Since the first reports of coronavirus disease 2019 (COVID‐19) in Wuhan, China, in December 2019, there have been 198 million confirmed cases worldwide as of August 2021. The scientific community has joined efforts to gain knowledge of the newly emerged virus named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the immunopathological mechanisms leading to COVID‐19, and its significance for patients with allergies and asthma.Based on the current literature, recent advances and developments in COVID‐19 in the context of allergic diseases were reviewed.In this review, we discuss the prevalence of COVID‐19 in subjects with asthma, attacks of hereditary angioedema, and other allergic diseases during COVID‐19. Underlying mechanisms suggest a protective role of allergy in COVID‐19, involving eosinophilia, SARS‐CoV‐2 receptors expression, interferon responses, and other immunological events, but further studies are needed to fully understand those associations. There has been significant progress in disease evaluation and management of COVID‐19, and allergy care should continue during the COVID‐19 pandemic. The European Academy of Allergy & Clinical Immunology (EAACI) launched a series of statements and position papers providing recommendations on the organization of the allergy clinic, handling of allergen immunotherapy, asthma, drug hypersensitivity, allergic rhinitis, and other allergic diseases. Treatment of allergies using biologics during the COVID‐19 pandemic has also been discussed. Allergic reactions to the COVID‐19 vaccines, including severe anaphylaxis, have been reported. Vaccination is a prophylactic strategy that can lead to a significant reduction in the mortality and morbidity associated with SARS‐CoV‐2 infection, and in this review, we discuss the proposed culprit components causing rare adverse reactions and recommendations to mitigate the risk of anaphylactic events during the administration of the vaccines. In a study from Stanford University, allergic asthma was found to mitigate the risk of hospitalization for COVID‐19 compared to patients with non‐allergic asthma .Intriguingly, the prevalence of allergic diseases showed heterogeneous patterns during the COVID‐19 pandemic. Childhood asthma presented better outcomes with fewer asthma exacerbations leading to a reduced number of emergency visits and hospitalizations. Moreover, 66% of pediatric asthma patients had improved asthma control as measured by the improved forced expiratory volume in one second (FEV1) and the peak expiratory flow (PEF).A survey based on 14 member countries commissioned by the Asia Pacific Association of Allergy Asthma and Clinical Immunology (APAAACI) indicated an increased prevalence of allergic diseases among healthcare workers during the COVID‐19 pandemic, for example, ocular and airway allergy with extended use of surgical masks and eye protection; skin allergy due to prolonged use of gloves, protective equipment and frequent handwashing.Systemic allergic reactions during the pandemic seemed to be drastically reduced as indicated by the number of patients attending an Emergency Department Unit in the UK, from 62 in the first half of pre‐pandemic 2019 to 10 in the same period of 2020. The clinical manifestations presented before the pandemic by 52% of patients were classified as mild, according to the Brown grading system, whilst during the pandemic the majority of patients (80%) experienced moderate systemic allergic reactions.2ACE2 expression was reduced in differentiated airway epithelial cells treated with interleukin‐13 (IL‐13) or after exposure to cat allergen. Its expression was negatively correlated with allergic sensitization in nasal epithelium and Immunoglobulin E (IgE), IL‐13, fractional exhaled nitric oxide (FeNO) and other type 2 signatures. Similarly, transcriptomic data analysis suggested decreased ACE2 expression in the nasal epithelium of children with allergic asthma and allergic sensitization or with asthma and/or allergic rhinitis.TMPRSS2 expression was found in ex vivo airway epithelial cells in respiratory allergic subjects and was positively correlated with type 2 cytokines.Allergy or atopy is characterized by a type 2 (T2) immune response against external antigens in individuals, in which genetic predisposition plays a major role.ACE2 gene expression was downregulated in the airway epithelial cells, including in the nasal polys, and the olfactory neuroepithelium. Chronic rhinosinusitis with nasal polyps (CRSwNP) was characterized by a type 2 immune signature with increased eosinophil counts in the olfactory mucosa. Moreover, ACE2 expression was negatively correlated with the number of eosinophils.+ T cells against respiratory virus infection. Eosinophil‐derived enzymes have been proposed to neutralize the virus via a ribonuclease‐dependent mechanism.+ T cell depletion, which might be related to a Th17 inflammatory pattern in severe COVID‐19 patients.Although type I and III interferons (IFN‐ α/β and ‐λ respectively) are essential to abrogate viral infection,TMPRSS2 (encoding TMPRSS2), PPIA (encoding cyclophilin A), SLC7A5 (encoding CD98), and other molecules involved in COVID‐19 pathophysiological mechanisms, both in lesional and non‐lesional skin.Various skin manifestations have been reported in SARS‐CoV‐2 positive patients,Treatment of allergic and non‐allergic asthma with ICS might also have an impact on the susceptibility to infection and COVID‐19 severity.Even though the preliminary mechanistic data might suggest the protective role of type 2 inflammation against SARS‐CoV‐2 infection and COVID‐19 severity, further studies are urgently needed to evaluate these observations and to understand in detail these mechanisms in humans. This might also lead to the further development of new targets for pharmacological interventions against COVID‐19.3Although the COVID‐19 pandemic significantly impaired health care, management of allergic diseases was still ongoing by adhering to preventive measures. For example, there has been a shift of chronic urticaria consultation from face‐to‐face (decreased by 62%) towards telemedicine (increased by over 600%).Based on a European Academy of Allergy & Clinical Immunology (EAACI) survey, nearly half of newly referred asthma patients received face‐to‐face consultation with telemedicine follow‐ups. The majority of lung function tests were temporarily postponed. Initial asthma diagnosis and therapy were largely based only on clinical manifestations. All these factors contributed to the lower quality of healthcare during the lockdown. In general, the performance of lung function tests together with efficient remote monitoring presented the biggest challenges for asthma management in both children and adults.The small droplets  generated during pulmonary function tests contain SARS‐CoV‐2 and pose a significant risk of viral transmission in a healthcare setting.Telemedicine ensures the safety of both patients and healthcare professionals during the COVID‐19 pandemic, for example, quantitative measuring of olfactory dysfunction using psychophysical analyses were remotely performed.EAACI launched a series of statements and position papers providing official recommendations for the treatment of drug hypersensitivity, allergic rhinitis, asthma, and other allergic diseases during the COVID‐19 pandemic (Table 4An APAAACI survey reported a decrease in AIT (46.1%) and immunosuppressive therapies (23.1%) in allergic patients during the COVID‐19 pandemic.Biological therapeutics targeting type 2 inflammation pathways have been adopted in a wide range of allergic diseases.Anti‐IgE antibody omalizumab has been reported to enhance the anti‐viral immune response by downregulating the high‐affinity IgE receptor on pDC.In summary, according to an evidence‐based EACCI statement, treatment with biologicals should be maintained in noninfected cases with continuous evaluation of atopic disease progression. In the case of active SARS‐CoV‐2 infection, biologicals need to be postponed until clinical resolution is established.5Novel COVID‐19 vaccines are being developed as an indispensable prophylactic strategy to reduce the morbidity and mortality associated with COVID‐19.2CH2O) n, which has been suspected as a causal excipient of hypersensitivity reaction to human papillomavirus vaccine.Polyethylene glycol (PEG) has been identified as a potential trigger of allergic reactions. PEGs are found in many daily products and are an integral part of the micellar delivery system of the Pfizer‐BioNTech BNT162b2 and the Moderna mRNA‐1273 vaccines containing mRNA coding the spike protein of SARS‐CoV‐2.There are also other potential culprit agents such as distearoylphosphatidylcholine (DSPC) in the Pfizer‐BioNTech and Moderna vaccines, formerly attributed to hypersensitivity to pollen. Trometamol is added as a buffer in the Moderna vaccine and there has been a case report of anaphylaxis to this excipient in an intravenously administered radiocontrast agent.It is worth noting that allergic patients without a previous history to the vaccine components are not contraindicated for COVID‐19 vaccination.In conclusion, current evidence shows that allergy might play a protective role in COVID‐19 with the involvement of eosinophilia, SARS‐CoV‐2 related receptors expressions, and IFN responses, and other immunological events. Further research is warranted to improve our understanding of the underlying pathophysiological mechanisms. Allergy care has continued during the COVID‐19 pandemic and official recommendations from EAACI have been developed to inform healthcare professionals on the management and treatment of allergies during this time. Patients with a previous history of allergic reactions to a component of the COVID‐19 vaccines should be referred to an allergy clinic for a diagnostic workup.There is a current need to improve our understanding of the underlying mechanisms involved in the vaccines adverse reactions and their culprit components to ensure their safety and public compliance.None of the authors has any conflicts of interest to declare.


We explore the evidence for this.A recent single‐cell RNA sequencing study by Wilk We downloaded the original data and code used by the authors in their study to replicate their findings and explore the possibility that regressing out variables may have led the authors to overfit their data.The lineage relationship between plasmablasts and developing neutrophils breaks down when key features are not regressed out, and the data are not overfitted during the analysis.Plasmablasts do not transdifferentiate into developing neutrophils. The single‐cell RNA sequencing is a powerful technique for biological discovery and hypothesis generation. However, caution should be exercised in the bioinformatic analysis and interpretation of the data and findings cross‐validated by orthogonal techniques. Single cell RNA sequencing can generate unexpected hypotheses. However, these should be rigorously tested and cross‐validated by orthogonal techniques. In this case, there is no evidence that plasmablasts differentiate into neutrophils in severe COVID‐19 disease. Indeed,s Figure . MoreoveTo test these hypotheses, we first noted that, in addition to regressing out mitochondrial genes, ribosomal RNA and ribosomal genes, the authors also regressed out the number of UMIs (nCount_RNA) and the number of expressed genes (nFeature_RNA) from the gene expression data in their analysis. This step is unnecessary in Seurat when using the SCTransform normalisation method as the number of UMIs is explicitly modelled using a regularised negative binomial regression.et al., developing neutrophils appear to occupy a similar manifold space as plasmablasts. However, from this viewpoint the plasmablasts appear to be distantly related to the B cells from which they are derived. In addition, these types of analyses make the a priori assumption that all the cell types being studied are developmentally related and linked by a cell lineage tree. This may hold true when analysing in vitro‐differentiated cells that share a common ancestor cell of origin that is present in the cell culture, but may not apply to PBMCs that contain a heterogeneous collection of cell types that have arisen from different committed progenitors.Accordingly, we believe that orthogonal approaches, such as single‐cell DNA sequencing to detect rearranged immunoglobulin heavy‐ and light‐chain variable genes in the developing neutrophils, are needed to provide evidence of their B‐cell origin and support the hypothesis that terminally differentiated plasma cells transdifferentiate into developing neutrophils. Furthermore, unless the data visualisation parameters are carefully chosen, the 2D UMAP embeddings may give the misleading impression that cell clusters are closer or further than they actually are. For example, in Figure Since plasmablasts are derived from B cells, we decided to subset neutrophils, developing neutrophils, B cells and plasmablasts to further explore any similarities between these cell clusters. This analysis revealed that counter‐intuitively, neutrophils were not related to developing neutrophils and that B cells were not related to plasmablasts Figure . This exet al. point out that the developing neutrophils were unlikely to be doublets and also unlikely to be granulocytes that had phagocytosed B cells as there were no clinical features of haemophagocytic lymphohistiocytosis (HLH), a well‐characterised state of immune hyperactivation. However, HLH is often difficult to diagnose clinically and severe COVID‐19 disease has been linked in some cases with secondary HLH.In their analysis, Wilk et al. is a timely and valuable contribution to the understanding of how severe COVID‐19 disease reshapes the composition and activation state of cell clusters in PBMCs. Neutrophils have a high density and are not normally present in Ficoll preparations of blood used to isolate PBMCs. The developing neutrophils may therefore be similar to the pro‐inflammatory low‐density neutrophils that have been described in a number of autoimmune diseasesNevertheless, the paper by Wilk et al. indicates that there is not enough evidence to suggest that developing neutrophils transdifferentiate from plasmablasts.In summary, scRNA‐seq is becoming an increasingly popular tool for dissecting cellular heterogeneity in complex biological systems. However, it does have its limitations and, in the example shown here, may mislead and generate interesting hypotheses that risk being erroneously accepted as conclusions without further cross‐validation. Our analysis of the data from Wilk et al. by data origin to obtain individual batches and applied SCTransform normalisation to each batch and the Wilk et al. data set. Next, we determined 3000 highly variable genes (HVGs) for integration across all data sets. We identified mutual nearest neighbour cells between data sets to define integration anchors and integrated the data by computing anchor integration matrices. Finally, we performed a principal component analysis (PCA) using the implicitly restarted Lanczos method and computed the first fifty principal components and embedded the principal components into a two‐dimensional space using the Uniform Manifold Approximation and Projection (UMAP).Before data integration, we split the Schulte‐Schrepping et al.: percentage of ribosomal  and mitochondrial (percent.mt) expression and the number of counts (nCount_RNA) and genes (nFeature_RNA) per cell. Finally, we performed PCA and UMAP following the same strategy described in the previous section. All analyses and graphs were generated using the Seurat v4.To test the effect of covariates on the low‐dimensional data representations, we applied SCTransform normalisation and included different covariates in the second non‐regularised linear regression as follows: (i) using the percentage of mitochondrial expression and (ii) using the percentage of ribosomal and mitochondrial expression. Next, we performed PCA and UMAP dimensionality reduction as described above. Additionally, we subsetted all B cells, plasmablasts , neutrophils and developing neutrophils. Next, we applied SCTransform using the original covariates considered by Wilk http://pantherdb.org/).We calculated cell cycle scores with the CellCycleScoring function using a set of 43 genes associated with the S phase and 54 with the G2 M phase. Similarly, for the assessment of covariate effects, we regressed out the scores for both phases from the gene expression data within the SCTransform function. Gene ontology enrichment analysis was performed using the PANTHER over‐representation test with Fisher’s exact test and the Bonferroni correction for multiple testing (The authors declare no competing interests.Jose Alquicira‐Hernandez: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐original draft; Writing‐review & editing. Joseph Powell: Conceptualization; Formal analysis; Supervision; Validation; Writing‐original draft; Writing‐review & editing. TriPhan: Conceptualization; Formal analysis; Supervision; Writing‐original draft; Writing‐review & editing.


Efforts to generate organomanganese reagents under ball‐milling conditions have led to the serendipitous discovery that manganese metal can mediate the reductive dimerization of arylidene malonates. The newly uncovered process has been optimized and its mechanism explored using CV measurements, radical trapping experiments, EPR spectroscopy, and solution control reactions. This unique reactivity can also be translated to solution whereupon pre‐milling of the manganese is required. A manganese‐mediated reductive dimerization of arylidene malonates by ball milling is reported. The process has been optimised and its mechanism explored by CV measurements, radical trapping, and EPR spectroscopy. Control experiments identify the action of ball milling rather than mortar and pestle is necessary to realise the effective activation of manganese, which can then be used in either solution or a ball mill for subsequent reactions. In recent times mechanochemistry by ball milling has been widely explored for a range of synthetic transformations including metal or organo‐catalyzed processes;II salts being the preferred strategy. Nonetheless, the chemistry and reactivity of organomanganese reagents appears to be relatively unique compared to alternative organometallic species and harnessing a reliable method for the preparation of these materials could well pave the way to further developing these species.1, as a pro‐nucleophile with diethyl 2‐benzylidenemalonate (2) as electrophile  iodide leading to reductively dimerized material in good yield and short timescales  is required for reactivity. It is already well observed in the literature that the composition of liquid and solid form reagents within the ball‐milling reactor can significantly alter the product distribution.1 omitted, an extra equivalent of THF was used, which led to an improved yield of 80 % , methyl (9), methoxy (10), methyl ester (11), nitrile (12) and chloro (13) functionality  in 77 % isolated yield and demonstrates a tolerance of the reaction mechanism to unactivated double bonds. The mixed nitrile and ester system, ethyl‐2‐cyano‐3‐phenylacrylate also underwent dimer formation to give the desired product (14) in 55 % yield. Notably, the nitrile malonate substrate led to a single diastereomer of product, in contrast to every other substrate which gave both syn and anti‐products. Substrate scope studies demonstrated that rather specific electronic activation is required to permit reductive coupling by manganese, including two strong electron withdrawing groups and benzylic stabilization. Further, substrates which could permit trapping of a radical by ring formation , or ring fragmentation (cyclopropyl) did not furnish any of the corresponding reductively coupled products. With this information in hand, together with prior methods for the reductive coupling of arylidene malonates we proposed and investigated plausible reaction mechanisms  suggesting that it is not possible for Mn to reduce these substrates without additional factors at play.E1/2=−0.76 V vs. SCE.Suprisingly, the measured reduction potentials are greater than the reducing power of Mn metal  in the form of a two‐electron reduction to generate the organometallic intermediate , or ii) via a series of single electron transfer (SET) reduction events to generate radical intermediates that could themselves then act as the nucleophile before further reduction. As a test for the presence of radical intermediates (i.e. pathway (ii)), experiments were performed in the presence of the radical inhibiters TEMPO and 1,1‐diphenyl ethylene Scheme . ReactioII species. The EPR spectrum of an ethyl acetate solution of the crude paste obtained directly from the milling jar pre‐workup contained a sextet multiplet feature, with six equal‐intensity hyperfine lines separated by 260 MHz (9.3 mT), which is characteristic of a d5 MnII species with I(55Mn)=5/2 . The possibility of a concerted [3+2] dipolar cycloaddition followed by ring opening is also proposed and electrophile trapping experiments have not resulted in any isolatable intermediates along this pathway. Finally, we were drawn to further explore the comminution of manganese by the action of milling to enable this newly observed reactivity  are returned in 86 % and 78 % respectively. This yield is comparable, if not marginally better than when the reaction is run purely by milling for three hours. As a final investigation, mortar and pestle grinding (or pre‐activation) for two minutes was compared to milling for two minutes at 30 Hz for both manganese pieces and powder. In the former case, pieces returned 3 % of the reductively coupled product following 24 hour reaction in THF at room temperature and powder returned 2 % yield. In the ball‐milled case the pre‐activated pieces afforded 69 % reductively coupled product and the powder afforded 65 % yield.Interrogation of the reaction by electron paramagnetic resonance (EPR) spectroscopy, demonstrates a characteristic signal for Mn2 Scheme . Observay Scheme . This waII at the end of the reaction; measured reduction potentials point towards activation by addition of lithium chloride and radical trapping experiments rule out the intermediacy of interceptable radical species. The exact mechanism remains unclear. However, control experiments have confirmed that ball milling of manganese is essential to unlock this mode of reactivity, this finding delivers a particularly exciting prospect for the future of earth‐abundant manganese chemistry.In conclusion, a novel dimerization of electron poor alkenes by manganese has been discovered. This new reaction has been optimized and explored to develop a deeper understanding. EPR measurements confirm the presence of MnThe 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.


COVID‐19 continues to periodically surge in many parts of the world. Even though highly effective vaccines were identified and granted emergency use authorization in 2020, most middle‐ and lower‐income countries have yet to obtain and distribute sufficient doses to afford large‐scale protection against new infections. Furthermore, viral variants that arise during replication threaten to diminish the protective efficacy of first‐generation COVID‐19 vaccines [As the end of 2021 nears, the world continues to battle the COVID‐19 pandemic. SARS‐CoV‐2, the etiological agent of COVID‐19, has infected over 200 million individuals and claimed the lives of over 4 million of them , the RV144 phase 2b trial in Thailand, and studies of passively administered monoclonal antibodies in NHP and, more recently, humans , 6, 7, 8Journal of the International AIDS Society (JIAS) was conceived as an opportunity to take stock in progress on HIV research and development, outline novel scientific and organizational approaches that might lead to success, and learn from HIV and SARS‐CoV‐2 vaccine studies.This special issue of the 1Efforts to identify HIV prevention approaches beyond barrier methods have resulted in several options for populations with access to HIV testing and antiretrovirals (ARVs). As summarized by Fauci et al.  in this Since all existing prevention methods require adherence, product developers and trialists need to design and evaluate approaches that individuals will effectively use. Acquiring community perspectives and input into product development and testing and understanding what individuals at risk are most likely to consistently use, are imperative to success. Given the wide diversity of at‐risk populations, including men who have sex with men, sex workers, people who use drugs, adolescent girls and young women in sub‐Saharan Africa, providing some choice in prevention interventions will likely be required to achieve population‐level impact on virus spread. As attested to by Luba et al.  in this 2Advances made in the past 25 years have been facilitated by the creation of new organizations and collaborations designed to bring additional resources and energy into the HIV vaccine development field. One such organization, International AIDS Vaccine Initiative (IAVI), has made significant contributions over the years, as described in Feinberg . PerhapsA significant shift in the field took place following the 2003 Science publication calling for creation of a Global HIV Vaccine Enterprise (the Enterprise), which received support from the G8 the following year , 14. TheThe Global HIV Vaccine Enterprise also evolved over time and has served to bring together stakeholders to discuss and address roadblocks such as how HIV vaccine efficacy trials might be conducted in the face of expanded access to other prevention approaches. Tatoud and co‐authors provide a valuable perspective on the past and potential future role of the Enterprise .The UNAIDS has also made significant contributions to the field, most notably the creation of a consensus document on the ethical considerations in HIV prevention trials . Almost 3The lack of an effective vaccine has not been due to a lack of effort. Over $800 million USD was spent on HIV vaccine research and development in 2019 alone . To dateOne novel vaccine design still in efficacy testing at the time of this writing is based on mosaic antigens, which were designed to afford broad coverage across HIV clades. The Imbokodo and Mosaico trials are evaluating a prime‐boost regimen that combines an rAd26 vector and protein. Unfortunately, following a recommendation from its data and safety monitoring board, the Imbokodo study will not be moving forward to its second phase of follow‐up due to inadequate efficacy although no safety concerns have been raised. Another study referred to as PrEPVacc is evaluating PrEP and two vaccine candidates: rDNA with gp120 boost and rDNA/gp140 followed by MVA/gp140 boost. This novel multi‐arm, multi‐stage adaptive trial will employ an averted infection ratio method to determine efficacy. These trials, other on‐going and past vaccine studies, as well as innovative studies of bNAbs, native envelope trimers, germline targeting immunogens, mRNA and new adjuvants are reviewed in Kim et al. . Most reVaccine design has benefited from improved structural biology tools, as reviewed by Derking et al. . StructuThe sera of a small percentage of individuals living with HIV contains bNAbs, which has provided the proof‐of‐concept that a bNAb‐inducing vaccine is possible. Detailed studies of how both the HIV envelope and the body's Ab response evolve over time in those individuals have led to a strategy that attempts to replicate that evolutionary process through sequential immunization with multiple immunogens . The firSchief and collaborators designed a germline targeting immunogen, eOD‐GT8, that was able to bind to VRC01‐class precursor naïve B cells from uninfected individuals . SubsequAn alternative to the induction of bNAbs through active immunization is the passive administration of bNAbs, referred to as AMP, or antibody‐mediated protection. Two studies of a passively administered bNAb, VRC01, to volunteers at risk for HIV infection were completed recently. As summarized in Miner et al. , infusio4Despite the disappointment of Imbokodo the Mosaico efficacy study, which is being conducted in men who have sex with men and transgender people, and which employs a boost with two glycoproteins, is continuing. If Mosaico shows a modest or better level of efficacy, then hope remains that induction of antibodies that bind to but do not necessarily neutralize HIV, along with HIV‐specific T cells could prove beneficial. If the results are “modest” , then efforts to improve upon those results will likely fall to the corporate sponsor, governments and non‐profit partners. Whether such a collaborative effort will be undertaken will likely depend on the progress made with other passive and active immunization strategies as well as the uptake of existing prevention measures. If, however, efficacy is sufficiently high to warrant licensure, then attention will need to turn to tackling implementation issues including bridging to other populations and other virus clades.Similarly, if passive administration of bNAbs or active immunization with a series of immunogens, as proposed by Corey et al.  and WillThe authors declare no competing interests.MIJ prepared the initial draft; co‐authors provided editing and content revisions; all authors approved final draft.LGB is on the Expert Advisory Group of the Global HIV Vaccine Enterprise.


In this Commentary, we discuss the recent advances in the field that suggest that the kinetome may be more conserved than expected. A conserved kinetome will accelerate the development of future kinetic models of integrated cellular functions and expand their scope and usability in many fields of biology and biomedicine.Computational biologists have labored for decades to produce kinetic models to mechanistically explain complex metabolic phenomena. The estimation of numerical values for the large number of kinetic parameters required for constructing large‐scale models has been a major challenge. This collection of kinetic constants has recently been termed the  This Commentary highlights recent discoveries suggesting that the kinetome (a collection of kinetic parameters) can be conserved and discusses how a conserved kinetome will accelerate the development of future kinetic models. Once whole‐genome sequences became available in the mid‐1990s, the reconstruction of metabolic networks reached the genome‐scale. Over the past two decades, genome‐scale metabolic models (GEMs) built from these network reconstructions have contributed to our understanding of systems biology of metabolism, with applications ranging from metabolic engineering to bacterial evolution to infectious disease. Genetic variation can now be incorporated into genome‐scale models of metabolism, transcription, translation, proteostasis, and cellular stresses. GEMs are often simulated using flux balance analysis that requires a minimal number of parameters estimated from empirical data.Kinetic models, on the other hand, have traditionally been limited by the need for extensive parameterization. If the kinetome can be estimated from high‐throughput data, then we can develop large‐scale—even genome‐scale—kinetic models. Since GEMs have a direct genetic basis, a new generation of kinetic models can be directly rooted in protein structures and sequence variation. The kinetome of such models would be large, requiring the estimation of many unmeasured parameters. However, if segments of the kinetome are conserved, then parameterization will be simplified. Reference kinetomes can be estimated for well‐characterized strains and applied to less well‐known strains.in vivo turnover rates of bacterial enzymes have been characterized using ratios of proteomic and fluxomic data  studies. ALE allows for the generation of strains that have adapted to high growth rates following the deletion of genes that encode specific metabolic enzymes. This approach results in the generation of “metabolic specialist” strains whose pathway usage has been rewired by ALE following the loss of a key metabolic enzyme. A large study resulted in an estimation of turnover rates for the same enzyme in multiple metabolic specialists Fig . These eE. coli with orthogenes from a diverse range of other species, from hyperthermophilic archaea to humans  models), or they specify the enzyme abundances based on measurement (enzyme‐constrained models). Ultimately, these models enable the study of the principles that underlie how the limited number of protein molecules that a cell can carry are optimally allocated to metabolic enzymes that have an inherent set of kinetic constants , providing insight into whether different omic data types can be used together to infer kinetic parameter values.et al, ex vivo erythropoietic models might be used in conjunction with gene editing of the adult hematopoietic stem cells to engineer specialist RBCs.The RBC is the most abundant cell in the human body, thus representing an important starting point for the development of personalized medicine in human systems. In a recent study, personalized RBC kinetic models were constructed through parameterization based on metabolomic, fluxomic, and genotyping data  from individual tumor samples to constrain maximum flux rates for cancer patients  and protein concentration (protein molecules per unit volume) through the well‐known equation, in vivo flux (iv), determined via fluxomics, is given by iv = app,ik × ie, where app,ik is the apparent catalytic rate for enzyme i in vivo, and ie is the in vivo enzyme concentration as determined by proteomic measurements. The term app,maxk (the maximum appk observed across multiple conditions) is a proxy for kcatinvivo and can be used in simulation models. When working with a large number of enzymes, ie becomes constrained by limited proteome size (∑i=1nei), and thus proteome allocation becomes important (Nilsson et al, The


Cripavirus isolates across experimental infections of 45 Drosophilidae host species. We find positive correlations between every pair of viruses tested, suggesting that some host clades show broad susceptibility and could act as reservoirs and donors for certain types of viruses. Additionally, we find evidence of virus by host species interactions, highlighting the importance of both host and virus traits in determining the outcome of virus host shifts. Of the four viruses tested here, those that were more closely related tended to be more strongly correlated, providing tentative evidence that virus evolutionary relatedness may be a useful proxy for determining the likelihood of novel virus emergence, which warrants further research.Virus host shifts are a major source of outbreaks and emerging infectious diseases, and predicting the outcome of novel host and virus interactions remains a key challenge for virus research. The evolutionary relationships between host species can explain variation in transmission rates, virulence, and virus community composition between hosts, but it is unclear if correlations exist between related viruses in infection traits across novel hosts. Here, we measure correlations in viral load of four  Many new infectious diseases are caused by viruses jumping into novel host species. Estimating the probability that jumps will occur, what the characteristics of new viruses will be, and how they are likely to evolve after jumping to new host species are major challenges. To solve these challenges, we require a detailed understanding of the interactions between different viruses and hosts, and metrics that can capture some of the variation in these interactions. Previous studies have shown that the evolutionary relationships between host species can be used to predict traits of infections in different hosts, including transmission rates and the damage caused by infection. However, the potential for different viruses to influence the patterns of these host species effects has yet to be determined. Here, we use four viruses of insects in experimental infections across 45 different host species of fruit fly to begin to answer this question. We find similarities in the patterns of replication and persistence between all four viruses, suggesting susceptible groups of related hosts could act as reservoirs and donors for certain types of virus. However, we also find evidence that different viruses interact in different ways with some host species. Of the four viruses tested here, those that were more closely related tended to behave in more similar ways, and so we tentatively suggest that virus evolutionary relatedness may prove to be a useful metric for predicting the traits of novel infections, which should be explored further in future studies.Virus host shifts, where viruses jump to and establish onward transmission in novel host species, are a major source of outbreaks and emerging infectious diseases  and recipient hosts is associated with decreased likelihood of cross‐species transmission  across a panel of 45 host species of Drosophilidae. Drosophila are a well‐established invertebrate model of innate immunity, responsible for major immunological discoveries including Toll, and possess both Dicer‐mediated antiviral RNAi responses and genotype‐specific immune memory  and is a widely used model insect pathogen  are correlated between viruses, using experimental infections of four Drosophila species in the wild—D. melanogaster and D. simulans  at 22°C, in a 12‐hour light‐dark cycle. Each bottle contained 50 ml of one of four varieties of food media (Supporting Information Methods), which were chosen to optimize rearing conditions of parental flies. Changes in the macronutrients available to adult Drosophila have been shown to have little effect on the outcome of viral infection . Gene sequences were aligned in Geneious version 9.1.8 (https://www.geneious.com) using a progressive pairwise global alignment algorithm with free end gaps and a 70% similarity IUB cost matrix. Gap open penalties, gap extension penalties, and refinement iterations were kept as default.The method used to infer the host phylogeny has been described in detail elsewhere , mitochondrial , and nuclear  groups. The mitochondrial and nuclear groups were further partitioned into groups for codon position 1+2 and codon position 3, with unlinked substitution rates and base frequencies across codon positions. Each group was fitted to separate relaxed uncorrelated lognormal molecular clock models using random starting trees and four‐category gamma‐distributed HKY substitution models. The BEAST analysis was run twice, with 1 billion Markov chain Monte Carlo (MCMC) generations sampled every 100,000 iterations, using a birth‐death process tree‐shape prior. Model trace files were evaluated for chain convergence, sampling, and autocorrelation using Tracer version 1.7.1  , DCV‐EB (MK645239), DCV‐M (MK645243), and CrPV (NC_003924) were retrieved from the NCBI Nucleotide database. Annotations of open reading frames (ORFs) for the replicase polyprotein  and structural polyprotein  were collected from the UniProtKB database and used to separate the coding and noncoding regions of each virus. ORF sequences were concatenated and aligned using the Geneious progressive pairwise translation alignment algorithm with a Blosum50 cost matrix and default parameters. Alignments were manually checked for quality and sequences aligning to CrPV ORF1 nucleotides 1–387 and 2704–2728 were removed due to the presence of large indels.Full genome sequences for DCV‐C . These needles were bent approximately 250 μm from the end to provide a depth stop and dipped in virus solution before being pricked into the pleural suture of each fly. Inoculation by this method has been shown to follow the same course as oral infection but is less stochastic  and were transferred to fresh media every 2 days for 1 week. Male flies were chosen to avoid any effect of sex or of female mating status that has been shown to influence the susceptibility of females to infection with other pathogen types  using chloroform‐isopropanol extraction, and reverse transcribed using Promega GoScript reverse transcriptase (Sigma) with random hexamer primers. qRT‐PCR was carried out on 1:10 diluted cDNA on an Applied Biosystems StepOnePlus system using Sensifast Hi‐Rox Sybr kit (Bioline). Cycle conditions were as follows: initial denaturation at 95°C for 120 seconds, then 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds.RpL32 housekeeping gene primers varied by species (Tables RpL32).DCV isolates were measured using the same primer pair  that targeted a conserved location and had similarly high efficiencies across all isolates. For CrPV, the following primers were used: forward, 5′‐TTGGCGTGGTAGTATGCGTAT‐3′; reverse, 5′‐TGTTCCGTCCTGCGTCTC‐3′. s Tables  and SC. tC values was estimated and corrected for using a linear model with plate ID and biological replicate ID as parameters, as described elsewhere  in either the 0 or 2 dpi samples were used as exclusion criteria for biological replicates. In total, of the 180 unique combinations of host species and virus measured, three biological replicates were obtained for 161 combinations, two replicates for 18 combinations, and one replicate for one combination . Power analysis based on the downsampling of previous data has shown that this provides adequate statistical power to detect interactions between different experimental treatments and host species  also includes a species‐specific random effect that is independent of the host phylogeny (μs:hv). This explicitly estimates the nonphylogenetic component of between‐species variance and allows the proportion of variance explained by the host phylogeny to be calculated. μs:hv was removed from model (2) as model (1) struggled to separate the phylogenetic and species‐specific traits. Wing size, measured as the length of the IV longitudinal vein from the tip of the proximal segment to the join of the distal segment with vein V . This was done to ensure that any phylogenetic signal in body size did not explain the differences seen in viral load between species v, where pv and sv represent the phylogenetic and species‐specific components of between‐species variance v, where ev is the residual variance of the model cov. If correlations between viruses are close to 1 (with no change in the variance while the means remain constant), it would suggest there are no host species‐by‐virus interactions  using the equation Falconer . Intersphttps://doi.org/10.6084/m9.figshare.13750711.v1.The data files and R scripts used in this study are available in an online repository: Drosophilidae with four virus isolates: DCV‐C, DCV‐EB, DCV‐M, and CrPV. The DCV isolates formed a distinct clade , with the closest relationship between DCV‐C and DCV‐EB. CrPV formed an outgroup to the DCV isolates  explaining a high proportion of the variation in viral load with little within‐species variation or measurement error (rv). We found no significant effect of wing length (a proxy for host body size) on viral load for any of the included viruses, with all estimates having credible intervals overlapping 0 , with the strongest correlation between DCV‐C and DCV‐EB (r = 0.97). Correlations between DCV isolates and the more distantly related CrPV were positive (r = 0.52–0.59) but weaker than the correlations between the DCV isolates. The fact the DCV:CrPV correlations (and their 95% CIs) are not close to 1 is consistent with virus‐by‐host species interactions on viral load  Fig. . We founr = 0.04; 95% CI: >0.001, 0.09; PMCMC = 0.04), and more strongly correlated to DCV‐M than to CrPV , consistent with an increase in the strength of correlation between viruses with closer evolutionary relatedness. Point estimates imply a similar pattern for DCV‐EB, but the evidence for a stronger correlation with DCV‐C than DCV‐M was not well supported (Table DCV‐C appears to be slightly more strongly correlated to DCV‐EB than to DCV‐M (Δed Table .Cripavirus isolates across 45 host species of Drosophilidae, to look for similarities between related viruses as well as evidence of virus‐by‐host species interactions on the outcome of infection. We found positive correlations between every pair of viruses tested, indicating broad similarities in the outcome of infection across host species, but also evidence for interactions between virus and host species with changes in the rank order of host species susceptibility between the different viruses (Fig. Closely related host species present similar environments to novel viruses (Freckleton et al. ses Fig. . This hiZaprionus davidi, DCV‐EB shows a decline in viral load, suggesting it is failing to replicate and persist in this host species, whereas the other isolates show an increase in viral load in the same host. Similarly, Scaptodrosophila pattersoni is among the least susceptible to DCV‐M but has relatively high viral loads for the other virus isolates.The strong positive correlations between DCV isolates are likely due to relatively high levels of sequence conservation resulting in only small differences in their ability to infect different host species. However, in other viruses a small number of mutations have been shown to allow successful infections in novel hosts (Allison et al. Drosophila ananassae and Drosophila sturtevanti are within the five most susceptible species to DCV‐C, but also the eight least susceptible to CrPV. The weaker correlations that exist between DCV and CrPV may be due to interactions with different host traits that vary in their patterns across the host phylogeny. CrPV and DCV are known to have distinct methods of suppression of the host antiviral RNAi pathway (van Rij et al. A greater number of these effects can be seen when comparing hosts infected with DCV isolates to those infected with CrPV, where multiple species have markedly different susceptibilities depending on the virus infecting them. For example, both Drosophilidae host species. Host species that are permissive to multiple viruses and virus genotypes may allow for the persistence of increased genetic diversity in the virus population, allowing viruses to generate and maintain mutations that make them more likely to emerge in novel host species (Woolhouse et al. The existence of correlations between viruses suggests that host susceptibility is not specific to individual viruses and that certain host clades may be broadly susceptible to infection. These hosts may share cell surface receptors with high affinity for both DCV and CrPV surface proteins, have a low efficiency or easily suppressed antiviral RNAi response, or have functionally diverged forms of other cellular processes linked to viral replication and persistence. Divergences in these immunological traits are possible candidates driving the large amount of variation in susceptibility we have detected across Drosophila parasites (Perlman and Jaenike The differences in correlation strength between pairs of viruses tended to follow differences in their evolutionary divergence, such that more closely related pairs of viruses were more strongly correlated in the outcome of infection across host species. This provides some tentative evidence that the ability of a virus to infect a novel host may be inferred based on its evolutionary relatedness to other viruses. A greater number of more diverged isolates from this virus family would have allowed this potential phylogenetic effect to be investigated more conclusively, although to our knowledge the viruses included here represent the most diverged viruses of this genus that are readily available for study. The pathogen phylogenetic effects seen here have also been observed in other pathogen and parasite systems (Vienne et al. Nevertheless, virus phylogenetic effects may still prove to be a useful proxy for determining the likelihood of novel virus emergence. Further work is now needed to expand the findings of this study to broader groups of viruses, and to test the importance of the virus phylogeny in determining the potential outcomes of virus host shifts.Supplement MaterialClick here for additional data file.


While a wide range of treatments exist for actinic keratosis and skin field cancerisation, the long‐term benefits of the most common topical therapies are poorly defined. This report reviews the efficacy of the most commonly used topical therapies to treat regional or field lesions. Limited clinical and histopathological data are available on clearance rates at 12 months post‐treatment for the most commonly used agents, with varied outcome measures making any comparison difficult. In general, total field clearance rates at 12 months are suboptimal for the most commonly employed agents. Given the increasing incidence of actinic keratosis and skin field cancerisation due to an ageing population, further research into the efficacy of therapies is critical to guide treatment choice. Skin field cancerisation describes areas of solar damage involving actinic keratosis, the clinical signs of photoageing, and variable numbers and types of skin cancers. These areas also commonly have a history of previous skin cancers and are prone to develop new malignancies, including nonmelanoma skin cancer, melanoma and rarely Merkel cell carcinoma. Current topical treatment options include 5‐fluorouracil (5‐FU), imiquimod, ingenol mebutate, diclofenac sodium and aminolevulinic acid photodynamic therapy (PDT). The goal of these therapies is to reduce the numbers of actinic keratosis and to prevent future cancer development. However, despite the burden and prevalence of actinic keratosis and skin field cancerisation, there is little understanding of treatment outcomes, as measured by recurrence or cancer prevention.https://www.ncbi.nlm.nih.gov/pubmed/), Cochrane Library and Web of Science were searched from January 1962 to February 2019 to identify relevant English language publications using the following specific search terms: ‘(skin field cancerisation OR skin field cancer) AND (actinic keratosis OR solar keratosis) AND  OR ’. The retrieved titles and abstracts were screened for review on the efficacy for actinic keratosis and skin field cancerisation. For the purpose of this review, data from the face and scalp were selected for inclusion, and the treatment regimens analysed approximated the common treatment schedules. This review also provides an overview on actinic keratosis, skin field cancerisation, current methods of grading skin field cancerisation and limitations of the studies analysed.To address this point, we conducted a literature review to elucidate the effectiveness of commonly used topical therapies. The PubMed/MEDLINE database (Actinic keratosis is a keratotic lesion occurring on chronically light‐exposed adult skin presenting as a dry, rough, sometimes pigmented lesion of variable thickness and size.Actinic keratosis is also a visible hallmark of skin field cancerisation and often helps to define its extent.Most historical studies have graded field damage on simple actinic keratosis counts, but measured in a variety of ways.Actinic keratosis numbers alone have been criticised for unreliability of grading because of significant differences in investigator counts and for being nonreproducible with significant count variations over time.Numerous studies have tested the efficacy of common topical therapies for actinic keratosis. Despite this, there is a significant lack of high‐quality randomised controlled trials and the durability of outcomes for multiple therapies has not been established.A major review of topical 5‐FU identified only 13 out of 29 randomised controlled trials with meaningful data for analysis. Of these, only three reported on patients at 12 months.In this review, the percentage reduction of actinic keratosis counts measured at 3–6 and 12 months was compared to baseline counts (Table Field therapies aim to reduce actinic keratosis for several reasons. There is undoubted benefit in lessening skin irritability and improving appearance, but the primary goal has always been to reduce the incidence of new sun‐related cancers.All the common topical therapies reviewed provided some short‐term reduction in actinic keratosis with 5% 5‐FU and PDT being the most effective formulations for short‐term clearance and ingenol mebutate being marginally less effective. Overall clearance at 12 months was marginally better for PDT compared to other agents but all had significant recurrence in a field of cancerisation, with approximately 50% of the baseline numbers evident at one year post‐treatment. Treatment combinations appear to improve efficacy compared to monotherapies but at significant additional cost.The natural history of actinic keratosis demonstrates a very high turnover, with large numbers developing, regressing and recurring over time.In summary, current topical therapies provide good short‐term clearance of actinic keratosis but significantly lower clearance rates by 12 months. The short follow‐up periods reported for many studies may reflect a desire to maximise apparent benefits while satisfying minimal regulator review requirements. The FDA approvals process have had to accommodate significant differences in study protocols.


Obesity in adolescence presents a major public health challenge, often leading to obesity in adulthood with associated chronic disease.This study aimed to perform a population pharmacokinetic and exposure‐response analysis of liraglutide by meta‐analysis of data from trials conducted in children, adolescents and adults with obesity.The population pharmacokinetic analysis investigated the effect of covariates body weight, age group  and sex on liraglutide exposure in adolescents compared with previous results in adults. The exposure‐response relationship of liraglutide for the change from baseline in body mass index standard deviation score (BMI SDS) was evaluated in adolescents and compared to that in adults.Body weight was the main covariate affecting liraglutide exposure, with lower exposures at higher body weights, whereas age group was of no importance and sex was of little importance. An exposure‐response relationship was demonstrated for liraglutide in both adolescents and adults as the decrease in BMI SDS from baseline increased in an exposure‐dependent manner with increasing liraglutide exposure.The population pharmacokinetic analysis supported similar liraglutide exposures in adolescents and adults; body weight was the most important covariate affecting exposure. An exposure‐response relationship was established for liraglutide. The safety, tolerability, pharmacokinetics and pharmacodynamics of liraglutide in adolescents aged 12‐17 yearsA previous population pharmacokinetic analysis performed with liraglutide using adult data from the 56‐week phase 3a SCALE Obesity and Prediabetes trial (1839)22.1The objectives of the population pharmacokinetic analysis were (1) to investigate body weight, age group  and sex as covariates to determine whether their impact on liraglutide exposure was in accordance with previous results in adultsData from trials 4180 and 3967 (in adolescents), 4181 (children) and 3630 (adults) were included in the pharmacokinetic assessment of liraglutide (Table a (absorption rate constant)kCL/F (apparent clearance)V/F (apparent volume of distribution)A standard one‐compartment model with first‐order absorption and elimination was the starting point for the description of liraglutide pharmacokinetics, and the model was developed and validated according to the US FDA and European Medicines Agency guidelines.The first‐order conditional estimation with interaction (FOCE‐I) method was used for the population pharmacokinetic analysis, implemented in the non‐linear mixed effects modelling (NONMEM) software. Due to pharmacokinetic sampling in steady‐state conditions, exposure levels were expected to be much higher than the LLOQ . Observed values close to or below the LLOQ were therefore believed to be due to missed doses. Data records with missing concentration values or concentration values below the LLOQ were excluded from the population pharmacokinetic analysis in order not to overestimate CL/F. Stricter exclusion criteria were tested in sensitivity analyses but were found not to be relevant.An analysis of the influence of covariates on exposure was carried out including all tested covariates in one step, using a confirmatory approach,th subject (shown for CL/F only):The covariate effects of baseline body weight, sex and age group were investigated for CL/F. Due to the sparseness of data, as well as the focus on average exposure, only the effect of baseline body weight was investigated with respect to V/F. The CL/F and V/F were parameterized as follows for the iSpecific steady‐state exposures for individual participants were based on the full model, including all covariates, and derived from the subject‐specific post‐hoc CL/F estimates, the maintenance dose and the dose interval (24 hours):The CIs for full model parameters were estimated using bootstrapping. For each investigated covariate, differences were considered relevant if the 90% CI of the estimated mean of the relative exposure fell outside the standard bioequivalence limits (0.80‐1.25).a. Within‐subject variability  was described by a proportional error model. Standard graphical quality analyses, including goodness‐of‐fit plots  to investigate the exposure‐response relationship of liraglutide in adolescents with respect to the change from baseline in BMI SDS and (2) to determine whether the exposure‐response relationship for the change from baseline in BMI SDS was similar in adults and adolescents.An evaluation of the exposure‐response of liraglutide in adults has been published previously using data from the phase 2 trial (1807), the phase 3a SCALE Obesity and Prediabetes trial (1839) and the phase 3a SCALE Diabetes trial (1922).avg) estimates from the population pharmacokinetic analyses of the four trials . For the primary exposure‐response analysis of change from baseline in BMI SDS, BMI SDS was calculated according to the World Health Organization (WHO).The exposure variable used for the analysis was individual model‐based average liraglutide concentration  and clear . Based on the reduced model from the previous step, covariates for Emax were investigated via forwards inclusion. Based on plots of exposure‐response stratified by covariates, each covariate factor was considered. If the data indicated differences in the treatment effect, the covariates were investigated by forwards inclusion and included if statistically significant (P < 0.05). If the covariates appeared to be relevant based on the plots, they were tested in the following order: predefined covariates for Emax were investigated first, then other clear covariates for Emax.A modified exposure‐response model based on the previous meta‐analysisStandard goodness‐of‐fit plots were made for checking and evaluating the model.0 was the placebo effect, Emax was the maximal drug effect, EC50 the exposure leading to half‐maximum effect, Ecov covariate effects on the overall response  and e the normal distributed residual error. Sex, baseline response parameter, age group and trial factors were considered as covariates. Baseline BMI SDS and age group were included as covariates in the final model.The final models were parameterized as follows:adolescent was the covariate representing a lower Emax in adolescents compared to adults, where adolescent was an indicator variable for adolescents. Imale was the covariate representing a lower Emax in males compared to females, where male was an indicator variable for males. ɣ was the Hill coefficient. In the final models, Ecov covariate effects for body weight and BMI included age group, while for waist circumference the covariate effects were baseline waist circumference (cm), age group and diabetic state . The “non‐diabetes” also included individuals with prediabetes. The other expressions were as defined above.The exposure‐response evaluation for changes from baseline in body weight, BMI and waist circumference were evaluated using similar models as for the BMI SDS:The exposure‐response relationship for the proportion of participants with at least 5% reduction in body weight (body weight responder rate [BW.RR]) was estimated using logistic regression analyses parameterized as:2.3The software program NONMEM  version 7.3 was used for the population pharmacokinetic analysis. R version 3.2.3  was used for data file processing, explorative data analysis and plotting. Exposure‐response analyses were implemented in R.33.13.1.1A base model without covariates was developed based on prior knowledge of liraglutide pharmacokinetics. Subsequently, a full model, which was the base model with all investigated covariates included, was estimated for the covariate analysis. All investigated covariates were significant, except for age group. The full model could therefore not be reduced and, therefore, the full model was adopted as the final model.A one‐compartment model successfully described the pharmacokinetics of liraglutide. The parameter estimates from the base and final models are presented in 3.1.2A total of 176 individuals was included in the analysis: 13 children from trial 4181, 121 adolescents from trial 4180, 13 adolescents from trial 3967 and 29 adults from trial 3630 ; 22 samples from two individuals were excluded during data cleaning due to timing being missing or an inadequate dosing history. There were also many lower‐than‐expected individual liraglutide values in trial 4180 . Data cl3.1.3The effects of intrinsic covariates on liraglutide exposure are shown in Figure avg values appeared to be similar in adolescents and adults when adjusted to the 3.0 mg dose, whereas children had slightly higher liraglutide concentrations  imputation was used if the observation was missing at the end of treatment, consistent with the original analysis.3.2.2An exposure‐response relationship was demonstrated for liraglutide in both adolescents and adults with a greater decrease from baseline in BMI SDS as liraglutide exposure increased Figure . Similarmax) was not significant in these analyses. The exposure‐response for adolescents in trial 4180 who gained height of at least 0.5 cm during the trial was not significantly different from the exposure‐response for those who had reached their final height. Approximately 64% of the adolescents in the exposure‐response analysis grew more than 0.5 cm during the trial. Of these, some received liraglutide and some received placebo. For those receiving placebo treatment, the average growth was 1.8 cm, while for those receiving liraglutide treatment, the average growth was 1.4 cm (data not shown).For all response variables, similar exposure‐response relationships were seen in both adolescents and adults. The two age groups differed in the placebo group response, with lower mean responses in adolescents; this difference was consistent across the exposure range as well. There was a tendency for a steeper exposure‐response in adolescents for all response variables, leading to overlapping effects with adults at the highest liraglutide exposures. However, the effect of age group on the maximal effect in the model , body weight (%), waist circumference and the proportion of participants achieving at least a 5% weight loss increased in an exposure‐dependent manner.The recommended 3.0 mg liraglutide dose was shown to provide similar exposures in adolescents and adults, even without adjusting for baseline body weight, which supports using the same dose as approved in adults for weight management in adolescents. The added benefits of increasing liraglutide exposures, in terms of a greater reduction in BMI SDS and greater changes in other response variables, were similar in adolescents with obesity and adults with overweight or obesity, thus further supporting the 3.0 mg treatment dose. While individual and mean exposures appeared to be slightly higher in children than in adolescents or adults, exposures were similar across age groups after adjusting for body weight.Although body weight was found to be the most important covariate affecting exposure and an exposure‐response relationship was demonstrated, the maintenance dose for liraglutide 3.0 mg is not dose‐adjusted according to body weight.A previous population pharmacokinetic analysis of liraglutide dosed up to 3.0 mg also identified body weight as a key covariate affecting exposure in adults with overweight or obesity with or without type 2 diabetes mellitus; sex was additionally identified as a key covariate in this large phase 3 dataset.max model with baseline BMI SDS and age group as covariates on the placebo response. As the BMI SDS tends to be skewed at higher BMI SDS values,max models of exposure‐response.The primary response variable used for the present exposure‐response analysis of liraglutide for weight management was BMI SDS, which is a measure of the number of SDs from the population mean BMI, matched for age and sex.The difference in the weight‐loss responses observed between adults and adolescents in the placebo group, where adults achieved a greater mean weight loss than adolescents, could have been due to lower adherence to active treatment and/or the diet and exercise program in some adolescents, although linear growth (height increase) in a part of the adolescent population might also have contributed. There were several lower‐than‐expected individual liraglutide concentration measurements in trial 4180 in adolescents, which could also potentially indicate a reduced adherence to the trial medication for some trial participants. Low liraglutide concentrations were also observed in four children in trial 4181, although the reason for this was unknown.In contrast, for adolescents with the highest liraglutide exposures, there was a tendency for weight‐loss responses being comparable to the adult responses. Although the tendency for a steeper exposure‐response in adolescents was not significant in this small data set, the exposure estimates in trial 4180 were based on sparse pharmacokinetic sampling over a long time period, and the estimates could be influenced by samples obtained during periods with reduced or varying adherence to treatment. Thus, the individual exposure estimates also could reflect the adherence level.Limitations of the present exposure‐response analysis include a relatively low number of paediatric participants as well as some variability in the populations included in terms of inclusion criteria and background lifestyle therapy. Trial design differed between the trials and included counselling in healthy nutrition and physical activity for all participants, but for the adult participants in trials 1839 and 1922 a calorie‐restricted diet was also included. Moreover, the reduced adherence to treatment in adolescents compared with adults as speculated above could not be confirmed in practice. In the exposure‐response analysis, the value for a 19‐year‐old female or male was used as the adult value for the BMI SDS response variable,In summary, the population pharmacokinetic analysis indicated similar exposures in adolescents and adults, with similar body weight ranges between the studied adolescents and adults. Body weight was identified as the most important covariate affecting exposure. An exposure‐response relationship with a larger response with increasing liraglutide exposure was established for BMI SDS change from baseline as well as the percentage change from baseline in body weight and BMI, change from baseline in waist circumference, and for the proportion of participants with at least 5% weight loss. While a lower mean response was observed in adolescents compared to adults, consistent with the placebo response across the exposure range, the added benefits of increasing liraglutide exposures were similar in adolescent and adults with overweight or obesity.KCCP, PMH, DH and NR are employees of Novo Nordisk and KCCP, PMH and DH own stocks in Novo Nordisk. LDM has received grant support paid to the University at Buffalo from AstraZeneca and Novo Nordisk and fees for serving as a healthcare professional consultant from Novo Nordisk.Appendix S1 Supporting informationClick here for additional data file.


We report a case of percutaneous epicardial left atrial appendage exclusion in a patient with the atrial septal closure Trans‐septal access following an atrial septum closure (ASC) is challenging and is generally positioned in contra‐indication of endocardial LAA closure. Successful implantation of endocardial LAA occlusion after the ASC has reported on a few cases. We report the first experience of percutaneous epicardial LAA exclusion in a patient with the ASC.A 51‐year‐old man with paroxysmal AF  underwent percutaneous ASC  for a patent foramen ovale following a cryptogenic stroke two years before. In addition, an implantable loop recorder (ILR)  was implanted. During the follow‐up, frequent AF episodes were documented and transient ischemic attack was observed under oral anti‐coagulation (OAC) therapy. Although lifelong OAC therapy was recommended with mild bleeding risk , the patient refused long‐term OAC therapy and requested LAA occlusion for stroke prevention instead of a second foreign body. After a great deal of consideration for a procedural limitation of trans‐septal puncture following ASC and a potential risk of the recurrent intra‐atrial shunt, we chose epicardial LAA exclusion using the LARIAT device , potentially favorable to electrical and structural LAA remodeling decreasing the AF recurrence rate and a smaller sheath reducing the risk for post‐procedural intra‐atrial shunt. Following dry epicardial puncture, a trans‐septal puncture was carefully performed under fluoroscopic guidance posteroinferior to the device using an 8,5F SL 1 sheath  Figure . ChickenLAA closure is an alternative therapy for patients with AF and a high bleeding risk for long‐term anticoagulation therapy. Epicardial LAA occlusion has several potential advantages as compared to endocardial devices. First, the LAA is one of the important substrates and triggers for AF. LAA ligation results in favorable electrical and structural remodeling of the LAA and has been shown to decrease the AF recurrence rate associated with the LAA trigger. Second, a smaller trans‐septal sheath (8F) is required for the LA access thereby reducing the risk for intra‐atrial shunt. Third, no foreign body remains in the heart. LAA ligation is a therapeutic option of LAA closure in a case with the difficulty of trans septal puncture and rhythm management for AF.R. R. Tilz received travel grants from SentreHeart. The other authors have nothing to disclose.


Osteosarcoma (OS) is the most common primary malignant bone tumour with a peak in incidence during adolescence. Delayed patient presentation and diagnosis is common with approximately 15% of OS patients presenting with metastatic disease at initial diagnosis. With the introduction of neoadjuvant chemotherapy in the 1970s, disease prognosis improved from 17% to 60%‐70% 5‐year survival, but outcomes have not significantly improved since then. Novel and innovative therapeutic strategies are urgently needed as an adjunct to conventional treatment modalities to improve outcomes for OS patients. Angiogenesis is crucial for tumour growth, metastasis and invasion, and its prevention will ultimately inhibit tumour growth and metastasis. Dysregulation of angiogenesis in bone microenvironment involving osteoblasts and osteoclasts might contribute to OS development. This review summarizes existing knowledge regarding pre‐clinical and developmental research of targeted anti‐angiogenic therapy for OS with the aim of highlighting the limitations associated with this application. Targeted anti‐angiogenic therapies include monoclonal antibody to VEGF (bevacizumab), tyrosine kinase inhibitors  and human recombinant endostatin (Endostar). However, considering the safety and efficacy of these targeted anti‐angiogenesis therapies in clinical trials cannot be guaranteed at this point, further research is needed to completely understand and characterize targeted anti‐angiogenesis therapy in OS. Mechanistic insights into the OS targeted anti‐angiogenesis therapy. It is characterized by a high incidence of metastasis at the time of diagnosis most commonly with early haematogenous lung metastasis, a poor prognosis and a high incidence of recurrence. The use of neoadjuvant chemotherapy with multiple agents has improved the 5‐year event‐free‐survival (EFS) of non‐metastatic patients to over 70% while reducing the incidence of amputation by 10%‐20%.Compared with traditional treatment methods, targeted therapy is a type of high selectivity and low toxicity cancer treatment that uses drugs or other substances to precisely identify and attack certain types of molecules that are involved in the growth and progression of cancer .In 1971, Folkman et al2Angiogenesis is a complex and highly adaptive process that is crucial for tumour growth and metastasis. The biological processes involved in angiogenesis include endothelial cell proliferation, differentiation and migration, recruitment of smooth muscle cells and maturation of blood vessels, with the processes being strictly controlled by vascular regulatory factors.Possible targets for anti‐angiogenesis therapies include transcription factors or signal pathways that directly or indirectly affect VEGF, PDGF, fibroblast growth factor (FGF), hepatocyte growth factor, integrin, cyclooxygenase (COX‐2), metalloproteinases  and HIF‐1.As the most studied marker of tumour neovascularization, VEGF and VEGFRs regulate both the development of blood vessels from precursor cells during embryogenesis (vasculogenesis), and the formation of blood vessels from pre‐existing vessels (angiogenesis). It has also been shown to promote endothelium proliferation, inflammation and vascular permeability.Over‐expression of VEGF and the resultant increase in angiogenesis have been reported in a number of human cancers including OS.3Current management of non‐metastatic OS is multimodal consisting of aggressive chemotherapy combined with radical surgical resection. Various combinations of adjuvant chemotherapeutic agents have been used, though the majority of recent trials still use high‐dose methotrexate, doxorubicin, and cisplatin (MAP).3.1Monoclonal antibodies (mAb) are laboratory engineered proteins which serve as substitute antibodies that can restore, enhance or mimic the immune system's attack on specific target cancer cells. mAb targeted drugs, which recognize and bind specific receptors or associated antigens on the surface of tumours, have been shown to improve the efficacy and reduce the toxic side effects of drugs in normal tissues.Several pre‐clinical OS studies have confirmed the anti‐angiogenesis and anti‐cancer effects of bevacizumab in OS cell lines. Zhong ZY et alMany distinct escape mechanisms have been shown to protect malignant cells from immune recognition and destruction in the tumour microenvironment, and these mechanisms make the response from single‐target drugs like bevacizumab short‐lived. As a result, it is often used in combination with other chemotherapeutic drugs in clinical trials. In a single‐centre trial using docetaxel, bevacizumab and gemcitabine for the treatment of high‐risk sarcomas with metastasis and/or recurrence in adolescents/young adults, Kuo et alBevacizumab has been shown to normalize tumour vasculature both in the laboratory and in patients with other types of cancers and therefore improve access for chemotherapy. In combination with other chemotherapeutic agents, it has been shown to be efficacious in the treatment of several cancers clinically by increasing tumour response and prognosis. However, there is a need for further investigation to the use of bevcizumab alone or in combination with other agents, to demonstrate its efficacy in OS patients.3.2Tyrosine kinases are important mediators of the signalling cascade.3.2.1Sorafenib is the first oral multi‐kinase inhibitor that targets VEGFR‐1, VEGFR‐2 and VEGFR‐3 to inhibit angiogenesis, the RET gene also known as RET/PTC rearrangement, RAF (including BRAFV600E) and platelet‐derived growth factor receptors to inhibit tumour progression.Three studies to date have evaluated the clinical efficacy of sorafenib alone for the treatment of OS. A phase II trial of sorafenib by the Italian Sarcoma Group in 2012Oncogenic activation of mTOR signalling significantly contributes to the progression of different types of cancers including OS, and the PI3K/Akt/mTOR pathway has been implicated in the metastatic behaviour of OS.Cathomas et alAlthough sorafenib is a relatively new drug, it is now considered first‐line treatment for advanced hepatocellular carcinoma and renal cell carcinoma. Its effect on OS has yet been studied on a large scale, and more clinical data are needed to properly assess its efficacy as a possible treatment option particularly for patients with metastatic disease and refractory OS. Current studies have demonstrated that the effect of sorafenib as a monotherapy for OS patients was unsustainable. Different pathways involved in the pathogenesis of OS warrant further investigation looking at multiple‐targeted therapeutic approaches which may yield major improvements in the management of the disease.3.2.2Apatinib is a small molecule tyrosine kinase inhibitor that highly and selectively targets VEGFR‐2 and also inhibits the activities of VEGFR‐1, Kit, c‐SRC and RET,Apatinib is reported to be safe and efficacious to use in patients with advanced OS. Clinical trials by Zhou et alFor patients with advanced OS non‐responsive to chemotherapy, apatinib proved to be effective in prolonging PFS in previous multi‐centre trials and was consequently added to the National Comprehensive Cancer Network guidelines in the United States as a second‐line therapy. However, it must be noted that these promising results have been limited to mostly retrospective studies with small population sizes without controls. Additional studies are needed to fully calculate the efficacy of apatinib for the treatment of OS.3.2.3Pazopanib, a second‐generation multi‐target TKI, is approved for the treatment of soft tissue sarcomas and has a high affinity for VEGF receptors including PDGFR‐a/3 and c‐Kit receptors.A few studies have suggested that the use of pazopanib as an inhibitor of these tyrosine kinases can lead to an improved clinical response. A study by Danish researchersThese reports indicate that pazopanib has strong anti‐tumour activity and may contribute to improved survival. The response rates, PFS and overall survival are comparable with the efficacy shown using sorafenib alone or in combination with everolimus. The toxicity profile of pazopanib also appeared to be acceptable for treatment. Recently, paxopanib was approved for second‐line treatment in non‐adiopocytic soft tissue sarcoma (STS) after failure of standard chemotherapy based on the PALETTE study—a double‐blind, randomized, phase 3 trial which was conducted globally comparing the efficacy of pazopanib versus placebo for the treatment of metastatic STS.3.2.4Regorafenib is a small molecule multi‐kinase inhibitor, which inhibits the activity of VEGFR1‐3, KIT, PDGFRB, RAF, RET and BRAF, and has been demonstrated to increase the overall survival of patients with US FDA approval for use in metastatic colorectal cancer, advanced gastrointestinal stromal tumours and hepatocellular carcinoma.Phase I clinical trials conducted by Klaus et al3.3Endostatin, isolated from a culture medium of rat endothelioma by O’Reilly MS et al in 1997,Pre‐clinical studies reported that the combination of ES and chemotherapy agents significantly inhibited tumour angiogenesis and growth. Zhang XL et alThe anti‐tumour activity of ES has been demonstrated in clinical trials. Xu et al3.4In recent years, the synergistic effects of traditional Chinese herbal medicine (CHM) combined with radio‐ and chemotherapy have been gaining increasing significance as its effectiveness as an anti‐angiogenetic agent are being explored. Huang YM et al4There is no established systemic treatment option for advanced or unresectable OS progressing after standard chemotherapy, and as such, the survival rate of patients with OS has not improved significantly in recent years. Angiogenesis is a key factor affecting tumour growth and metastasis, and theoretically, anti‐angiogenic therapeutic agents present potentially novel therapies for various cancer types.Recently, progress has been made in the development and application of targeted anti‐angiogenic drugs, providing much‐needed relief in the search of therapeutic alternatives for OS patients. Much of this research has focussed on cases of refractory and/or metastatic disease following failed traditional chemotherapy and surgical resection. However, the search for alternative targeted anti‐angiogenic regimens is still in its infancy and a long way from achieving desired clinical applications. New research has generated plenty of excitement, but still faces a myriad of questions before it can be widely adopted for clinical use. For instance, how do we predict the therapeutic effect of an anti‐angiogenetic targeted therapeutic agent for OS, and which combination of new agent with traditional therapies would be most effective. More detailed clinical studies are needed to establish reasonable norms and guidelines for the application of these plausible therapeutic alternatives. With much‐needed technological developments and extensive research, targeted anti‐angiogenesis therapy could become a potent and effective weapon in our fight to effectively manage patients with OS.All authors declare that they have no conflict of interest.Yun Liu and Nenggan Huang conducted research and drafted the manuscript. Shijie Liao, Emel Rothzerg, Yihe Li and Felix Yao provided assistance in the process of revised drafting manuscript and figure and tables construction. David Wood and Jiake Xu contributed to conceptual framework, supervised the study and revised the manuscript.