Influenza superinfections with GAS have been described to occur regularly and to show a severe clinical picture with high mortality. We aimed to study the extent to which influenza A and B viruses (IAV and IBV), respiratory syncytial virus (RSV) and rhinovirus circulation contribute to iGAS incidence and severity.Invasive infections by group A STime‐series regression models were built to explore the temporal associations between weekly laboratory counts of IAV, IBV, RSV and rhinovirus as independent variables and weekly counts of GAS disease notifications or laboratory GAS cultures as dependent variables.The weekly number of IAV detections showed a significant temporal association with the number of notifications of streptococcal toxic shock syndrome (STSS), a severe complication of iGAS. Depending on the season, up to 40% of all notified STSS cases was attributable to IAV circulation. Besides STSS, none of the other iGAS manifestations were associated with a respiratory virus.Our study found an ecological temporal association between IAV and STSS, the most severe complication of iGAS. Future studies are needed to confirm this association and assess the possible preventability of STSS by influenza vaccination, especially in the age group 60 years and older. As a complication of invasive GAS infection (iGAS), streptococcal toxic shock syndrome (STSS) may develop, which has a mortality rate of around 40%.Streptococcus pneumoniae also included GAS.In 2010‐2011, an increased iGAS incidence was observed in the United Kingdom during a high influenza season.Streptococcus species (Streptococcus pneumoniae),Therefore, the extent to which influenza contributes to iGAS seasonality or severity remains unknown. Importantly, in the case of such an association, a proportion of iGAS cases is potentially avertable by influenza prevention. Patients with iGAS disease are not usually tested for influenza infection, which complicates investigation of a link between the two infections. However, comprehensive surveillance systems both of respiratory pathogens and of iGAS disease separately offer an opportunity to explore the association between iGAS and respiratory pathogens through modelling of their time series. In the absence of individual‐level data on co‐infections, such time‐series analyses using population‐level data are an important tool for hypothesis generation and directing future in‐depth research. While time series have been used to estimate the association between respiratory virus infections and infections with another Our research question was whether influenza (A or B) seasonality was associated with iGAS disease in the Netherlands, and if so, whether this association was specific to influenza or whether it existed for any respiratory virus with a winter season. We constructed time‐series regression models to estimate the temporal, population‐level associations between weekly respiratory virus counts  and weekly counts of iGAS disease notifications or microbiological GAS cultures.22.12.1.1Weekly numbers of respiratory virus detections from week 1 2008 to week 26 2018 were extracted from the Virological Weekly Reports (VWR), a voluntary and sentinel laboratory surveillance system established by the Dutch Working Group on Clinical Virology. Participating laboratories report the weekly counts of detections for 46 pathogens. No patient‐level data are collected. Participation has been consistent over the study period, with a median of 20 (range 17‐21) laboratories reporting weekly, and the data were shown to be representative of epidemiological trends on the national level in a recent evaluation comparing the VWR to mandatory notification data.2.1.2Notifications of iGAS disease per week (week 1 2011‐week 26 2018) were extracted from the national database for notifiable diseases “OSIRIS.” In the Netherlands, three manifestations of iGAS disease are notifiable since 2011: STSS, necrotising fasciitis and puerperal sepsis. Since June 2016, also puerperal  fever with a GAS culture from a non‐sterile site  is notifiable. Puerperal sepsis or puerperal fever will henceforth be referred to as puerperal GAS. STSS and necrotising fasciitis are notifiable if GAS is cultured from a normally sterile site, or when GAS is cultured from a normally non‐sterile site, in the absence of another micro‐organism that can cause the disease. Since notifiable by law, notifications of iGAS diseases STSS, necrotising fasciitis and puerperal GAS should include all cases occurring throughout the Netherlands, some underreporting notwithstanding.2.1.3Weekly numbers of GAS‐positive cultures (week 1 2008‐week 52 2017), by specimen type, were derived from the national Infectious Diseases Surveillance Information System—Antimicrobial Resistance (ISIS‐AR).2.2P‐values were used to determine whether a parameter was associated with the dependent variable. Parameters with P‐values <0.1 were included in a stepwise model selection.We used time‐series regression to estimate the temporal association between respiratory virus detections and both iGAS notifications and GAS cultures. Negative binomial models were constructed with weekly counts of respiratory viruses as independent variables and weekly counts of iGAS disease notifications  or GAS cultures (per specimen type) as dependent variables. Building a separate model per each dependent variable, we thus built eight models. For estimating the association between the weekly counts of respiratory virus detections and iGAS disease notifications, the period of week 1 2011 to week 26 2018 was included. The GAS culture data were available from week 1 2008 up to week 52 2017; therefore, this period was included in the GAS culture models. For ease of interpretation, we used an identity link, which assumes the effects of different respiratory viruses on GAS incidence to be additive rather than multiplicative. In model selection steps, To allow a baseline seasonal variation in GAS, independent of respiratory virus activity, first a sine wave was modelled before including the respiratory virus variables. For each GAS outcome, initially a model was constructed with four sets of sine/cosine terms, and the number of significant sine/cosine pairs was determined by backward selection. Linear time trends were added only when significant. For all four respiratory viruses, we also explored whether they were significantly associated with GAS when a time lag was taken into account . Time lags of zero up to 4 weeks were explored in this manner. Negative (inverse) associations between respiratory viruses and GAS were disregarded, as we assumed a protective effect of respiratory viruses on GAS disease to be biologically implausible.If the number of IAV detections showed an overall significant association with GAS, we explored whether a time‐dependent approach improved the model fit, that is separating IAV detections into separate variables per season (defined as week 27‐week 26 the next year). This was done because IAV strain dominance and antigenic drift are known to result in season‐specific influenza epidemiology .To estimate the fraction of iGAS disease attributable to respiratory viruses, beta coefficients from the final model were multiplied by the observed count of virus detections. Analyses were performed in Stata version 15 (StataCorp).3Weekly numbers of detections of respiratory viruses between January 2008 and June 2018 reflected the typical seasonality of respiratory viruses IAV, IBV and RSV, that is with clear annual peaks around winter and almost absent outside of their season Figure , but ret3.1In all models, one set of sine/cosine terms (of 1‐year periodicity) showed the best fit. A secular time trend improved the fit of (and was therefore included in) all models with GAS culture numbers as dependent variables, likely reflecting the increasing number of laboratories contributing to the data over the study period. Detections of respiratory viruses IAV, IBV, RSV and rhinovirus were not significantly associated with counts of any of the GAS culture specimen types, or notifications of necrotising fasciitis or puerperal GAS, either in the same week or with any of the four explored time lags.P = 0.007, Table P‐value was below 0.1, and beta coefficients differed notably by season. The model with season‐specific IAV variables was selected as final model GAS are scarce, some studies have also reported that influenza‐iGAS coinfection increases iGAS disease severity. For example, in a mouse model of influenza A‐GAS superinfection, Okamoto et alA time‐series regression similar to ours found influenza B rather than influenza A to be associated with iGAS detections in Montréal during 1996‐2008.The current study has important limitations. First of all, it is an ecological study; only associations in time on a population level were estimated which has the risk of finding spurious associations as well as missing actual associations. Further, for the respiratory viruses, the population coverage of the VWR surveillance system is not precisely known and may vary over time as reporting by laboratories is voluntary and laboratories can merge. Other unknown surveillance artefacts regarding potential variations over time in laboratory test request behaviour, and technical testing practices may also play a role. For the GAS cultures, the coverage of the ISIS‐AR surveillance system increased during the study period. Further, the notification criteria for puerperal GAS changed in 2016, which is reflected in the increased number of notifications. The secular trend added to the puerperal GAS and ISIS‐AR models crudely adjusts for these increases but may have left residual surveillance artefacts in the data. Also, the formal iGAS surveillance of notifiable disease presentations is partially limited in the Netherlands as national surveillance data on GAS pneumonia are absent (because not notifiable), while it is strongly associated with STSS.Our study supports the notion that iGAS burden may be reduced by influenza prevention. Indeed, a case‐control study among US military found a strong protective effect of influenza vaccination on GAS disease.Our finding of a specific association between IAV and STSS seasonality and not other (i)GAS manifestations should be interpreted with caution as it is an ecological association and needs further confirmation in future studies. However, we believe our study adds a population perspective to the evidence from experimental studies and clinical case reports that influenza significantly impacts iGAS severity. Replication of our study in other settings or systematic influenza testing of iGAS patients may provide more insight into the burden attributable to influenza‐iGAS superinfection. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Escaped farmed Atlantic salmon interbreed with wild Atlantic salmon, leaving offspring that often have lower success in nature than pure wild salmon. On top of this, presence of farmed salmon descendants can impair production of wild‐type recruits. We hypothesize that both these effects connect with farmed salmon having acquired higher standard metabolic rates  during domestication. Fitness‐related advantages of phenotypic traits associated with both high SMR and farmed salmon  depend on environmental conditions, such as food availability. We hypothesize that farmed offspring have an advantage at high food availability due to, for example, dominance behavior but suffer increased risks of starvation when food is scarce because this behavior is energy‐demanding. To test these hypotheses, we first compare embryo SMR of pure farmed, farmed‐wild hybrids and pure wild offspring. Next, we test early‐life performance  of hybrids relative to that of their wild half‐siblings, as well as their competitive abilities, in semi‐natural conditions of high and low food availability. Finally, we test how SMR affects early‐life performance at high and low food availability. We find inconclusive support for the hypothesis that domestication has induced increased SMR. Further, wild and hybrid juveniles had similar survival and growth in the semi‐natural streams. Yet, the presence of hybrids led to decreased survival of their wild half‐siblings. Contrary to our hypothesis about context‐dependency, these effects were not modified by food availability. However, wild juveniles with high SMR had decreased survival when food was scarce, but there was no such effect at high food availability. This study provides further proof that farmed salmon introgression may compromise the viability of wild salmon populations. We cannot, however, conclude that this is connected to alterations in the metabolic phenotype of farmed salmon. All fertilizations were performed within the same day (8 November 2012 and 29 November 2013). Eggs from each wild and farmed female were fertilized with sperm from one wild and one farmed male so that each pure wild (ww) and pure farmed (ff) family were half‐siblings with two hybrid families; one with a farmed mother and a wild father (fw), and one with a wild mother and a farmed father (wf). The crosses are hereafter called “types”. The crosses performed using gametes from Surna and AquaGen in 2012 gave 40 full‐sibling family groups. This part of the fish material will be referred to as “Surna‐AquaGen”. The crosses made with AquaGen and Imsa gametes in 2013 resulted in 44 full‐sibling family groups, which are hereafter referred to as “Imsa‐AquaGen”.2 consumption was measured at the embryo (eyed egg) stage using both the Surna‐AquaGen and the Imsa‐AquaGen material, whereas tests of juvenile performance under semi‐natural conditions were undertaken only with the Imsa‐AquaGen material.Eggs and juveniles originating from these crosses were used to conduct two different sets of experiments: ODue to low fertilization success or high mortality at the egg or juvenile  stage, some families are not represented in the experiments that constitute this study. See the descriptions of experiments below for information on numbers of families included in the different parts of the study.2.22 consumption in a closed system, the Surna‐AquaGen material during 7–10 February 2013 and the Imsa‐Aquagen material during 26 February to 3 March 2014. Total O2 consumption in each sample was measured with a micro‐cathode oxygen electrode (model 1320) connected to an oxygen meter .Eyed embryos used in the SMR measurements were shipped from Ims to the Norwegian University of Science and Technology (NTNU) on 5 February 2013 and 24 February 2014. At NTNU, the families were kept in separate petri dishes at 5°C. Representatives from all families had their SMR measured as the rate of O2 consumption in the Surna‐AquaGen and the Imsa‐AquaGen materials was measured with similar procedures. Individual embryos from the Surna‐AquaGen material were placed inside 2 ml syringes containing 10°C oxygenated water. The water had been transported from Ims and was filtered with Sterivex 0.2‐µm filters. Syringes were sealed with warm wax and kept for 2.2–4 hr at 10°C. Sixteen syringes were not loaded with embryos, and these functioned as controls to account for microbial metabolism. For the Imsa‐AquaGen material, five embryos per family were placed inside separate 13 ml glass vials that contained aerated 10°C synthetically prepared water  of the embryos was calculated from the measured decline in O2 concentration and the water volume  in the containers. Control measurements were used to correct for microbial metabolism. The results showed a decline in estimated O2 consumption h−1 with increasing duration of the measurement period, likely caused by diffusion of O2 into the containers. We could therefore not use O2 consumption h−1 as a direct measure of SMR. To account for differences in duration of the measurement period among samples, and variation in embryo size, we estimated mass‐specific SMR as residuals from linear models where total O2 consumption (mg) was regressed against the duration of the measurement period and egg mass . Oxygen consumption and egg mass were log10‐transformed prior to these regressions to linearize the relationship between them  that were spread by the water current so that food was available throughout the full length of each stream channel. Pre‐trial tests confirmed that this procedure ensured a consistent distribution of food across the stream channels. Salmon juveniles from six families of the wild type as well as six from each of the two hybrid types were used  and both types of hybrids  at different levels of food availability, we set up an experiment in 40 semi‐natural stream channels. The stream channels were 4.5 m long, 24 cm wide, had a water level of 10–15 cm, and gravel substratum suitable for salmon juveniles . Ninety‐six SNPs  and male type (MT) and their interaction. Since each female and male was represented by several offspring , we added female identification (femID) and male identification  as random intercepts. Thus, the initial LMM models for embryo SMR can be represented as:Differences in mass‐specific standard metabolic rate (SMR) of embryos resulting from different crosses between farmed and wild adults  at the eyed embryo stage were tested using LMMs. Different LMMs were fitted for the Surna‐AquaGen and the Imsa‐AquaGen data. Both models included the main fixed effects of female type  and if any such effect depended on food availability (high and low), within each of the two competition treatments . At the same time, we tested if the mean family mass‐specific SMR (family‐level SMR) or mean family embryo mass had an effect on survival, and whether any such effect depended on food availability. This was done by modeling the survival (S) of families from the different types in allopatry and in sympatry in two separate binomial GLMMs that included the main effects of type (T), family‐level SMR (fSMR), family‐level embryo mass , food availability treatment (F), and the interaction between F and the other main effects. Family (fam) and stream channel (ch) were included as random intercepts to take into account possible block effects and that each family was represented in several replicates. Thus, the structure of the starting GLMMs was as follows:Juvenile survival was modeled in two different ways. First, we tested whether there were differences in survival within each type. This was done by modeling the effect of competition and food treatment on survival (S) of wild offspring and each of the two types of hybrids in three separate binomial GLMMs, one for each type. All initial models included the main effect of the food availability treatment (F), competition (C), family‐level SMR (fSMR) and the interaction between these, as well as the main effect of family‐level embryo mass  and its interaction with F and C. Random factors for family (fam) and stream channel (ch) were also included. The structure of these models was:Secondly, we tested if the competition  , food treatments, family‐level SMR or any of the interactions between these main effects had an effect on survival FM, ln‐transformed) was the response variable. Family‐level embryo mass  was included as a co‐variate to take into account variation in start weight of juveniles from different families. Also included were the main effects of type (T), food availability treatment (F), competition , the final number of surviving juveniles (N) in each stream channel and family‐level SMR (fSMR), as well as the interactions between T and F and T and C, as well as their interactions with fSMR. Similar to the GLMM models, stream channel (ch) and family (fam) were included as random factors. Thus, the initial LMM model can be represented as:Variation in final mass among juveniles of different types was modeled in a LMM where final individual mass . Furthermore, family‐level SMR and embryo mass had no significant effect on survival across.According to the model selection, only the main effect of the food availability treatment influenced survival in allopatry. Thus, the survival of wild and hybrid juveniles did not differ significantly in allopatry but was significantly lower in the low food treatment (64%) than in the high food treatment (87.5%) . There was no significant difference in survival between the food availability treatments or among the types, and there was no effect of family‐level SMR.The corresponding best model for survival in sympatry included only the main effect of family embryo mass, with a positive effect of large mass , the corresponding best models included only the main effect of food availability. According to these models, survival was significantly lower at the low food treatment  than at the high  for wf and fw families, respectively Table . Thus, t3.2.3According to model selection, only the main effects of family embryo mass (ln‐transformed) and the final number of surviving fish in each stream channel had an effect on the individual final mass in the stream channel. Specifically, there was a positive relationship between family embryo mass and final mass and a negative relationship between the final number of survivors in each stream channel and final mass Table . Hence, 4This study advances the concern that feral domestic animals impact wild populations negatively since we found that the presence of farmed‐wild Atlantic salmon hybrids led to decreased early survival of their wild half‐siblings under controlled, semi‐natural conditions. This negative influence of hybrid offspring on survival of wild juveniles was, at least partly, caused by genes associated with farmed salmon since it prevailed irrespective of whether the wild juveniles competed with half‐siblings from wild or farmed mothers. That is, irrespective of maternal effects due to, for example, egg size differences between farmed and wild mothers. Combined with results from earlier studies (Fleming et al., As expected, the survival of both wild and hybrid fry was lower at low food availability than at high food availability. Contrary to our predictions, food availability did not influence the effect that hybrids had on the survival of wild fry, thereby indicating that hybrids were as strong competitors at low food availability as they were at high. Furthermore, the relative survival of wild and hybrid fry in sympatry and in allopatry did not differ between the food availability treatments. Thus, despite the farmed salmon strain used in this experiment having adapted to high food availability for 11 generations, their hybrid offspring were able to perform as well as, and even outcompete wild salmon, under low food availability.The impact of SMR on survival among wild families depended on food availability. At high food availability, family‐level SMR had no effect on survival, while at low food availability there was a negative relationship between family‐level SMR and survival. Thus, in accordance with findings in Bochdansky et al. , there wUnder conditions with high competition, the large juveniles that hatch from large eggs often have a competitive advantage (Hutchings, In contrast to several published results showing that hybridization between farmed and wild Atlantic salmon results in offspring that display lower survival in nature than wild offspring (McGinnity et al., We found no difference in growth among the farmed‐wild hybrids and the wild juveniles. It appears that hybrid offspring were not able to utilize the higher growth potential from their farmed ancestry Gjedrem,  under thOur laboratory tests of the hypothesis that farmed Atlantic salmon have acquired increased mean levels of SMR compared to that of wild salmon gave inconclusive results. Embryos of farmed males had significantly higher SMR than embryos of wild males in the crosses between farmed salmon and wild salmon of the Imsa population, but there was not a similar finding in the Surna population. Moreover, SMR did not differ significantly between embryos of farmed females and females of the wild populations. This is, however, in line with the previously reported finding that phenotypic effects of farmed introgression vary among wild Atlantic salmon populations (Bolstad et al., 2, a decline in the O2 concentration due to embryo metabolism would continuously be counteracted by O2 diffusing in, leading to an overall underestimation of the O2 consumption. This tendency would be more pronounced in containers containing embryos with high SMR since the O2 concentration in these would decrease at a faster rate than in containers containing embryos with low SMR. Thus, if a more precise methodology had been employed, larger differences among the Atlantic salmon types in this study could possibly have been detected.We emphasize that the methodology we employed to measure SMR is coarse. Yet, it should provide conservative results. For example, since the containers used to measure SMR were not totally impermeable to OOur results show that descendants of domesticated organisms can induce increased mortality of genetically wild individuals in early life. The resulting decrease in production of recruits may obviously impact the viability of wild populations negatively. In addition, presence of domesticated descendants and an accompanying increase in mortality of wild‐type juveniles could affect the adaptive landscape, potentially resulting in unforeseen changes to the wild genotype.None declared.GR, DR, SE, IAF, OU, EK, and KH contributed significantly to the conception and design of the work. GR, DR, TA, and LSH performed different parts of the experiments. SK planned and led the work with parentage assignment. GR, SE, and IAF contributed significantly to the analyses and interpretation of the data. GR, SE, and IAF drafted the manuscript. All authors revised the intellectual contents of the manuscript critically and agree to be accountable for all aspects of this work.https://doi.org/10.5061/dryad.17bq5d8Data available from the Dryad Digital Repository:  Click here for additional data file.


Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes  exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation—most notably in the emergence and evolution of tumors. Recent technical advances in single‐cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems. The cell is thus a fundamental unit of biology, and the capacity to analyze the behavior of organs and organisms at the single‐cell level is critical to developing and understanding of the emergent behaviors of these communities of cells.Increased biological complexity is enabled by the ability of cells to differentiate and attain distinct “identities” within a system—reflecting a divergence in form or function from precursor cells. This identity has largely been defined in terms of cell type and cell state. Precise definitions for these terms remain elusive: cell type has historically been described by observing reproducible functional distinctions in vivo or in vitro (often coupled with expression of a set of marker genes), while cell state refers to dynamic, responsive changes that alter the phenotype and function of the cell, but not so significantly that a new cell type is acquired.As an example, the murine hematopoietic system Figure  consistsHSCs must enter into different states to fulfil their functional roles. The hematopoietic system is highly dynamic and responsive, and at all levels, changes in cell state are possible—and indeed essential—HSCs are typically quiescent, but must enter a cell cycle “state” to produce new progeny to maintain the blood supply and the stem cell pool. Since this pool is maintained, cell type is based on cell‐intrinsic properties that can be passed on to daughter cells during mitosis; however, since HSCs are pluripotent, cell types can enter into intermediate states as they transition into committed and mature cell lineages.Cell type and state, as defined in this example, are a product of the cell's molecular profile—including genomic, epigenomic, transcriptomic, proteomic, and metabolomic aspects—which in turn emerge from cell intrinsic and extrinsic factors. The recent development of approaches sensitive enough to assess the molecular profiles of individual cells offers the opportunity to gain a new perspective on our definitions of cell identity, and to more clearly delineate the processes by which a single stem cell can generate an entire hematopoietic system, or indeed how a complex multicellular organism can arise from a single zygotic cell.2Key to the study of single cells is the capacity to effectively isolate them to enable analysis of the cell's unique molecular identity, and there are numerous methods for doing so Figure . While mDemand for higher throughput—from hundreds to millions of cells—has driven the development of microfluidic approaches for cell isolation  platforms require that the minute amounts of DNA or RNA present within a single cell be amplified to generate sufficient material to sequence. Due to the accessibility of approaches to replicate nucleic acids in vitro, a multitude of methods have emerged to perform this amplification on single‐cell genomes, epigenomes, and transcriptomes, while the embryonic field of single‐cell proteomics relies on the development of methods which either translate protein abundance into amplifiable nucleic acid signatures or the direct measurement of protein abundance by sensitive mass spectrometry Figure .2.1It is widely thought that the genome is relatively stable throughout life and remains the same independently of cell type—with the exception of post‐meiotic germ cells, vertebrate immune cells, and in pathological conditions such as cancer. However, during the generation of maintenance of the extraordinary number of cells that comprise a complex multicellular organism—in adult humans, the total cell number is estimated to be 38 trillionGenomic heterogeneity between individual cells of the same organism can occur on multiple scales, including single nucleotide variants (SNVs), indels, inter‐ and intra‐chromosomal fusions, copy number variants (CNVs) and aneuploidies of whole chromosomes, as well as insertion sites of viruses or mobile elements . By sequencing the genomes of single cells, each of these phenomena can be studied with greater resolution than by sequencing a “bulk” DNA specimen. Since these events are acquired through cell division and errors in DNA replication, it is possible to use patterns of somatic variation to reveal cell lineages in both normal and diseased tissues, by examining the sequential acquisition of particular mutations,Several approaches to amplify single‐cell DNA have been applied, each with advantages and disadvantages Table . The tecMultiple displacement amplification (MDA)–based methods remain the most applicable where base‐level events  are to be investigated, as the phi29 enzyme utilized in the amplification has an extremely low error rate.The vast majority of single‐cell genome sequencing has explored only tens to hundreds of cells, but recent developments have applied combinatorial indexing2.2Epigenomic aspects of cell identity, including DNA methylation, histone modification, chromatin accessibility, and chromosome conformation, are dynamic, regulatory mechanisms that enable cells with identical genotypes to have distinct gene expression profiles. Thus, by representing the “functionalization” of the genome, these different mechanisms are key aspects of how cell type and state should be defined. Epigenomic diversity is also often heritable through cell division, and as such, offers a mechanism by which cells within a specific type can proliferate and maintain their identity. There has been a substantial proliferation in the diversity of epigenetic techniques aimed to study single cells , hydroxymethylation (5hmC) and formylcytosine (5fC) can regulate gene expression in the short and long term, with 5mC associated with transcriptional repression, while 5hmC and 5fC are associated with transcriptional activation. Assays to detect each of these modifications across the genomes of single‐cells have been developed, though they still present several technical challenges. Single‐cell bisulfite sequencing (scBS‐seq),Overall, this sparseness of data limits the analysis of cellular variation and thus it is necessary to use methylation frequency data from single‐cell populationsChromatin accessibility, which is a regional marker of “activity” within the genome, can be assessed through treatment of permeabilized cells or nuclei with enzymes which cleave at sites where DNA is not protected by chromatin. Single‐cell DNAse‐seqSingle‐cell droplet–based chromatin immunoprecipitation (drop‐ChIP) has been demonstrated, although using extremely sparse data (1000 reads per cell) to generate population distributions of histone 3 lysine 4 trimethylation (H3K4me3), enabling detection of discrete epigenetic states in populations of mouse ES cells, fibroblasts, and a hematopoietic cell line.DNA/laminin associations can be probed using the Dam‐ID protocolAs with single‐cell genome analysis, each of these epigenetic methods tend to generate sparse data which preclude true “genome‐wide” identification of DNA modifications, open chromatin sites, and chromatin conformation, and while imputation approaches are available to complete missing information,2.3The transcriptome is highly dynamic, reflecting cell type and cell state. Cells committed to particular type would be expected to express selections of marker genes which differentiate them from other cells within the population, and within each type, variation in gene expression may be indicative of cell state transitions—for example, specific gene expression profiles can be associated with cell cycle status.Perhaps the greatest technical and experimental advances using single‐cell techniques have been in the study of the transcriptome. This is largely due to the accessibility of the transcriptome, which in contrast to the genome is present at relatively high copy number per cells—transcript copy numbers can range from 1–100 000 copies per transcript per cell, although the majority are estimated to be present at less than 100 copies per cell.While many single‐cell transcriptomics approaches can readily be applied in medium throughput on FACS‐sorted cells isolated into multi‐well plates, the scale and ease with which single‐cell transcriptomics experiments can be undertaken has benefited considerably from the introduction of combinatorial indexing,Smart‐seq/Smart‐seq2, which enables full‐length amplification of single‐cell cDNA can readily be applied to FACS isolated cells in multi‐well platesCEL‐seq/CEL‐seq2, which relies on IVT‐based amplification and 3′ enrichment, generates 3′‐end cDNA libraries, incorporating unique molecular identifiers (UMIs)—random barcodes added to the sequence during the first reverse transcription step.High‐throughput single‐cell gene expression profiling has been revolutionized by the introduction of microfluidics approaches which enable 3′ transcript counting from thousands of cells in parallel. The Drop‐seqCaenorhabditis elegans larvae, representing over 50‐fold coverage of all of the cells in a single organism.Nanowell approaches, such as Seq‐Wellt‐distributed stochastic neighbor embedding (t‐SNE) plots.Throughput in single‐cell transcriptomics experiments has reached astonishing levels, with experiments now detailing thousands to millions of cells now becoming routine. However, there is minimal change in the total amount of sequencing performed in a single experiment, and thus the transcriptional profiling of these large numbers of cells focusses on enumeration of 3′ tag sequences and shallow coverage of the whole transcriptome. The majority of single‐cell transcriptomics analysis uses 3′ tag sequencing approaches and assigns cell types as a result of clustering—for example, using principle components analysis (PCA) or The transcriptomes generated at this throughput are currently necessarily superficial, and nuanced changes in lower expressed genes cannot be detected. Furthermore, due to the 3′ selectivity of these methods, UTR sequences, alternative splicing, RNA editing, mutations, and allelic expression can only fractionally be considered. Similarly, small RNA molecules, including microRNAs will not be detected using these approaches, although protocols for plate‐based small RNA‐sequencing have been described.2.4The functional identity of a cell is largely a product of its proteome—it is through proteins and post‐translational modifications that cells sense and respond to virtually all extrinsic and intrinsic stimuli. A comprehensive overview of a cell's proteome would perhaps give the most detailed definition of cell type and state possible by molecular means, but the limitations of current approaches make such observations impossible. Neither antibody nor mass spectrometric–based detection or quantification of proteins has the throughput or sensitivity required for proteome‐wide screening. However, advances in antibody labeling and detection, microfluidics, and recently, sensitive mass spectrometry approaches, are beginning to show the potential of proteomic analysis of single cells, and protein level detection remains an important tool for validation of single‐cell RNA‐seq results.Western blotting approaches sensitive enough to detect proteins from single cells have been developed.High‐throughput, multiplexed analysis of protein expression in single cells has been carried out by FACS analysis for several decades. The detection of immunofluorescently tagged proteins in thousands of single cells is routine for FACS analysis, with modern high parameter instruments capable of analyzing up to 50 parameters in parallel. This is, however, technically challenging due to the potential for overlap between fluorescent spectra and these high parameter applications likely represent the upper limit of the capability of FACS. The CyTOF approach is a variant of FACS in which antibodies are labeled with heavy metal ion tags rather than fluorophores; the abundance of each metal ion labeling the cell is read out using time‐of‐flight mass spectrometry.However, antibody‐based methods will always be dependent on the specificity and availability of the antibody. Unbiased proteomic analysis of single cells is challenging due to limitations on the sensitivity of mass spectrometry techniques, and the lack of a PCR‐like method to amplify protein signals. The phase‐enhanced sample preparation (SP3) method was describedRecently, Single‐Cell ProtEomics by Mass Spectrometry (SCoPE‐MS)Microfluidics technologies constitute another class of tools available to collect highly multiplexed measurements of proteins from single cells. Microchip‐based proteomics analysis enable simultaneous quantification of up to 40 nuclear, cytoplasmic, membrane and secreted proteins across thousands of single cells, with the sensitivity threshold of as low as a few hundred protein copies per cell.A “microengraving” approach using small volume microwells in an array format can be used to isolate and culture single cells. Using an antibody‐coated substrate to cap the microwell array and capture secreted proteins, an ELISA immunoassay can be performed to enable protein quantification.A related approach is the single‐cell barcode chips (SCBCs) that patterns a capture antibody array in a single‐cell microwell so that different proteins can be detected at designated array spots. Cells are lysed on‐chip, and the levels of released proteins are assayed using the antibody arrays. SCBC data have been used to examine altered signal transduction networks in tumor and immune cells.2.5The metabolome, defined as the full collection of all low‐molecular‐weight metabolites that are produced by a cell, could be a key indicator of cell state—reflecting the precise metabolic activity and condition within the cell. However, the metabolome is challenging to measure at the single‐cell level, largely due to the diversity and rapid dynamics of the system, coupled with the lack tagging and/or amplification approaches for small molecules.Advances in optical tools  together with improved expression systems and in vivo imaging have made possible the measurement of metabolites in real time and in single cells.Given these developments, and the significance of cell metabolism in the definition cell state and function, it is anticipated that further technical advances will lead to more complete coverage of the metabolome, accurate and fast metabolites identification, and nondestructive measurement in single cells.2.6Cellular identity does not materialize from the isolated activities of the genome, epigenome, transcriptome, or proteome, but rather as a property of the interaction of each of these aspects with each other and other aspects of cell biology. As such, there is a considerable interest in capturing as much information from a single cell as possible—which has led to the emergence of single‐cell “multi‐omics” approaches  approach labels cells with antibodies tagged with barcode sequences flanked by polyadenine and PCR handle sequences compatible with transcriptome‐wide amplification.Integration of metabolite assays with proteomics assays might directly resolve connections between protein signaling networks and functional small molecule metabolites within the cell, reflecting the cell's state and type. The single‐cell barcode chip (SCBC) platform described above can be used to integrate quantitative measurements for intracellular metabolites with functional protein immunoassays into a microarray format.Approaches for multi‐omic analysis of single cells continue to develop at pace, and as methods improve and sequencing throughput increases, it seems likely that methods to capture as much as possible of a single cell's identity will become increasingly important in focused studies of cellular heterogeneity.3Single‐cell transcriptomic studies have been widely applied to “atlassing” studies of tissues, organs, and even whole organisms.3.1Stem cells are characterized by both being capable of unlimited self‐renewal and having the potential to differentiate into specialized types of cells,Application of single‐cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease.3.2Cancer is one of the most common manifestations of genomic mosaicism in humans. The study of tumor heterogeneity is further complicated by their often polyclonal nature, with single‐cell derived clones harboring genetic and epigenetic alterations that differ from the host genome and from other cells within the tumor. Although the genetic heterogeneity and the evolutionary principles governing resistance are actively being discovered, tools enabling the study of molecular processes that govern tumor progression are lacking. The single cell is the fundamental substrate upon which mutational mechanisms and the principles of selection act to evolve the complex structure that is a tumor mass.Single‐cell technologies able to characterize a single‐cell genome, transcriptome, epigenome, and proteome could provide a clearer picture of tumor biology complexity at every phase of tumor development, with potential applications in new therapeutic approaches, cancer treatment, and clinical management. The application of bulk exome sequencing, targeted deep‐sequencing, and parallel single‐cell DNA sequencing to study clonal evolution during metastatic dissemination in two colon cancer patients, demonstrated that, in contrast to bulk sequencing methods, single‐cell analysis was able to distinguish between a tumor self‐seeding later dissemination and an early dissemination models of metastasis. Moreover, single‐cell sequencing identified a rare ancestral subpopulation, composed of three diploid cells carrying a mutation in the APC gene that initiated the tumorigenesis and subsequently gave rise to the primary tumor and liver metastasis.Single‐cell sequencing can also be applied to the molecular phenotyping of circulating tumor cells (CTCs),3.3Novel preimplantation genetic screening (PGS) for chromosomal abnormalities has recently been developed to improve clinical outcomes in patients undergoing in vitro fertilization (IVF) as aneuploidy in one of the most prevalent genetic abnormality in human embryos. Fluorescence in situ hybridization (FISH)–based screening is unable to provide a comprehensive analysis of all chromosomes, while the development of WGA technologies has enabled the analysis of whole chromosomes aneuploidies in single cells. Performing WGA on oocytes, one of the blastomeric cells from day‐3 embryos, or from trophectoderm cells from day‐5 blastocyst embryos, enables comprehensive chromosome analyses on various genome analytical platforms, such as a comparative genomic hybridization array3.4Arabidopsis thaliana have been successfully performed.Examples of plant single‐cell analysis are relatively uncommon, although the technology has a number of exciting potential applications. As with animal and human samples, genotyping, developmental studies, and cell‐typing using single‐cell approaches are all highly relevant, as is the use of these approaches for the analysis of biomolecule synthesis and interactions.Single‐cell genome sequencing may have immediate and highly beneficial application in pollen typing, applicable in both basic molecular genetics and agricultural breeding. During the meiotic cycle, chromatids recombine resulting genetic differences in each of the daughter cells. The frequency of segregation of different alleles into different pollen grains then determines the genetic diversity and distribution of beneficial traits  of the offspring plants. Currently, studies of plant population genomics are performed using low‐throughput cytological assessment of the pollen grains and conventional breeding, with large numbers of offspring plants needed per study. Often these plants have long generational times, for example, wheat can take up to 9 months to mature in the field, making the process slow and costly.Hordeum vulgare) pollen in order to assess the number and location of recombination sites along the length of each chromosome, testing the cytological hypothesis that the majority of the sites are located at the distal ends despite the “peri‐centromeric” regions.By sequencing the genomes of single pollen grains, it may be possible to haplotype the parental chromosomal contribution and understand factors regulating the frequency of crossing‐over, and thus population genetic diversity. Pollen‐typing has advantages which work to help with some of these issues. It is high‐throughput, often using FACS, and only one plant is needed for studies such as those looking at quantitative‐trait loci (QTL) association or mapping which usually require thousands of replicates.3.5Actinobacteria and Crenarchaeota.Since microbial populations are often complex and consist of a community of multiple species, single‐cell analysis may be vital in dissecting molecular heterogeneity between cells.4Approaches for the study of the molecular identity of single cells have emerged and been adapted at a rapid pace over the last 5 years. Through application in large scale, multi‐center studies of whole organism biology, such as the Human Cell Atlas,However, continued technical development is essential to maximize the amount of information that can be retrieved from a single cell. Each of the methods described in this review has limitations, particularly in the coverage they provide of the analyte of interest, which is particularly important where base‐level events  are to be considered. Improvements in molecular biology and microfluidics may resolve some of these issues, and computational approaches for imputation of missing data are also increasingly being applied.The authors declare no conflict of interest.


Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Herbicides classified as synthetic auxins have been most commonly used to control broadleaf weeds in a variety of crops and in non‐cropland areas since the first synthetic auxin herbicide (SAH), 2,4‐D, was introduced to the market in the mid‐1940s. The incidence of weed species resistant to SAHs is relatively low considering their long‐term global application with 30 broadleaf, 5 grass, and 1 grass‐like weed species confirmed resistant to date. An understanding of the context and mechanisms of SAH resistance evolution can inform management practices to sustain the longevity and utility of this important class of herbicides. A symposium was convened during the 2nd Global Herbicide Resistance Challenge  to provide an overview of the current state of knowledge of SAH resistance mechanisms including case studies of weed species resistant to SAHs and perspectives on mitigating resistance development in SAH‐tolerant crops. © 2017 The Authors.  Beauv.], E. crus‐pavonis [(Kunth) Schult.], E. zelayensis [(Kunth) Schult.], and E. colona (L.) Link), have evolved resistance to quinclorac, which has a proposed cyanide‐mediated mechanism of action on grasses that is distinct from that of SAHs.In 1957, the first cases of 2,4‐D resistance were reported in climbing dayflower (Raphanus raphanistrum L.) in Australia, phenoxy herbicide‐resistant corn poppy (Papaver rhoeas L.) in Europe, dicamba‐resistant kochia [Kochia scoparia (L.) A.J. Scott] in Canada and the USA, prickly lettuce (Lactuca serriola L.) resistant to 2,4‐D, dicamba, and MCPA in the USA.Amaranthus tuberculatus (Moq.) Sauer] biotypes from Nebraska and Illinois and smooth pigweed [A. hybridus L. (syn.: A. quitensis Kunth)] in Argentina were determined to be resistant to certain SAHs.Daucus carota L.) in Canada and the USA, 2,4‐D‐resistant musk thistle (Carduus nutans L.) and Italian thistle  in New Zealand, and multiple SAH‐resistant wild mustard (Sinapis arvensis L.) and quinclorac‐resistant false cleavers  in Canada.Weeds of economic importance  exhibiting resistance to SAHs include 2,4‐D‐ and MCPA‐resistant wild radish ‐ and acetolactate synthase (ALS)‐inhibiting herbicides Fig. . There aThis low incidence of resistance can be attributed to several factors including: (1) potential multiple sites of action of these herbicides;33.1Papaver rhoeas) is the most common broadleaf weed in winter cereals in Europe.P. rhoeas biotypes have been reported over the last 10 years in Spain, France and Greece.Corn poppy  per year in lost crop yield and increased weed control expenses.14C‐labeled 2,4‐D out of the treated leaf.Phenotypic characterization of two wild radish populations collected from the field in 2001 and 2002 showed about a 20‐fold increase in resistance compared with a susceptible population. The traits conferring 2,4‐D resistance in these two populations segregated at one major locus, and were nuclear‐inherited and incompletely dominant.et al., unpublished). This is likely a second resistance mechanism, since some populations that were highly resistant to 2,4‐D translocated 14C‐labeled 2,4‐D out of the treated leaf as efficiently as susceptible plants .The 2,4‐D‐resistant biotypes collected from populations in 1999, 2001 and 2002 were also resistant to MCPA.Biomass of plants grown in competition with wheat and treated with the recommended rate of 2,4‐D was reduced by 75% compared with 2,4‐D‐treated plants grown without competition.Finding alternative molecules that interfere with the genes or gene products involved in altering SAH response of resistant plants or those capable of re‐activating normal translocation of the herbicide could prolong the utility of the SAHs in wild radish‐infested crops. To date, there is no evidence of metabolic detoxification of 2,4‐D (either cytochrome P450‐dependent or ‐independent) in resistant wild radish populations.44.1In the case of SAHs, the candidate mechanisms of resistance can include target site modifications (auxin receptors or auxin‐specific transporters) and non‐target site mechanisms such as other transporters and enzymes that metabolize SAHs. One major source of resistance to herbicides is target site resistance. Target site resistance frequently arises from mutations in the active sites of the proteins to which the herbicide normally binds, rendering the protein insensitive to certain herbicides as non‐native compounds. No incidence of resistance arising from SAHs applied in the field has been traced back to any of the core signaling target sites. However, mutations in many of these proteins can confer resistance to SAHs, as demonstrated by the fact that most auxin signaling and transport proteins were identified from screens for Arabidopsis mutants tolerant to applied SAHs.Scientists have identified a number of putative auxin receptor proteins,The TIR1 receptor structure was determined by crystallization.The TIR1 and AFB1–5 auxin receptors vary little in their sequences and there is a high degree of sequence conservation across the plant kingdom, especially in amino acids that line the binding site.Co‐receptor AUX/IAA proteins should be considered because they bind on top of auxin once it is inside the recognition pocket to complete the co‐receptor complex Fig. .51 Diffe4.2Transport proteins can confer both target‐site and non‐target‐site resistance Fig. .55 For aSelective auxin efflux gives rise to polar auxin transport and contributes to the establishment and maintenance of plant cell polarity.It is more challenging to evaluate the potential of efflux proteins as sites for resistance on the basis of available biochemical knowledge. Upregulation of either efflux carrier would likely accelerate extrusion of substrate auxins from cells, which may confer resistance by removing herbicide. The PINs are dominant when considering endogenous auxin concentrations and gradients.The ABCB transport proteins are well characterized in non‐plant eukaryotic systems and there is a growing understanding about the importance of ABCB transport proteins in plants.4.3Non‐target site resistance to agrochemicals has frequently been associated with their metabolism by enzymes into harmless products.Oxidation of small molecules, often by cytochrome P450‐type enzymes, may be a mechanism for detoxifying herbicides5The discovery of aminopyralid in the late 1990s prompted an intensive structure–activity relationship (SAR) assessment of the pyridine‐carboxylate SAHs at Dow AgroSciences. This led to the discovery of a novel chemical family of SAHs, the arylpicolinates, which are derivatives of pyridine carboxylic acids. The acidic functionality of SAHs is critical for their mobility in the phloem and for accumulation inside cells through ion trapping. For the pyridine‐carboxylate herbicides clopyralid, picloram, aminopyralid, and arylpicolinate herbicides [halauxifen‐methyl (Arylex™ Active) and florpyrauxifen‐benzyl (Rinskor™ Active)], the carboxylic acid functionality is involved in key binding interactions at the TIR1 and AFB1–5 receptors.The arylpicolinates have unique herbicidal activities that include increased efficacy at relatively low use rates /ha or less) compared with pyridine carboxylic acid herbicides. The analog DAS402 Fig. , exhibitEchinochloa crus‐galli, and some sedges (Cyperus spp). The first registration for florpyrauxifen‐benzyl was approved in 2017.During the exploration of the halauxifen‐methyl SAR, several important features of the picolinate scaffold were identified. The 2‐carboxylic acid and the 4‐amino functional groups of the molecule were found to be essential for the expression of potency and broad‐spectrum herbicide activity and the 3‐chlorine functional group contributed further to herbicide potency. Exploration of the SAR of the 5‐position of the picolinate head of halauxifen‐methyl led to the discovery of a second arylpicolinate herbicide with commercial utility. This 5‐fluoro analog of halauxifen‐methyl exhibited potent and broad‐spectrum herbicide activity with excellent selectivity for use in rice. Its corresponding carboxylic acid (4‐amino‐3‐chloro‐6‐(4‐chloro‐2‐fluorophenyl‐3‐methoxy)‐5‐fluoropicolinic acid) had a soil half‐life in the range of 10 to 30 days. Further optimization of the ester eventually led to the development of florpyrauxifen‐benzylThe rise of the arylpicolinate herbicides provides compelling evidence that there are rich opportunities for developing new SAHs. The high diversity of activity of the arylpicolinates adds to the long lineage of SAHs that have brought broad utility to weed management market segments including cereals, rice, rangeland, pastures, and rights‐of‐way.6New herbicide formulations have been developed for use in recently developed crops tolerant to 2,4‐D, developed by Dow AgroSciences, and dicamba, developed by Monsanto. Enlist Duo™  and Enlist One™  herbicides were developed for the Enlist™ Weed Control System and Xtendimax (dicamba) with VaporGrip® was developed for the Roundup Ready® Xtend Crop System. Dow AgroSciences and Monsanto have developed stewardship programs designed to ensure the longevity and sustainability of these herbicide‐tolerant crop systems and to provide guidance on their integration into herbicide resistance management programs. In addition, the US Environmental Protection Agency (USEPA) is developing a regulatory framework to emphasize the need for grower adoption of herbicide resistance best management practices to further the sustainability of these crop systems.With the introduction of SAH‐tolerant crops, there has been extensive consideration of herbicide resistance management from regulatory and stewardship perspectives. These tools will aid in weed management programs and the control of some species of herbicide‐resistant weeds. Best management practicesFrom the 1950s to 2001, herbicide registrants had to fulfill only a few requirements in the USA regarding herbicide resistance management. After 2001, registrants were required to report confirmed herbicide‐resistant weed species to USEPA using the Adverse Effects Reporting [6(a)2] process.Strong collaboration among stakeholders to develop and deploy effective herbicide stewardship programs are needed to limit the onset of field‐evolved resistance and to ensure herbicide longevity and sustainability. Along with herbicide developers and registrants, farmers, regulators, extension specialists, crop advisors, dealers and retailers have roles to play in technology stewardship. The industry encourages weed management stakeholders to continue to promote the use of herbicides as part of a comprehensive weed management approach so that these valuable tools can be preserved by delaying onset of field‐evolved herbicide resistance.7Our purpose is to synthesize the state of the knowledge about plant resistance to SAH and to reinforce the compelling opportunities for future innovation within the SAH mode of action. The SAHs are unique and complicated in their pharmacology. Their complex mechanism of action, beneficial impacts on agricultural production, and durability are a testament to their utility in weed management systems.The SAHs continue to be viable after more than 70 years of commercial use, partly because they have not commonly been used as the sole instrument for weed control. Selection for resistant biotypes does occur when the diversity of the tools employed is limited. Recent insights into the mechanism and site of action of SAHs, including redundant auxin receptors and complex molecular interactions between auxin‐responsive genes and cascading disruption of homoeostasis on multiple biochemical pathways, help explain why simple hypotheses for many resistant field biotypes have yielded unsatisfactory answers to questions about mechanisms of resistance. This knowledge has given credence to the proposed reasons why target site resistance has been slow to develop and may be associated with significant fitness cost .A simple altered site of action with reduced affinity for SAHs has been difficult to demonstrate. Hypotheses of increased metabolism or decreased uptake into the plant often do not adequately explain biotype responses to SAHs. With insights into the interactions of chemistry, proteins, and gene regulation, and with improved analytical tools, we now understand that reduced transport can potentially explain resistance to SAHs. However, enhanced metabolism may  or may not  be associated with SAH resistance. Understanding the impacts of each of these mechanisms has proven difficult because of the multiple and redundant variables in the complex auxin responsive pathways.inter alia) may develop and spread. A significant concern could be the selection of traits causing an altered transport for SAHs potentially resulting in co‐evolution of resistance to other herbicide modes of action, as seen with multiple drug‐resistant bacteria.In our collective view, the risk of additional herbicide‐resistant biotypes developing with altered target sites  remains relatively low. We also believe the complex interaction with the auxin perception and responsive pathway makes resistance through overexpression of the target proteins  a low probability. As SAH use continues to grow, the risk for resistance selection will also increase. Our concern is that additional sequestration and/or altered transport mechanisms of resistance (e.g. ABCB transporters, These insights have been made possible by improved knowledge and analytical capabilities. Yet, the pharmacology of cellular and long‐distance transport mechanisms in plants are poorly understood. Pursuing greater understanding of these mechanisms is a clear opportunity for weed science and allied disciplines. The fact that after more than 70 years of use, new SAHs demonstrating unique weed control spectra and different chemical attributes continue to be discovered supports the proposition that the future is rich with opportunities for further innovation with SAHs.Another lesson is that the entire community of weed control experts including industry scientists, academia, regulatory agencies, and farmers need to be aware and fully supportive of the implementation of best herbicide resistance management practices.


HDPs) or gestational diabetes mellitus (GDM) is highly predictive of women at increased risk of developing chronic hypertension, Type 2 diabetes, and cardiovascular disease. This study investigates perceptions of women and healthcare providers in rural India regarding these long‐term risks.A diagnosis of hypertensive disorders during pregnancy , primary care physicians, obstetricians, laboratory technicians, and healthcare officials.Pregnant and postpartum women, community health workers  postpartum management, and (4) knowledge of long‐term sequelae of high‐risk conditions. A thematic analysis was undertaken.In‐depth interviews and focus group discussions explored: (1) priorities for high‐risk pregnant women; (2) detection and management of n = 71 participants) were performed. The key priority area for high‐risk pregnant women was anaemia. Blood pressure measurement was routinely embedded in antenatal care; however, postpartum follow up and knowledge of the long‐term complications were limited. GDM was not considered a common problem, although significant variations and challenges to GDM screening were identified. Knowledge of the long‐term sequelae of GDM with regard to an increased risk of Type 2 diabetes and cardiovascular disease among doctors was minimal.Seven focus group discussions and 11 in‐depth interviews . The George Institute for Global Health Global Women's Health programme provided financial support for the research assistant and fieldwork costs in India.Improved education and postpartum care of women with hypertension and diabetes in pregnancy in rural India are needed to prevent long‐term risks. Improved education and postpartum care of women with hypertension and diabetes in pregnancy in rural India are needed to prevent long‐term risks. India is undergoing a major epidemiological transition towards non‐communicable diseases (NCDs).Two‐thirds of the Indian population live in rural areas.We undertook a qualitative study in rural areas of two culturally and socio‐economically diverse states (Haryana and Andhra Pradesh). Located in northern India, Haryana has above‐average rates of gender and social inequality.We undertook semi‐structured interviews and focus group discussions with key stakeholders involved in women's health during and after pregnancy, in rural parts of two diverse states in India (Haryana and Andhra Pradesh). Ethical approval was obtained from the Oxford Tropical Research & Ethics Committee (OxTREC), and The George Institute Ethics Committee, India. Patients were not involved in the development of the research.Key stakeholders in the health system  were recruited through purposive sampling and snowballing of key contacts.District government representatives responsible for healthcare in the state were approached through The George Institute for Global Health (TGI) in Delhi and Hyderabad, for Haryana and Andhra Pradesh, respectively. Primary care physicians at two PHC facilities in each study area were approached to participate in in‐depth interviews. Obstetricians were identified through snowballing of key secondary care referral contacts known to the primary care physicians. CHWs were identified and recruited through the primary care physicians. Laboratory technicians were interviewed opportunistically during PHC visits through snowballing of contacts.Women with both complicated and uncomplicated pregnancies, and postpartum women who had delivered a baby in the last 2 years were recruited for focus group discussions by their CHWs. All participants gave their written informed consent to participate in the study.Topic guides were developed for interviews and focus group discussions. The guides were reviewed by the research team prior to use but were not piloted. All interviews and focus group discussions were audio‐recorded with consent for transcription and translation. Field notes were made during and immediately after the interviews or focus groups. Interviews and focus group discussions were conducted by a female researcher (SN), trained in qualitative methods, with the help of a research assistant from TGI. Each in‐depth interview or focus group discussion lasted 30–60 minutes, and was conducted in English when possible or in the local languages of Haryanvi (Haryana) and Telugu (Andhra Pradesh), with simultaneous translation by a research assistant.Transcripts of the recordings were translated into English (from Haryanvi and Telugu) by a professional transcriber in India. Transcripts were validated for accuracy by a local research assistant based at TGI, India. Data were coded by the lead researcher (SN), with the assistance of an experienced qualitative researcher (LH). An inductive, modified‐grounded theory approach was adopted, using guidance for systematic, theory‐driven data analysis outlined by Corbin and Strauss.To ensure triangulation of results, we collected data from several sources (key stakeholders) in different ways (interviews and focus groups) to explore how the results converged and complemented each other and any outlying cases.The lead researcher (SN) is a DPhil student with a clinical background in primary care. She entered each interview or focus group with an understanding of the principle of reflexivity,A total of 71 participants ; 24 pregnant/postpartum women (aged 20–35); and 3 laboratory technicians) participated in seven focus group discussions and 11 in‐depth interviews. All the participants completed the study. Please see Table A summary of the main themes and subheadings arising from the qualitative analysis is given in Table Stakeholders prioritised anaemia in pregnancy, followed by HDPs, as the most important priorities for high‐risk pregnant women in their areas. These conditions were chosen for differing reasons: anaemia because of its high prevalence and hypertension because of the serious impacts on maternal and neonatal mortality and morbidity.Anaemia was the most common condition affecting pregnant women at both study sites Table , Quote 1“The patient gets pregnant multiple times, the pregnancy‐induced hypertension, gestational diabetes will increase in each and every pregnancy…Anaemia will increase….And sometimes the patients are such stubborn patients that even ASHA workers say – please come to the hospital and I will go with you to be treated – No… The patient will not come…She will come with 3 g Hb [haemoglobin of 3 g/dL], when she is going to die….then she will come.”Interview: Obstetrician.Community health workers and doctors were well informed about the causes of anaemia in pregnancy, including dietary factors, household poverty, multiple pregnancy, and helminth infection. They counselled women accordingly, regarding lifestyle changes and adherence to oral iron. Healthcare workers expressed their frustrations with regard to preventing anaemia in pregnant women.Pregnant women, however, reported facing several difficulties in following the advice provided by their healthcare workers, including long‐established cultural and household dietary practices, and intolerable side effects of oral iron.“Even the baby might suffer if mother has high BP…the sac may dry up.”…“Due to high BP, IUD [Intrauterine death] can happen.” Focus group: ANMsHypertension was regarded as a priority because of the potentially serious consequences to the mother and baby. Knowledge of HDPs, including pre‐eclampsia, was embedded in ANC, and the training and education of all healthcare workers. Pregnant women also understood the importance of blood pressure (BP) measurement as part of routine care. Healthcare workers demonstrated knowledge of the diagnostic criteria, symptoms, and complications of pre‐eclampsia.“…at the time of delivery, there will be increase in BP in most of them [the women] and that too due to apprehension, stress, and due to the labour pains and all of the women will have that…”Interview: ObstetricianDuring the focus groups, CHWs discussed cases of women who had reported normal BP throughout pregnancy, and presented in labour with a high BP, with serious consequences, including stillbirth Table , Quote 3Although recognised as a high‐risk condition, GDM was considered low priority at both study sites, with very few cases reported by women, healthcare workers, and government officials Table , Quote 4Blood pressure measurement was embedded in routine ANC, and the experiences of healthcare workers and pregnant women alike Table , Quote 5Obstetricians reportedly provided advice on when to stop antihypertensives. One woman described taking medication for a short period until her BP normalised, which later had to be restarted. ASHAs conducted most home visits but home‐based BP monitoring was not conducted. The lack of home BP measurement was explained by women and healthcare workers because “ASHAs don't take BP”.“We can do [the OGTT]…but it will take time…Those two hours of waiting, taking a sample again… and all that… I've seen a patient tested there… it takes two hours for each patient… waiting for hours is difficult in villages… There will be people who go to work.”Focus group: ANMThere were significant variations in testing for GDM and the diagnostic criteria used Table , Quote 8Alternatives to the OGTT were offered: pregnant women were asked to attend the PHC with a roti (Indian bread), and capillary or venous blood samples were taken before and after eating it. These practices did not follow any established guidelines, but had been locally devised.Pregnant women described having their blood sugar measured; however, there was a lack of understanding about why, how, and when the test needed to be performed in pregnancy. In contrast with how BP measurement during ANC had become routine practice, there were not similar expectations for GDM screening.“There is a lot of awareness here… this is a rich village… there is no one who doesn't have a fridge…. Already older patients have glucometers with them… most of them have relatives staying in America.”Interview: Laboratory technicianThe management of GDM varied between study sites. In Andhra Pradesh, insulin was first‐line treatment, although this was difficult to implement in the villages because refrigeration and home blood glucose monitoring were not available. In one particular village, with a more affluent population, the laboratory technician made reference to everyone having a refrigerator and using glucometers at home. This finding was not replicated in other villages.“Metformin is free of cost here for patients…. the patients that come here are poor, first they want to come here as the services are free.”Interview: ObstetricianIn contrast, in Haryana, the first line management of GDM was metformin, which was free of charge through government schemes, and in this setting, did not require blood glucose monitoring.When asked about the new Government of India 2018 GDM guidelines (which suggest CHWs perform OGTTs and capillary blood glucose measurements in the villages),Following delivery, it was cultural practice for women at both study sites to remain at home for the first 40 days. Their main point of contact during this period was ASHAs. Women would not leave their house unless the baby had a problem requiring hospital care or they were advised to do so by the ASHA.Although obstetricians counselled women about the chance of recurrence of HDPs and GDM in subsequent pregnancies, and recommended postpartum checking of BP and blood glucose in high‐risk women, they acknowledged that postpartum blood glucose measurements were rarely performed. However, they presumed BP was checked postpartum, although the women with experience of HDPs reported their BP was not measured after returning home  had witnessed the long‐term sequelae of both HDPs and GDM in their villages.This study was rigorous in its methodology and adopted a theory‐driven approach to data analysis. A representative sample of all stakeholders ensured a wide range of data sources from which to draw conclusions. Although data saturation was reached at these two sites, the views expressed may not be representative of these communities as a whole. Whilst most of the in‐depth interviews were conducted individually as per study protocol, at one site, primary care physicians/obstetricians/laboratory technicians were interviewed in pairs at their request.Anaemia was identified as the most common and important high‐risk condition affecting pregnant women at both study sites. This finding is supported by several studies,Gestational diabetes mellitus has been the key priority area for funding and research within the Indian context.Postpartum management and long‐term follow up of high‐risk pregnant women were found to be lacking in our study. As socio‐cultural practices around childbirth in rural India often require the woman to stay at home for the first 6 weeks postpartum, CHWs are ideally placed to deliver community‐based interventions to engage and follow up women in their homes, and ensure continuity of care. Home‐based BP monitoring by CHWs has been shown to be feasible and effective in several studies, both within and outside the context of pregnancy.Given the high rates of hypertension, CVD, and conversion of GDM to T2DM for women in India, there is a need for improved education of health providers, as well as standardisation of testing and diagnosis of HDPs and GDM in rural Indian settings. Further work is particularly needed to raise awareness among rural women and healthcare workers at all levels of the postpartum period and long‐term health of women with a history of hypertension and GDM. Early identification and counselling of women at high risk of cardiometabolic disorders through innovative community‐level interventions may offer opportunities to address the escalating burden of these conditions.There are no competing interests declared. Completed disclosure of interests forms are available to view online as supporting information.SN was responsible for the conception, planning, delivery, analysis, and writing up of the paper. LH contributed to the planning of focus groups/interviews, qualitative analysis and provided feedback on the draft paper. DP contributed to the planning and delivery of the study, and provided feedback on the draft paper. JH contributed to the planning of the study and provided feedback on the study design, results, and drafting of the paper. RN and SK helped revise the paper.Ethical approval for the study was obtained from the Oxford Tropical Research Ethics Committee (OxTREC), UK ; and The George Institute Ethics Committee, India .SN is supported by a Medical Research Council Clinical Research Training Fellowship (MR/R017182/1). The George Institute for Global Health Global Women's Health programme provided financial support for the research assistant and fieldwork costs in India. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


A major challenge in the development of cancer nanomedicine is the inability for nanomaterials to efficiently penetrate and deliver therapeutic agents into solid tumors. Previous studies have shown that tumor vasculature and extracellular matrix regulate the transvascular and interstitial transport of nanoparticles, both critical for successfully delivering nanomedicine into solid tumors. Within the malignant tumor microenvironment, blood vessels are morphologically abnormal and functionally exhibit substantial permeability. Furthermore, the tumor extracellular matrix (ECM), unlike that of the normal tissue parenchyma, is densely packed with collagen. These pathophysiological properties greatly impede intratumoral delivery of nanomaterials. By using an antivascular endothelial growth factor receptor antibody, DC101, and an antitransforming growth factor β1 (TGF‐β1) antibody, normalization of the tumor vasculature and ECM is achieved, respectively, in a syngeneic murine glioma model. This normalization effect results in a more organized vascular network, improves tissue perfusion, and reduces collagen density, all of which contribute to enhanced nanoparticle delivery and distribution within tumors. These findings suggest that combined vascular and ECM normalization strategies can be used to remodel the tumor microenvironment and improve nanomedicine delivery into solid tumors, which has significant implications for developing more effective combinational therapeutic strategies using cancer nanomedicine. One of the major limitations of cancer nanomedicine is the inefficient delivery of nanomaterials into solid tumors.In this study, we hypothesized that simultaneously normalizing the tumor vessels and ECM would facilitate the delivery and distribution of nanoparticles into solid tumors. By using a syngeneic malignant glioma tumor model, we set out to determine whether a combined anti‐VEGFR and anti‐TGF‐β1 treatment could significantly normalize tumor vasculatures and reduce collagen density within the ECM. Using multiphoton imaging, our goal is to determine the effect of simultaneous tumor vascular and ECM normalization on nanoparticle delivery and distribution with the tumor interstitium in vivo, thereby providing a strong preclinical rationale for combining a microenvironmental priming strategy with nanomedicine for cancer treatments.FigureWe previously studied the effect of vascular normalization on improving nanoparticle delivery in murine syngeneic breast cancers, and found that the optimal intratumoral nanoparticle delivery relies on both transvascular and interstitital transport mechanisms, dictated by the tumor vasculature network and ECM, respectively.−1 every 3 days), or a murine anti‐TGFβ1 antibody (100 µg every 3 days)  Figure2a. By FigureWe next evaluated the effect of anti‐TGFβ antibody treatment on collagen density within the tumor ECM. TGFβ1 is a well‐known inducer of collagen deposition and promoter of stromal cell differentiation.FigureNext, to test whether the normalization of tumor blood vessels and ECM can improve intratumoral nanomedicine delivery, we treated tumor‐bearing mice with combined DC101 and anti‐TGFβ1 antibody and measured the distribution of nanoparticles within the tumor interstitium. We used water‐soluble semiconductor nanocrystals (QD) with surfaces modified by 10KD molecular‐weight polyethylene glycol (PEG) as fluorescence tracers. These monodispersing nanoparticles possess a final hydrodynamic diameter of ≈40 nm, with a slightly negative surface charge  and also exhibit a high degree of photo‐ and physical stability within biological environments , thus enable us to perform quantitative studies on particle accumulation using fluorescence intensity measurements. We found that the combined DC101 and anti‐TGFβ1 antibody treatment significantly enhanced the intratumoral delivery of the fluorescence nanoparticles, resulting in deeper tissue penetration and a more homogeneous distribution of these particles within the tumor interstitium Figure4a,d,e.Achieving efficient and uniform delivery of nanomedicine into solid tumors has been a major challenge facing cancer nanotechnology research. A recent metaanalysis further suggested that the inability to more effectively deliver nanomedicine from systemic circulation into the tumor parenchyma has severely impeded its potential for clinical translation.Abnormalities in the tumor microenvironment result in pathophysiological features that prohibit the efficient transport of nanomedicine and macromolecules into the interstitial space of the tumor.Even if nanoparticles are able to extravasate from the intraluminal space of blood vessels into the tumor interstitium, their transport is further restricted by diffusional hindrance, exerted by a densely packed ECM.Here, we investigated whether combined normalization of tumor vessels and ECM could result in improved delivery of nanoparticles into solid tumors, leading to a more uniform distribution pattern. We demonstrate that by using low‐dose anti‐VEGFR and anti‐TGFβ1 antibodies, we could normalize blood vessels and decrease collagen content within murine syngeneic gliomas. These physiologic and structural changes in the tumor microenvironment increased the depth of penetration of nanoparticles and the uniformity of their distribution within the tumor. Notably, although TGFβ has long been established as a potent inducer of collagen synthesis and differentiation of stromal cells such as fibroblasts, recent studies have found that TGFβ inhibition in itself can lead to a decrease in pericytes coverage, resulting in enhance tumor vessel permeability in pancreatic adenocarcinoma.Finally, it is important to realize the normalization of both tumor vessels and ECM is a transient event that is strongly dictated by the dose and duration of the therapeutic agents given and by the characteristics of the tumor itself.The present study suggests that simultaneous normalization of abnormal tumor vasculature and components of the ECM can enhance the delivery of nanomedicine into solid tumors. The finding that the combined anti‐VEGFR and anti‐TGFβ treatment resulted in enhanced and more uniform accumulation of nanoparticles within the tumor interstitium relative to either therapy alone further confirms that intratumoral transport of nanoparticles and macromolecules is a two‐step process, governed by distinctive cellular and pathophysiological properties of the tumor. The development of tumor microenvironment modulation strategies to improve the transvascular and interstitial transport of nanoparticles, coupled with a more complete understanding of how intrinsic properties of nanomaterials affect intratumoral delivery, can pave the way for identifying optimal strategies to deliver nanomedicine into solid tumors.The authors declare no conflict of interest.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.