface waters, but their effects on many aquatic organisms are still poorly understood. Species with thyroid‐dependent development, like amphibians, can be especially susceptible to PFAS effects on thyroid hormone regulation. We examined sublethal effects of aquatic exposure to four commonly detected PFAS on larval northern leopard frogs ( Per‐ and polyfluoroalkyl substances (PFAS) are a large family (~5000) of widely used legacy and emerging contaminants that are pervasive in aquatic habitats globally and thus present a risk to the health of humans and wildlife , American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum). Our secondary aim was to assess how aquatic PFAS exposure could affect thyroid form and function in toads and salamanders. We hypothesized that sensitivity would be driven by duration of larval stages, with American toads being the least sensitive due to their rapid developmental rate, followed by northern leopard frogs, and eastern tiger salamanders.Despite the prevalence of PFAS in aquatic habitats, their toxicity is not well characterized for most wildlife due to a lack of or limited toxicity data available for many relevant taxa including amphibians. Although basic PFAS ecotoxicological data are lacking for many amphibians, evidence is accumulating that PFAS can have adverse effects on this group. Indeed, PFAS exposure has been associated with altered growth, development, and body condition in larval and terrestrial forms of amphibians , eastern tiger salamanders (n = 63), and American toads (n = 15) were collected from an ephemeral pond with no history of PFAS inputs in West Lafayette, Indiana (USA) in March 2018, February 2017, and May 2017, respectively. We selected these three species for several reasons: (1) their broad distribution and high abundance across the United States means they are likely to occur at sites affected by PFAS . Egg masses of northern leopard frogs , PFOS , and PFHxS  from Sigma‐Aldrich and 6:2 FTS  from SynQuest Laboratories. We prepared primary stock solutions (200–1000 mg/L) by dissolving chemicals into 1 L of ultraviolet (UV)‐irradiated filtered well water. We mixed the solutions on a stir plate until the chemicals dissolved . The 6:2 FTS solution was further vacuum‐filtered to remove any remaining insoluble solids . Stock solutions were stored in polypropylene bottles and refrigerated until used to make test solutions.n = 4 blocks) and replicates of each treatment equally represented in each block. Animals were maintained at 20 ± 2 °C with a 12:12‐h light:dark photoperiod for the duration of the exposure. Water changes were conducted weekly for salamanders and twice weekly for frogs and toads. Larvae were fed  at 20% of the average larval body weight every other day . We determined feeding rations using extra‐experimental units and animals (n = 4 bins/species) that were independent of the experiment  but were set up identically to the control treatments of each species. These animals were weighed weekly to calculate food rations.Our study design included a no‐PFAS control and a factorial combination of four PFAS  crossed with three concentrations . For all species, our experimental units were 15‐L tubs; however, because of the cannibalistic nature of salamanders, they were kept in individual 1‐L cups inside the tubs. For frogs and toads, tubs were filled with 7.5 L of filtered, UV‐irradiated well water and assigned 20 individuals to each tub. For salamanders, cups were filled with 750 ml of filtered, UV‐irradiated well water and assigned one individual per cup, for a total of 15 cups/tub. Each treatment was replicated 4 times for a total of 52 experimental units/species. Experimental units were arranged in a randomized block design with shelf height as the blocking factor  immediately after initial exposure and immediately prior to the first water change for verification of PFAS concentrations. A previous study in this laboratory showed that our methods resulted in consistent PFAS exposure concentrations to animals throughout a 30‐day experiment  as the developmental endpoint for toads collected at the final sampling point.We sampled larvae to quantify snout–vent length (SVL), body mass, and developmental stage (frogs and toads only). The SVL and body mass were used to calculate the scaled mass index (SMI) as described in We also preserved the heads of five toads and five salamanders from each experimental unit in 10% buffered formalin for thyroid histopathology. The remaining animals were stored whole at −80 °C for thyroid hormone quantification. Because there were no substantial effects of PFAS on these response variables, the methods and detailed results for thyroid histology and hormone concentrations are presented in the Supporting Information.The methods used for quantification of PFAS in test water are described in detail in previous publications and their associated supplementary information . The body mass and SVL of individual i are given by Mi and Li, respectively. Finally, bSMA is defined as the allometric scaling exponent of the standardized major axis regression of log10‐mass on log10‐SVL of salamanders from the control treatment. We calculated scaling coefficients for SMI estimation using only animals from controls for each species, at each time point to ensure coefficients were not skewed by treatment effects.Analyses of morphological  and developmental (Gosner stage and time‐to‐metamorphosis) endpoints were conducted using linear mixed models. We calculated SMI as a measure of body condition and lipid reserves using the approach of Peig and Green :Mi^=Mip < 0.05, indicating a statistical difference from the control group.In all models, chemical treatment was included as a fixed predictor with 13 levels, and experimental unit was used as a random effect to account for the nonindependence of larvae sharing experimental units. Salamanders were reared in “individual” containers nested within experimental units. The effect of a treatment was considered significant if that treatment was associated with a Measured PFAS levels across treatment concentrations within a chemical were well separated and differed, as intended, by roughly a factor of 10 . Overall salamander survival was 99%. Although we did not observe an overall effect of treatment on salamander survival , survival in the 6:2 FTS 100‐µg/L treatment (96%) was reduced relative to the control . Survival in toads was the lowest of the three species (86%), but was still high and did not differ among treatments .Across all species and treatments, mean survival was more than 85% for the duration of the studies. Leopard frog survival was greater than 93% in every treatment and did not differ among treatments  by Day 30, but variation was substantial among species Figure . Frog SMThe effects of PFAS on larval development differed between species Figure . Frog deThyroid hormone levels and gland development, at the developmental stages examined, were only appreciable in toads  values of 10 µg/L for all PFAS in frogs and of 10 µg/L for salamanders for all PFAS except PFOS. Although the 100‐ and 1000‐µg/L treatment levels are not reflective of PFAS concentrations likely to be encountered by wildlife, PFAS can approach 10 µg/L in surface waters at heavily affected sites , although mean SVL tended to be reduced in tadpoles exposed to PFOS, PFHxS, and 6:2 FTS. The final study assessed the toxicity of PFOA and PFOS in simulated pond communities , in which tanks were dosed with PFAS‐spiked sediment, which allowed PFAS to equilibrate between water and sediment more naturally and become incorporated into the diet  exposure to PFOS can affect amphibian larvae. One study examined how development, thyroid morphology, and gene expression in The distinct responses among the three species offer insights into how PFAS can differentially affect amphibian taxa. Taking our data along with those of previous research, we were able to compare the effects of aquatic, dietary, and dermal PFAS exposure in eastern tiger salamanders. Dietary and dermal exposure were also associated with reduced body conditions driven by increases in SVL coupled with no changes in mass in salamanders . We note that the lack of substantial effects on thyroid endpoints in our study could be due to the small sample sizes, analytical methods, range of treatment concentrations, or other aspects of our methodology. The thyroid gland histology showed rapidly growing and developing tissue in both treatment and control salamanders. No significant differences were observed because the glands were variable in size, the follicles were consistently small, and the follicular cells were pleomorphic. Mature, colloid‐producing glands were found in toads, but again, no treatment amount influenced the histological appearance of the gland. This is further supported by the hormone data, with no treatment group significantly deviating from control animals for any of the three hormones examined. Given the lack of morphological differences between these groups, it perhaps follows that the thyroid hormone levels and the thyroid glands themselves did not show significant changes. Those species that did show toxic effects  may indeed show alterations to the thyroid gland once the gland has had more of a chance to develop .Our study offers important insights into the relative sensitivity of three common North American amphibian species to common PFAS. We determined that chronic PFAS exposure can negatively affect larval amphibian growth and development. However, the observed effects were highly species dependent. Our results indicate that evaluating the toxicity of PFAS using a single species is not sufficient for protective amphibian risk assessment. Future studies assessing the effects of PFAS on amphibians would be needed to determine whether these differences in susceptibility can be predicted from species' life histories . Given that amphibians already face substantial declines around the world (International Union for the Conservation of Nature, https://doi.org/10.1002/etc.5319.The Supporting Information is available on the Wiley Online Library at Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the authors or the US Government.This article includes online‐only Supporting Information.Supporting information.Click here for additional data file.