Indications were found that a diagnostic bias could have contributed to the National Cancer Institute’s (NCI) suggestion of a persistent increased mortality risk for nasopharyngeal cancer (NPC).NCI provided the cohort data updated through 2004. We computed local county rate-based standardized mortality ratios (SMRs) for NPC and all other entities of the pharynx for two time periods. Moreover, SMRs were calculated for pharyngeal cancer in relation to study site by cumulative exposure to formaldehyde (FA).Overall, our results corroborate the indications of a diagnostic bias by strong but contrary temporal trends for NPC and pharynx, not specified. Moreover, it was shown that mortality risks were increased in the Wallingford cohort for all pharyngeal cancer combined and for pharyngeal cancer excluding NPC. In contrast, no increased risks for these categories were found in the nine other study sites combined.Our re-analysis provided little or no evidence to support NCI’s suggestion of a persistent association between FA exposure and mortality from NPC. In October 2009, a working group of the International Agency for Research on Cancer (IARC) unanimously reaffirmed the classification of formaldehyde as Group 1, based on sufficient evidence in humans of nasopharyngeal cancer (NPC), although a group of different working group members concluded in March of the same year that exposure to formaldehyde is unlikely responsible for the increased risks of NPC in woodworkers reported in most case–control studies and in a pooled reanalysis of cohort studies  - is proA recent re-analysis of the NCI formaldehyde cohort showed that the observed overall increased risk for NPC was driven by results for only one study site , and the results for the other nine study sites Plants 2–9) were unremarkable . The str were unrIndications were also found in a second independent reanalysis that diagnostic bias could have contributed to the elevated risk estimators for NPC, and it was suggested to carry out an expanded analysis of the NCI formaldehyde cohort to explore these findings further . The anaThe current re-analysis was extended to all subcategories of pharyngeal cancer : 146–149). In order to recognize changes over calendar time, the time period of the initial analysis (1934–1979) was compared with that of the two extension periods for the cohort’s follow-up (1980–2004). Moreover, we compared results between the Wallingford site with those from all other study sites combined.2 = 4.40 (p < 0.05). In contrast, for pharynx, unspecified (ICD8: 149), a strong increase of the SMR from 0.23 to 1.52 was observed (χ2 = 3.54 (p = 0.06). These results give clear indications of a diagnostic bias or even death certificate coding errors.Table 2 = 8.37; p < 0.01) as did the statistically significant  interaction term computed in the recent re-analysis by Marsh and colleagues [Our analysis by NCI study site showed that mortality risks were increased in the Wallingford cohort for all pharyngeal cancer combined and for pharyngeal cancer excluding NPC Table . In contThe comparison of exposure categories with regard to cumulative exposure gives no indication of a possible exposure-risk relationship either for the original or for the extended follow-up period Table . A furthThe analyses presented here provide no any evidence for a relationship between exposure to formaldehyde and pharyngeal cancer mortality in Plants 2–10. The likelihood of a diagnostic or death certificate coding bias with respect to pharyngeal cancer appears be high, especially in the Wallingford cohort. Furthermore, the new findings support the hypothesis that occupational exposures external to the Wallingford cohort may be responsible for the excess of pharyngeal cancer in the Wallingford cohort. Our findings are consistent with other recent study results and reviews –20 and t


Since the discovery of DNA, the normal developing and functioning brain has been assumed to be composed of cells with identical genomes, which remains the dominant view even today. However, this pervasive assumption is incorrect, as proven by increasing numbers of reports within the last 20 years that have identified multiple forms of somatically produced genomic mosaicism (GM), wherein brain cells—especially neurons—from a single individual show diverse alterations in DNA, distinct from the germline. Critically, these changes alter the actual DNA nucleotide sequences—in contrast to epigenetic mechanisms—and almost certainly contribute to the remarkably diverse phenotypes of single brain cells, including single‐cell transcriptomic profiles. Here, we review the history of GM within the normal brain, including its major forms, initiating mechanisms, and possible functions. GM forms include aneuploidies and aneusomies, smaller copy number variations (CNVs), long interspersed nuclear element type 1 (LINE1) repeat elements, and single nucleotide variations (SNVs), as well as DNA content variation (DCV) that reflects all forms of GM with greatest coverage of large, brain cell populations. In addition, technical considerations are examined, along with relationships among GM forms and multiple brain diseases. GM affecting genes and loci within the brain contrast with current neural discovery approaches that rely on sequencing nonbrain DNA ). Increasing knowledge of neural GM has implications for mechanisms of development, diversity, and function, as well as understanding diseases, particularly considering the overwhelming prevalence of sporadic brain diseases that are unlinked to germline mutations. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol, 2018 Do all our body cells have the same DNA?The exquisite organization and complexity of cells within the brain have been recognized since the days of Golgi and Cajal Cajal,  at the tnormal cells of the body, with clear functional consequences through the generation of antigen receptors, and selection and survival of immune cells  it occurs somatically and therefore does not affect germline DNA sequences, contrasting with “genetic” mutations that enter the germline (hence the term “genomic” rather than “genetic” mosaicism here); (2) it produces nucleotide sequence changes, as differentiated from epigenetics   on nonmitotic cells   since they would not be produced by sectioning artifact, combined with a focus on sex chromosomes that provided positive, internal controls for identifying 1 (single X‐ and Y‐) chromosome in males or 2 (X‐) chromosomes in females in the face of aneuploid numbers of sex chromosome or autosome copies  , and analyses by either flow cytometry or fluorescence activated cell sorting (FACS)  and mouse cloning techniques involving mitral cells of the olfactory bulb to amplify single neuronal genomes, which also identified hundreds of SNVs within seven single neurons  samples. As noted above, the first was achieved by SCNT and clonal expansion of a single neuron—where all derivative cells have identical genomes—which allowed the collection of high resolution, whole genome coverage data, albeit with the previously noted limitations. A second approach involves capture of targeted genomic regions using bulk DNA combined with pulldown “bait” strategies. The smaller size of the genome under interrogation allows ultra high depth sequencing , which enables detection of somatic variants with lower allele frequency  an absolute requirement to amplify the single‐cell genomic template using polymerases, which itself may introduce a range of artifacts that could create or obfuscate mosaically present CNVs or SNVs; (2) different protocols and amplification techniques—including different DNA polymerases—limit direct comparisons; (3) an inability to replicate the results, since each single cell is destroyed by sequencing; (4) a current inability to assess sub‐megabase CNVs; (5) varied and often opaque technical details that obfuscate both failure rates and bioinformatics pipeline details; (6) limited individual brain interrogation that consists of a few or even just 1 brain; and (7) miniscule sample sizes—typically less than 100 cells. Considering the 1 trillion cells in the human brain, these samples constitute only 10The relative sizes and forms of GM Fig.  undersco3H‐thy) , ataxia telangiectasia (ATM), and Aicardi‐Goutières syndrome . MECP2 is a suppressor of LINE1 transcription , in which inactivating gene mutations in mitotic proteins causes an increase in forms and frequency of aneuploidy, ultimately results in microcephaly and mental retardation . DCV increases of ∼200 Mb over the normal 250 Mb within prefrontal cortical neurons indicate significant, subgenomic increases in DNA content that are not explained by cell cycle reentry   through both increased DCV and specific CNV amplification of the pathogenic gene, Amyloid Precursor Protein (RAG1 but not RAG2 within the brain vs. both in the immune system). All combined, these diverse, nonmutually exclusive and pervasive forms of neural GM could “barcode” each brain cell by creating a unique genome, thus representing a small universe of genome diversity residing within a single brain. Moreover, further evidence that this universe changes over time adds another dimension of complexity, representing a relatively unassessed variable contributing to neural diversity at all levels of brain development and function. This same genomically diverse universe is currently unrecognized by virtually all genetic models of brain disease, particularly those relying on statistical relationships of genes identified from cells outside of the brain, as is common for genome‐wide association studies (GWAS). The overwhelming prevalence of sporadic brain disease unaccounted for by defined familial genes—as observed in Alzheimer's disease—may be more fully explained by mosaic genomic changes that affect both the genes identified in rare familial cases, as well as new genes and nongenic loci (including mutations that may not be compatible with life if present constitutively), particularly within postmitotic neurons. Individual cells altered in sporadic disease could offer a rich, new source for discovery of meaningful disease targets. GM within the brain therefore represents a vast frontier awaiting further exploration and discovery, toward more fully understanding the developing and functioning brain and its diseases.Over the last 20 years, neural GM has advanced from a theoretical concept to a definitive experimental fact and now represents a vibrant field of active research. The proven forms of GM within single cells of the brain—aneuploidies and aneusomies, other CNVs, and SNVs—are no doubt the “tip of the iceberg” in considering the pervasive presence of DCV throughout the brain that captures virtually all forms of DNA sequence alterations, affecting both mitotic and postmitotic populations. The combination of these alterations contribute to increased GM over time Fig. a–c. As p


The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Recent advances have provided evidence for the involvement of neutrophils in both innate and adaptive immunity, robustly challenging the old dogma that neutrophils are short‐lived prototypical innate immune cells solely involved in acute responses to microbes and exerting collateral tissue damage. There is now ample evidence showing that neutrophils can migrate into different compartments of the lymphoid system where they contribute to the orchestration of the activation and/or suppression of lymphocyte effector functions in homeostasis and during chronic inflammation, such as autoimmune disorders and cancer. In support of this notion, neutrophils can generate a wide range of cytokines and other mediators capable of regulating the survival, proliferation and functions of both T and B cells. In addition, neutrophils can directly engage with lymphocytes and promote antigen presentation. Furthermore, there is emerging evidence of the existence of distinct and diverse neutrophil phenotypes with immunomodulatory functions that characterise different pathological conditions, including chronic and autoimmune inflammatory conditions. The aim of this review is to discuss the mechanisms implicated in neutrophil trafficking into the lymphoid system and to provide an overview of the immuno‐regulatory functions of neutrophils in health and disease in the context of adaptive immunity. Copyright © 2018 The Authors.  Pathological induction of NETs, such as that induced under conditions of sterile injury (e.g. ischemia/reperfusion injury) can also cause tissue damage and indeed NETosis has been implicated to the pathogenesis of a wide range of non‐infectious inflammatory disorders Neutrophils are short‐lived immune cells that are commonly accepted as being the first leucocyte sub‐type to be recruited in large numbers from the blood circulation into inflamed tissues. Their migration through the cellular and matrix components of blood vessel walls is a tightly regulated process involving intricate cellular and molecular interactions, as summarised in recent articles in vitro and the general and well‐accepted assumption that the neutrophil life expectancy in the blood circulation did not exceed 1 day. However, advancements in techniques for tracking neutrophils in vivo, and the overall better understanding of neutrophil biology, have unequivocally demonstrated that neutrophils can exhibit prolonged survival both in vitro and in vivoNearly 50 years ago, whilst studying the trafficking of immune cells in sheep, Smith and colleagues discovered that the neutrophil ‘endgame’ was not limited to apoptosis within inflamed tissues but that these cells could be detected in the peripheral lymph of the animals LysM‐GFP‐ki mice), enabled detailed analysis of the dynamics of neutrophil–lymphatic vessel interactions as well as the role of specific molecular cues involved in this process , CCR7 deficient mice have reduced numbers of neutrophils migrating into tissue‐associated lymphatic capillaries and into draining LNs, as compared to wild‐type control littermates in vivo expression and function of CCR7 on neutrophils. Other studies have suggested that the CXCR4/CXCL12 axis is critical for neutrophil entry into the lymphatic system Staphylococcus aureus infection, a specific inhibitor of CXCR4, AMD3100, was shown to significantly reduce the migration of neutrophils into afferent lymphatic vessels and draining LNs, whilst CCR7‐deficient neutrophils exhibited normal trafficking to the lymphatic system in vitroin vivoin vivo and in vitro – and species – scenarios. In addition to chemokines, adhesion molecules such as ICAM‐1 and VCAM‐1 have been reported to be expressed by stimulated LECs and to support human and mouse neutrophil–lymphatic vessel interactions via binding to leucocyte β2 integrins (e.g. Mac‐1) both in vitro and in vivoin vivoNeutrophil migration to lymphoid organs was first demonstrated in animal models through localising neutrophils within the draining LNs of tissues infected with microorganisms or following vaccine challenge ss Table . These sSeveral studies have also demonstrated the capacity of blood circulating neutrophils to enter LNs via high endothelial venules (HEVs) during infection, post immune complex activation and antigen sensitisation Collectively, there is now ample evidence to demonstrate the capacity of neutrophils to migrate during inflammation into LNs via two distinct routes, though the molecular pathways of such events require further exploration. Nevertheless, the fact that LN neutrophils can originate from either the blood circulation or inflamed tissues suggests potential differential modes of neutrophil‐mediated regulation of the adaptive immunity. The following section discusses the immuno‐modulatory functions of neutrophils in the context of lymphocyte activation.S. aureus infected animals in vivo and in vitroS. aureus infected animals Recent advances in neutrophil biology, including studies detailed in the previous section, have acknowledged these cells as key players at the interface of innate and adaptive immunity in both physiological homeostasis and pathological inflammation. The rapid and transient nature of their trafficking to LNs, with a dwelling time in these organs not exceeding 2–7 days following an initial inflammatory insult S. aureus infection, B‐helper neutrophils were shown to limit the production of IgM through release of TGF‐β1 In recent years, the homogeneity of neutrophil population has become increasingly questionable, with detection of neutrophils exhibiting distinct functions and profiles leading to the concept of different subsets Taken together, there is now unquestionable evidence supporting neutrophil trafficking into the lymphatic system and the ability of these cells to regulate lymphocyte functions. These works have promoted more interest into the potential role of neutrophils in chronic disorders, including autoimmune diseases, as discussed below.The notable and now widely accepted diversity of neutrophil functions has placed a spotlight on the potential existence of distinct neutrophil‐subsets and their association with a broad range of pathologies  was also associated with the rapid recruitment of neutrophils (i.e. within 4 h post PDT) into tumour‐draining LNs through HEVs in an IL‐17 and CXCR2/CXCL2 (but not CXCL1)‐dependent manner In human cancer, tumour‐associated neutrophils have been classified as having anti‐tumour (N1) and pro‐tumour (N2) properties  Neutrophils with immuno‐modulatory activities have also been detected in ageing, now regarded by some researchers as a low grade chronic inflammatory state (i.e. inflammageing). Accordingly, in aged mice neutrophils have been detected in high numbers in both T‐cell and B‐cell areas of the secondary lymphoid organs (i.e. LNs and spleen) In vitro, freshly isolated synovial neutrophils are capable of stimulating a proliferative response in T‐helper cells in vivo within the secondary lymphoid organs. Related to this caveat, using a murine model of lupus, Bird and colleagues demonstrated that neutrophils can preferentially form close interactions with T cells in the early phase of the disease, whilst in the advanced stage, they mainly accumulate in B cell areas of the spleen, reminiscent of a B‐helper phenotype In autoimmune pathologies such as RA, human synovial neutrophils express transcripts and proteins of MHC‐II and co‐stimulatory molecules CD80 and CD86 during the early phase of the disease The number of PMN‐MDSCs has also been reported to increase in RA and SLE patients Overall, whilst the current literature supports the concept of neutrophils orchestrating a pathogenic response during autoimmune and chronic inflammatory disorders, further investigations are required to determine if and how neutrophil trafficking into lymphoid organs might be contributing to the aetiology of these diseases.ex vivo and in animal models of inflammation in vivo has led to the identification of different sub‐types of neutrophils with distinct regulatory functions in health and disease. It is still debated whether neutrophil subsets might represent an acquired phenotype, and/or level of activation through environmental molecular cues, or are in fact ontogenically separate cell populations. Irrespective of this, renewed interest in this phenotypic diversity and associated varied effector functions, in conjunction with better understanding of the spatiotemporal localisation of neutrophils is opening challenging research opportunities. In particular, such works could pave the way towards addressing outstanding questions regarding the diverse functions of neutrophils during the development of autoimmune pathologies. Most importantly, association of different neutrophil sub‐types with the pathogenesis of defined disorders has the potential for stratifying patients in terms of disease severity as well as identify new therapeutic targets in chronic and autoimmune conditions and cancer.In addition to their essential role in protecting the host against acute harmful insults, recent studies have identified a broader role for neutrophils in both physiological and pathological immunity, and hence the development of numerous chronic and autoimmune inflammatory disorders. Specifically, it is now clear that, despite being short‐lived as compared to other immune cells, neutrophils can traffic into lymphoid tissues and contribute to shaping key adaptive immune responses. As such, current evidence supports the notion that in addition to dendritic cells and macrophages, neutrophils can act as a cellular bridge between innate and adaptive immunity in both health and disease. Whilst our understanding of the mechanisms of neutrophil trafficking and functions within lymphoid tissues is incomplete, it is now accepted that depending on the nature, duration or the site of the insult, neutrophils can enter the lymphatic system by using both afferent lymphatic vessels as well as venular portals within LNs. Once in secondary lymphoid organs, neutrophils can act as immune‐modulatory cells through cytokine production and via direct cell–cell interaction with other immune cells. Accordingly, neutrophils can orchestrate elaborate cellular and humoral responses as well as exert regulatory effects on lymphocyte functions. The development of new technologies applied to investigate the dynamics of neutrophil behaviour, phenotype and trafficking in both patient samples MBV performed the literature search and wrote the manuscript. SN contributed to the writing and revision of the manuscript. Both authors approved the final version.


Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE)  coupled with droplet‐based microfluidics allows the detailed analysis of millions of enzyme variants on ultra‐short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet‐based microfluidics, made in this context towards the high‐throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell‐based assays, as in the case of directed evolution experimentations. DEdirected evolutionFACSfluorescence‐activated cell sortingFADSFluorescence‐activated droplet sortingIVCin vitro compartmentalizationMTPmicrotitre platePFPEperfluoropolyetherw/owater‐in‐oilw/o/wwater‐in‐oil‐in‐water1Living organisms often take millions of years to evolve enzymes capable of metabolizing naturally occurring substances. Enzymes are exquisitely versatile and proficient catalysts. Optimized by evolution over time, they can initiate, accelerate, and control a diversity of reactions within living systems, whilst ensuring high substrate specificity as well as extraordinary selectivity. Despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to the processing of non‐living systems in industrial settings 13 unique combinations of amino acids , which exceeds the achievable library size using any common protein library creation method Directed evolution coheres with Darwinian evolution in two ways. First, a genetically diverse “population” (or gene library) is created and translated into a corresponding library of gene products (encoded enzymes). This is then followed by a selection process aimed at isolating variants with the most‐desired characteristics (the “fittest”) Although the realization of improved mutants via DE strategies has been reported, variants with even higher activities are in most cases anticipated, since the number of variants that have been screened is miniscule when compared to the scope of available gene sequence space. Indeed, the structural characterization of enzymes and mutant library design together represents only the first step en route to the discovery of novel enzymes. Given the vast libraries of mutants that can in principle result from all kinds of engineering activities, tools and technologies that allow for the creation of high‐throughput screening platforms are equally, if not more, valuable. Droplet‐based microfluidic technologies have been shown to hold enormous potential for high‐throughput screening applications 24 and 105 tests per day 6 compounds per day can be considered as ultra‐high‐throughput. Although assays for specific enzyme families are normally tailored according to the reactions involved, a common principle is followed. First, a reliable enzyme production system is required. Based on their high transformation efficiencies and well‐established genetic manipulation tools, Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Saccharomyces cerevisiae (S. cerevisiae) are the most widely used host organisms The scale of DE experiments is generally limited by access to high‐throughput screening methods, since the number of variants that can be realistically screened are orders of magnitude smaller than the typical library size. Put simply, the development of assays that allow rapid and cost‐effective selection of desirable mutants is demanded. High‐throughput screening is a poorly defined term, but is generally understood to describe platforms and methods able to perform between 107 per round of evolution) and inexpensive. However, such a method is only possible when enzyme activity offers a growth advantage in host cells, significantly limiting its application. Next in the complexity scale and throughput are agar plate assays, where substrate conversion creates a visual signal (such as a color or fluorescence variation) around active colonies within the agar medium 4 per round of evolution), making agar plate assays more widely used as a pre‐screening tool  screening is still the most commonly used format for library screening. It represents a miniature analogue of the cuvette system, where the balance between assay complexity and throughput is well‐distributed. In particular, the possibility to couple MTPs with many analytical tools and standardized equipment for detection has made it exceptionally practical. A standard plate reader can readily perform UV‐Vis absorbance and fluorescence detection of samples in a 96‐well plate. Analysis of the contents of each microwell using LC/MS or GC/MS can also be realized E. coli cells, Aharoni et al. reported the screening of > 106 glycosyltransferase variants from an error‐prone PCR library using FACS Pseudomonas aeruginosa esterase EstA by displaying enzyme variants on the surface of E. coli. Esterase activity results in the release of a phenolic compound which in turn is captured on cell surface via peroxidase catalyzed radical formation Further miniaturization of reaction systems has led to increased interest in using single host cells as reaction compartments, with the activity of each enzyme variant being assessed by fluorescence‐activated cell sorting (FACS). FACS is a powerful technique that allows screening and sorting of cells at rates of up to 400 000 cells per second. This means that, in principle, the screening of 1 million variants can be accomplished in less than 2 min. In addition, the assay volume in this case has effectively decreased from a few microliters (in a MTP well) to approximately the volume of single bacterial cell, which is typically a few femtoliters. Yet, unlike MTP‐based assays, where each well acts as a physical boundary to enclose the reaction mixture, the use of FACS requires the physical linkage between genotype (the enzyme‐encoding gene) and phenotype  to be confined at a single‐cell level. This ensures the possibility to trace back and identify beneficial gene mutations accordingly. In the context of an enzymatic reaction, the assay is restricted to two main scenarios. In the first case, the enzyme produced stays within the cytoplasm of the cell. The substrate should then have the ability to diffuse across the cell membrane for reaction to occur, whilst the fluorescent product accumulates and stays trapped inside cells. In the second scenario, the enzyme is displayed at the surface of the cell to facilitate reaction with substrate and at the same time, the fluorescent product is also captured on the cell surface Fig. C. By exp7‐fold excess of genes encoding another enzyme 10 compartments available in 1 mL of emulsion), high‐throughput screening and sorting of the created compartments is not possible, due to the incompatibility of the continuous oil phase with FACS. Bernath et al. later proposed further re‐emulsification of w/o emulsions in an aqueous phase to yield water‐in‐oil‐in‐water (w/o/w) double emulsions. This effectively provides the droplets with an external aqueous phase suitable for flow cytometry applications To address the product accumulation challenge, alternative methods for compartmentalization have to be devised to ensure genotype‐phenotype linkage. To this end, emulsion‐based technologies have emerged as potential tools, where the evolved enzymes, substrate as well as the product are all encapsulated in water‐in‐oil (w/o) microdroplets 3Droplet‐based microfluidics are flow‐based microfluidic systems that enable the generation and processing of monodisperse w/o droplets at kHz to MHz frequencies 3.1To date, a large number of substrate materials have been reported for the fabrication of microfluidic devices/chips, including glass, silicon E. coli) per droplet, such that each of them expresses a distinct enzyme variant of the designed library. In practice, cells are usually loaded together with the aqueous phase, a random process that is passive in nature, yet simple and rapid in operation. This in turn yields a population of droplets with Poissonian‐distributed cell occupancy During droplet generation, the encapsulation of various entities including cells , biomolecules such as enzyme or DNA, and beads is yet another significant task in droplet‐based microfluidics. Crucially, and relating to the DE of proteins, the goal is often to encapsulate one bacterial host cell  is made possible via merging of droplet pairs. Passive methods involve the use of dedicated structures such as pillar arrays Once formed, droplets can be incubated in delay lines, for periods from minutes to hours subject to the needs of the assay in hand. For example, enzymatic reactions could range from seconds to hours depending on activity, whilst monitoring cell growth may occur over days. To facilitate incubation at shorter time scales (up to a few seconds), an extended microfluidic channel is simply used, where droplets remain flowing in a single file The ability to perform quantitative analysis and then select specific droplets represents perhaps the most significant operation within a screening platform. An excellent review of analytical methods used in droplet‐based microfluidic systems can be found elsewhere Recovery of droplets of interest is carried out on‐chip by active sorting, i.e., by deflecting specific droplets into a dedicated outlet channel on‐chip. To perform sorting, droplets are typically reinjected and spaced by oil stream(s) before passing the point of detection and finally arriving a junction at which the main channel is split into two outlet channels: the “waste” and “collection” channel. Droplets flow per default into the “waste” channel owing to a lower hydraulic resistance. To deflect a chosen droplet, an AC field is applied, which results in a positive dielectrophoretic force on the droplet and hence movement towards the higher electric field region . Depending on the assay, integration of fluorescence or absorbance detection with dielectrophoresis‐based sorting gives Fluorescence‐activated droplet sorting (FADS) 3.3The success of any DE experiment is primarily conditioned by the confinement of the genotype‐phenotype linkage. From a microfluidics perspective, long‐term stability of droplets is thus essential; meaning that any uncontrolled coalescence or exchange of components between droplets has to be excluded. Certainly, biocompatibility of all materials and reagents involved, both in prototyping of devices as well as compartmentalization, has to be ensured. Otherwise, cross‐contamination, leakage from droplets or a change in droplet size can obscure assay readout.Long‐term stability of droplets is almost exclusively facilitated by the use of appropriate surfactants. Surfactants are amphiphilic molecules, bearing groups that have affinity for each phase (aqueous and oil). They are normally supplemented in the continuous phase and upon contact with the discrete phase partition at the interface to enhance stabilization of droplets through the reduction of surface tension. It was proposed that surfactants prevent droplet coalescence mainly by steric hindrance and the establishment of a Marangoni flow, counteracting oil drainage between droplets that are in touch with each other Finally, one must not overlook the importance of controlling channel surface properties. Despite the hydrophobic nature of PDMS, surfaces are usually treated with fluoroalkylsilanes to ensure efficient generation and processing of droplets 4Over the last two decades, it has become clear that droplet‐based microfluidic platforms  enable not only ultra‐high throughput screening of enzyme libraries at low cost, but also effective identification and isolation of novel, improved enzyme variants. Moreover, the breadth of enzyme classes that can be engineered has also been expanded, owing to the rapid increase in the number of chemistries made applicable in droplet formats. Recent examples of library screening campaign performed in microfluidic workflows involve enzyme classes including aldolases kcat) compared to the best previously evolved variant S)‐enantioselectivity. In contrast, MTP‐based assays have only allowed access to (R)‐enantioselective enzymes 8 protein variants have been screened. As emphasized by the authors, such a screen has clearly exceeded the capacity of standard MTP‐based assays (droplets containing fluorescent product can be sorted at frequencies up to 2 kHz, whilst screening 2000 aldolase variants in a conventional plate assay took about two weeks Nonetheless, it should be mentioned that in some cases, the odds of success are so small that isolation of new enzymes would have been extremely challenging without the throughput gains provided by droplet‐based platforms. For example, Colin et al. reported the application of FADS in the functional screening of metagenomics libraries Directed evolution of proteins using droplet‐based microfluidic technologies has undeniably demonstrated promise. Since most droplet assays still rely largely on fluorescence readouts, the next challenge is perhaps the development of new detection systems to target a wider range of activities, without compromising sensitivity. This could allow protein engineers to move away from fluorogenic substrates, which in many cases show only limited resemblance to the corresponding natural substrates. In light of providing additional readouts, biophysical methods based on surface‐enhanced Raman scattering 5The commercial enzyme market has grown exponentially in recent years due to improved production technologies, engineered enzyme properties and new application fields. Directed evolution has developed rapidly to become the method of choice for protein engineers to create enzymes having bespoke properties. Advances in the biochemistry of such enzymes, made possible through the development of new technologies, will enhance the knowledge of their structure function properties. For example the development of an efficient screening system for on‐the‐fly screening of novel dehalogenases combined with semi‐rational design will dramatically increase the number of practical applications employing dehalogenases, including biodegradation and biosensing of environmental pollutants, neutralization of warfare agents and industrial biocatalysis The authors have declared no conflict of interest.


This study examined facial expressions produced during a British Sign Language (BSL) narrative task :343–353, 2014) by typically developing deaf children and deaf children with autism spectrum disorder. The children produced BSL versions of a video story in which two children are seen to enact a language-free scenario where one tricks the other. This task encourages elicitation of facial acts signalling intention and emotion, since the protagonists showed a range of such expressions during the events portrayed. Results showed that typically developing deaf children produced facial expressions which closely aligned with native adult signers’ BSL narrative versions of the task. Children with ASD produced fewer targeted expressions and showed qualitative differences in the facial actions that they produced. Deaf people use facial expressions while they are using sign language to express their own emotions or to describe the emotions of others, through the use of the same range of emotional facial expressions used naturally by the general population e.g. happiness, anger, sadness etc.  has investigated both comprehension and production of emotional facial expressions. The majority of these studies have focused on emotion recognition, and while not all demonstrate impairment in emotion processing ; rate of speech being too fast; limited or unusual pitch ranges; poor volume modulation; and more frequent errors in residual articulation distortion  expressions were likely to be elicited.Twelve TD deaf individuals were recruited from deaf schools across the UK. Ten deaf individuals with ASD were recruited from the National Deaf Child and Adolescent Mental Health Service, where they had received a diagnosis of ASD from a specialist multidisciplinary social and communication disorders clinic for deaf children, using an adapted version of the Diagnostic Interview for Social and Communication Disorders , hearing aids (4) and unaided (1); control group: cochlear implant (4), hearing aids (5) and unaided (3)]. In order to meet the inclusion criteria for the study, participants needed to be able to communicate using sign language at least at a phrasal level. One child in each group was a native signer with deaf parents, the remaining participants were all from hearing families. All participants were in bilingual education environments and were exposed to BSL in the classroom at the time of testing, which ensured greater consistency in signing abilities.The groups were matched for chronological age, nonverbal intellectual ability using the Raven’s Standard Progressive Matrices . Ethical approval was obtained from the ethics committee at University College London and the National Research Ethics Service, which is the ethical body for the NHS.The Social Responsiveness Scale  was a native sign language user and was aware of the participant group status of the individuals. These ratings were then sampled by a deaf native sign language user who was blind to group membership. The second rater scored 20% of the BSLPT videos (10% from each group). Both raters were required to identify when each expression was produced and label it in the ELAN video file  score is five facial action targets for each ‘demand’ expression and this participant scored 11 out of 15 for ‘demand’ overall, missing four facial actions in total. Overall scores for each participant were then converted into percentages, by calculating the total number of facial actions divided by the maximum number of facial actions that could have been produced. So the participant described above produced 32/44 facial actions, and a facial action score of 72%. In this way, the child participants were compared on their number of overall facial actions produced  relative to the adult gold standard measures.The BSLPT includes three screening questions to ensure participants had attended to and understood what happened in the scenario. The questions are asked in BSL. The translated English equivalents are: (1) what was on the tray? (2) Why did the boy throw the spider? and (3) why did the girl tease the boy? Each question is worth 2 points, and there is a maximum obtainable score of 6 points.There were no differences between groups for story comprehension as measured by the BSLPT content questions [t(22) = − .69, p > .05, Non-parametric Mann Whitney tests were used to compare the groups. The groups did not differ significantly on the length of narratives produced, although the ASD group had shorter narratives [U(25) = 41, p > .05, Figure The relationship between SRS severity and facial expression production was explored. There were significant correlations between SRS scores and two expressions: ‘annoyance’ r (11) = − .82, p < .05, and ‘mischief’ r(11) = − .77, p < .05. That is, higher SRS scores (more reported autistic traits) were associated with fewer ‘annoyance’ and ‘mischief’ facial actions.To test whether differences in general sign language or narrative abilities might account for differences in emotional facial expression production, scores on the BSLPT were compared  = .132, p = .71), narrative structure (H(1) = .89, p = .018) or grammar (H(1) = 2.132, p = 1.44).This is the first study to investigate the frequency and quality of facial expressions produced during a sign language narrative in deaf children with and without ASD. No differences were found between groups in terms of the total number of facial expressions produced, but there was a significant difference in the quality of facial actions produced. Deaf children with ASD compared with TD deaf children produced fewer expressions which corresponded to those produced by adult deaf signers when they retold the story. When individual expressions were analysed, the groups did not differ significantly on four of the six facial expressions scored, however deaf children with ASD produced significantly fewer facial actions for ‘demand’ and ‘mischief’ relative to deaf controls.The reduced quality of facial expressions in the deaf ASD group was broadly consistent with the finding of emotion production impairments ) but also expressions which could involve both emotion and other cognitive and communicative features . This is likely to be because the narrative itself involved a series of communicative events including a scenario in which one character deceives another (a girl hiding a pretend spider inside a sandwich that she knows a boy will eat). Indeed, it could be argued that a theory of mind (ToM) is required to fully understand the narrative. Hearing individuals with ASD have been shown to have deficits with ToM, and often fail false belief tasks (Baron-Cohen et al. A strength of this study is the use of an ecologically-valid, narrative methodology to elicit facial expressions; this contrasts with more artificial techniques sometimes used where participants are asked to copy facial expressions. This did however mean that only a narrow range of expressions were explored, and did not include all of Ekman’s classic examples of facial expressions (Ekman and Oster One relevant caveat of the current study is the lack of assessment of imitation or motor skills in deaf children with ASD. It is well known that children with ASD often have a range of motor deficits (Ghaziuddin and Butler Facial expressions in BSL convey both emotion and linguistic meaning. The present study focused on emotional facial expressions. The production of BSL linguistic facial expressions will be reported in a future paper. We expect that there will be differences in how both groups use emotional and linguistic facial expressions since linguistic expressions are more rule-governed, and may be easier for individuals with ASD to learn.The findings from the current study on facial emotion production lend some support to the hypothesis that deaf children with ASD have difficulties similar to those of their hearing counterparts. However, there is still much more research to be done in this field. In particular, research carried out with hearing individuals with ASD exploring their ability to be taught and use facial actions in BSL could inform whether the use of sign language helps facilitate attention to the face and improve communication skills.Impairments in facial expression production in deaf individuals with ASD are likely to impact on interactions with other deaf signers which may further exacerbate social communication (Shriberg et al. This study shows that deaf children with ASD show subtle differences in terms of their production of emotional facial actions during narrative retelling. These data are an important first step in documenting differences in sign language production for deaf children with ASD, with affected aspects of communication relating to emotion and mentalising about the internal state of others.


The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell‐line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9‐day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12‐ and 24‐hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription‐ and promoter‐specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non‐coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA–DNA–DNA triplexes. Such triplex‐forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex‐forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA‐triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes. So far, most studies have focused on understanding ON/OFF mechanisms such as the complete silencing of one X‐chromosome  or long‐term (DNA methylation) modifications that bring about conformational changes in chromatin, thus activating or repressing transcription  cells have been known as workhorses for the industrial production of recombinant therapeutic proteins since 1987 . Samples for ChIP‐seq were taken every 12 hr , for RNA‐seq every 24 hr (1 × 106 cells into 1 ml Trizol), and for whole‐genome bisulfite analysis at mid‐exponential and mid‐stationary phase . For RNA extraction, the cells were centrifuged and lysed using TRI reagent  following the manufacturer's instructions: phase separation was done by addition of chloroform and the aqueous phase collected. After precipitation with 2‐propanol and washing with 70% Ethanol, pellets were air‐dried and resuspended in nuclease‐free water. Libraries for RNA‐seq were prepared using NEBNext Ultra Directional RNA library prep Kits (E7420L), starting from total RNA according to the instructions and analyzed by Illumina HiSeq 2000 PE100 . Libraries for ChIP‐seq and bisulfite sequencing were prepared as described , resulting in 25,541 genes with functional annotation (Supporting Information Tables 1 and 2) and 78,873 noncoding transcribed regions encoding for 80,973 transcripts, including 51,193 long noncoding RNAs (lncRNAs) or processed transcripts. Based on the presence of active chromatin marks and length distribution, 1,528 noncoding genes and 947 protein‐coding genes were annotated with an unknown function (Supporting Information Table 3).2.4p values with a threshold of 0.01 and an absolute value of log2 fold change >1.The design formula for the samples was created based on the PC analysis that separated samples from different TP corresponding to the gene expression values  considering TP1, TP3, TP5, and TP7 (17–90 hr) as the exponential phase; TP9 and TP11 (114–138 hr) as the stationary phase; and TP13, TP15, and TP17 (162–210 hr) as the decline phase. Phase‐wise comparison was done between exponential and stationary (ES) and exponential and decline (ED) phases. Differentially expressed genes were extracted based on DESeq.2 normalized read counts by the Benjamini–Hochberg method to adjust z‐score normalization (x−mean/standard deviation) using the R clusterSim package v0.45–2  allows computation of statistical significance of predefined gene sets that share common biological function, based on a ranked list of differentially expressed genes observed while comparing two distinct states or phenotypes. This was performed with the GSEA software (v2.2.4)  with all active transcription marks  calculated with the DiffBind R package  within their gene body were identified, and the corresponding coordinates were intersected for the presence of H3K36me3 peak coordinates as identified from the MACS2 peak caller  coding genes. The coordinates of TTSs were extracted and overlapping coordinates merged to avoid redundancy. The percentage length covered by TTSs within and around 500 DE coding genes with maximum fold change and NDE genes with minimum fold change was plotted in Violin plots showing the distribution of the percentage with the probability density of data. The correlation coefficient for changing expression levels across all TPs was calculated for the interacting gene pairs based on the 2.8.2The enrichment of TTSs was analyzed within the published chromatin states . qPCR was performed on a Rotor‐Gene Q  and transcript levels determined by the 2−ΔCt method against housekeeping genes PLEKHA5 (cgriseus1B003354) and CUL7  was reverse‐transcribed with the High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) including an RNase inhibitor. For each sample, a reverse transcription (RT) control was included, which was treated equally, but without the addition of reverse transcriptase. The generated complementary DNA samples and RT controls were 1:4 diluted with nuclease‐free water and analyzed in quadruplet reverse transcription polymerase chain reaction (RT‐PCR) reactions of 10 µl with SensiFAST33.1Batch culture is a perfect example for changing conditions, with significant environmental variation and altered media composition during different growth phases Young, . To addr3.1.1To explore the similarity of the transcriptomes analyzed at different time‐points, a principal component (PC) analysis was performed over the expression levels of all expressed coding and noncoding transcribed regions . A cumulative percentage variance of 94.2% was accounted for by PC1. No separation across PC1 shows the overall similarity across all samples. This is not surprising, as 96% of all expressed genes are constantly expressed. However, PC2, accounting for 1.9% of the variability, separates samples into three different growth phases: exponential, stationary, and decline. We found 14,547 protein‐coding genes to be expressed (rowSums >1), of which 188 genes were DE  between exponential and stationary phase (ES) and 1,381 between exponential and decline (ED) phase (Supporting Information Table 5). In total, 1,397 unique coding genes show DE between growth phases, comparable with a previous study   positive correlation between changes in expression levels and histone modifications for the three active histone marks, especially in early exponential and decline phases. Figure p < 0.01) correlation is observed for H3K36me3‐E4 marks . Read distribution for hyperacetylation of different phase‐wise comparisons  shows that the genome has more hyperacetylated sites in exponential phase , which agrees well with the higher transcriptional changes in this phase  correlation  with expression levels, the trends for promoter marks (H3K4me3 and H3K27ac) behave noisy  in comparison to protein‐coding ones were found to be expressed (rowSums >1). Such a high number suggests an important role in regulation. However, overall expression levels of noncoding RNAs were found to be much lower than that of protein‐coding genes . All expressed lncRNA genes were checked for homology against the 279 lncRNAs reported with known function in lncRNAdb, using default parameters from BLAST. We found 2,565 lncRNAs to have homology with only 56 lncRNAs in lncRNAdb, with alignment length ranging from 28 to 4,049 nucleotides and a minimum percentage identity of 71.35%. Such an overall low count of lncRNAs with known function shows the necessity of a better understanding of the functional relevance of these abundant regulators. Gene expression profiling and in situ hybridization provide various evidence that lncRNA expression is differentially regulated spatially, temporally or in response to stimuli . To analyze the trend, this list of 387 DE lncRNAs was separated according to the lncRNA clusters. As expected, Figure 3.3.2While many studies report co‐expression and localization in promoter regions as the mechanism for cis‐acting lncRNAs, a trans‐mode of action has long been known, but its mechanism is not yet well established. The homologous base pairing has been suggested as the general mechanism for posttranscriptional regulation by trans‐acting lncRNAs to gain locus specificity. Recent studies also report the presence of RNA–DNA triplex formation in the regulatory regions, and propose it as a plausible mechanism for the interaction of lncRNAs with coding genes  and RNAPII were observed by the GRID‐Seq method  and NEAT1 (nuclear enriched abundant transcript 1) were selected to evaluate the gene targets reported in our interaction list reporting triplex forming lncRNA‐coding gene pairs. MALAT1 has been reported to regulate gene sets associated with cellular proliferation, localization, apoptosis, and metabolic processes and thereby plays an important role in tumorigenesis  and NEAT1 (cgriseus1ncB038466) were highly upregulated in the later culture phases as compared with the early exponential phase. The coding gene pairs were extracted for these lncRNAs from our interaction list, and KEGG pathway enrichment was performed individually for coding genes corresponding to MALAT1 and NEAT1 homologues (Supporting Information Table 16). As shown in Supporting Information Figure 12, all the pathways enriched in our gene lists seem to be highly representative of the functional roles associated with MALAT1 and NEAT1. In addition, a recently developed technology—Capture Hybridization Analysis of RNA Targets (CHART)—was utilized by West J. A. et al to identify the genomic binding sites for NEAT1 and MALAT1 lncRNAs in human cell lines. Performing proteomic analysis over CHART‐enriched material (CHART‐MS), the authors reported proteins associated with NEAT1 and MALAT1 in vivo. Of the 885 genes reported by CHART‐MS, 727 genes were found to be expressed in our cell lines, and 65% of those were also identified in our triplex forming gene pairs corresponding to MALAT1 and NEAT1. In addition, a comprehensive review published recently describes MALAT1 as highly associated with human cancers and presents a list of 28 genes that regulate the expression of MALAT1 during transcriptional and posttranscriptional processing . Due to the high resolution of closely spaced analysis TPs, transient changes in expression and regulatory interactions could be captured. We could show a clear association of the major epigenetic marks with the expression levels of both protein‐coding and noncoding genes. While the enrichment or depletion of DNA methylation around the TSS determines whether a gene is actively transcribed or not, functioning as an ON/OFF switch, the effect is more pronounced in the presence of active promoter states . The second major determinant for the level of gene expression was the presence of an active transcription state mark on gene bodies decorated by H3K36me3, where a strong correlation between the trend in levels of active histone marks and expression levels across different TPs of the batch culture was observed Figure . NotablyThe deduced cross‐talk between the key epigenetic regulators with direct impact on the expression of protein‐coding genes provides a wealth of information on the cellular demands for sustenance under changing conditions. This study reveals different mechanisms of response and dynamics that provide cells with the tools to handle and adapt to both short‐term and long‐term changes by different, but interacting mechanisms. These mechanisms include: (a) ON/OFF mechanisms such as CpG island methylation in promoter regions, (b) the staged presence of activating and repressing histone marks that enable either stable expression or moderate upregulation or downregulation, and (c) the overlay of noncoding RNA regulation, that enables rapid and possibly transient DE to higher degrees. Further studies to obtain a more detailed understanding of how these regulatory mechanisms determine process behavior of cells and their ability to adapt to a variety of culture conditions will also increase our ability to control and manipulate gene expression towards more reliable process performance and outcome.As an example, the pathway analysis over time of genes that are DE during stationary and decline phase indicates the struggle of cells to maintain homeostasis. These results might be used to understand the changes in product quality or productivity during late production process stages and indicate alleviatory feeding strategies, to ensure proper processing of the product. For instance, in the context of the cells, it makes sense to mobilize lipids under nutrient limitation and to initiate degradation of dispensable cellular components (as observed in the upregulation of lysosome and lipid metabolism), however, for protein production, lipids are essential as they are required for the generation of organelles, such as the endoplasmic reticulum and the Golgi, which are known to be bottlenecks of secretion. Likewise, the fact that “galactose metabolism” pops up as DE in the decline phase is critical as galactose is an important sugar required for proper glycoprocessing and thus the quality of the product.For cell engineering approaches, the observed rapid response mechanism and control over gene expression levels exerted by lncRNAs also opens up the opportunity for completely new, so far unused tools for manipulating gene expression. Similar to microRNA (miRNA) engineering approaches, where the aim was to target the translation of multiple genes without burdening the overall protein production capacity of a cell, lncRNAs are no burden on the translational machinery and could be used to control gene transcription of target genes rather than their translation, thus intervening at an even earlier stage. While miRNAs can reduce translation of their target, engineering by lncRNAs could also be used to enhance transcription of individual genes, such as the product gene, an approach that has already been shown to work at the level of mRNA translation using lncRNAs . Chromatin states, genome sequence, and DNA methylation data are available at http://cho‐epigenome.boku.ac.at/The improved annotation is available for download at (DNA‐ and BS‐seq raw data: PRJEB9185; ChIP‐seq and RNA‐seq data: PRJEB9291.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Sentinel node (SN) mapping has been used to obtain important information for selective lymphadenectomy in patients with early‐stage melanoma and breast cancer.Regarding patients with upper gastrointestinal cancer, many studies have reported that SN mapping yields a good detection rate and diagnostic accuracy for determining lymph node status in patients with early‐stage esophageal and gastric cancer. Avoidance of unnecessary lymphadenectomy and reducing the extent of organ resection would inevitably result in a better quality of life postoperatively.Esophageal cancer is one of the most malignant of all gastroenterological cancers, and lymph node metastasis is a significant prognostic factor. Because of anatomical reasons, it is associated with a complicated pattern of lymph node metastasis, from the cervical to the abdominal region. Lymph node metastasis occurs in approximately 40% of cases of early‐stage (T1b) esophageal cancer. Therefore, esophagectomy with two‐ or three‐field lymphadenectomy is considered a standard surgical procedure, even for cN0 esophageal cancer.Annals of Gastroenterological Surgery, Takeuchi et al present a nice review of SN navigation surgery in esophageal cancer. As for SN mapping and biopsy procedures in esophageal cancer, a radioisotope‐guided method is recommended for the detection of SN in cases of thoracic esophageal cancer. 99mSn colloid solution, as the radioisotope tracer, is injected into the submucosal layer in the four quadrants surrounding the primary tumor. Preoperative lymphoscintigraphy and intraoperative detection of the SN using a handheld gamma probe are usually carried out. After intraoperative SN mapping and biopsy, all the SN in the resected specimens are confirmed using the handheld gamma probe and examined histopathologically. A dual‐tracer method using a radioactive tracer and blue dye is useful for SN detection in patients with abdominal esophageal cancer or adenocarcinoma of the esophagogastric junction. Dye‐only‐guided SN mapping is not recommended for thoracic esophageal cancer because of a poor detection rate. The radioisotope‐guided method is superior in terms of SN detection rate and accuracy at predicting lymph node metastasis as compared to the conventional dye‐guided method in patients with esophageal cancer.In this issue of the Regarding the influence of tumor depth, although cT1 esophageal cancers are suitable for SN mapping, T3/T4 tumors are not suitable, because the original lymphatic drainage routes may be altered in the latter. It remains controversial as to whether SN mapping is possible for patients who have received neoadjuvant chemoradiotherapy, considering the high false‐negative rate reported. Distribution of SN in cases of esophageal cancer is widespread, from the cervical to the abdominal areas. In cases of upper thoracic esophageal cancer, SN are identified in the lymph nodes along the bilateral recurrent laryngeal nerve chain and lymph nodes in the cervical area. In cases of middle thoracic esophageal cancer, SN are widely distributed from the cervical to the abdominal region. In cases of lower thoracic esophageal cancer, SN are mainly located in the lower and middle mediastinum and in the abdominal area. A systematic review and meta‐analysis showed that SN detection rate and accuracy of prediction of lymph node metastasis based on SN status were 93% and 88%, respectively, and not markedly different between squamous cell carcinoma and adenocarcinoma. It is considered that SN mapping is technically feasible in esophageal cancer.Some possible scenarios for omission of standard lymphadenectomy using SN mapping in patients with esophageal cancer are as follows. In patients with cT1N0M0 middle or lower thoracic esophageal cancer, when SN are detected in the mediastinum or abdominal area only and all the SN are histopathologically negative for cancer metastasis, cervical lymphadenectomy can be omitted. In patients with cT1N0M0 adenocarcinoma of the distal esophagus, when all the SN are identified in the abdominal area only and all the SN are histopathologically negative for cancer metastasis, limited resection of the distal esophagus can be carried out by a laparoscopic transhiatal approach without extensive mediastinal lymphadenectomy. Recently, transmediastinal esophagectomy has been carried out and been shown to be associated with a lower incidence of postoperative pneumonia.Sentinel node mapping may be useful for developing new surgical methods, in terms of carrying out appropriate lymphadenectomy, and in patients with cT1N0M0 esophageal cancer.Conflicts of Interest: Author declares no conflicts of interest for this article.


Weight perception and degree of confidence in achieving healthy lifestyle can be determinants of engagement in obesity interventions. This study explored patients' perceived need for weight loss and the degree of self‐confidence in ability to lose weight and sought to identify factors associated with patients' self‐confidence in ability to lose weight.The authors analysed data from a survey mailed to primary care patients within five sites of the Learning Health Systems Network that explored participants' prior experience with weight management.p value < 0.001). Reported desire to lose weight was also significantly higher among those with the highest BMI than those who were overweight . However, this same group had the lowest belief in ability to lose weight . In a multiple regression analysis, female gender, higher BMI and need to lose >10 lb were each independently associated with less belief in being able to lose weight.Among the 2,263 participants who completed the survey section on ‘Patients’ Experience with Weight Management’, perceived need to lose 51 lb or more was statistically significant among those with class III obesity compared with other body mass index (BMI) groups (Patients had varying perceptions on weight loss; those with category III obesity had the highest desire to lose weight but had the least confidence in ability to lose weight. Higher BMI, female gender and need to lose >10 lb were associated with decreased self‐confidence in ability to lose weight. The worldwide prevalence of obesity nearly tripled between 1975 and 2016; it has been linked to more deaths than has underweight.Weight perceptions and confidence in one's ability to achieve a healthy lifestyle can be determinants of engagement in overweight and obesity interventions. A prior study performed in a large institution showed that while individuals with higher BMI were more likely to report that their physician had told them that they had overweight, such conversations were less likely to change their personal view of their weight or motivate them to lose weight.Studies evaluating perceptions of individuals who have overweight or obesity had consisted of small size and had yielded contrasting observations, from those who had realistic perception of obesity and recognized the need to lose weight, to those whose perception of obesity was below its true prevalence.2The LHSNet is composed of nine sites and includes data on approximately 10 million patients. Details of the LHSNet structure and sites have been described elsewhere.2.1The Weight Management Survey was developed by the LHSNet weight cohort work group on the basis of previously developed questions/scales of patient beliefs, behaviours and experiences.The survey was purposefully developed to be brief and anonymous. Surveys were delivered and returned via US Postal Service. Only one mailing was performed.2.2−2), obesity class I , obesity class II , obesity class III , totalling 20,000 for the network.Each of the five participating LHSNet sites ran a query on its local CDM DataMart to identify patients who fit study criteria. Eligible patients were 18 years or older with available weight and height . The query randomly selected 4,000 patients per site who met the criteria with additional stratification of 1,000 patients per BMI category: overweight  were returned as completed with an additional 1,033 (5.2%) refusal to participate. The Mayo Clinic Survey Research Center received and entered the data from all returned surveys using a dual‐data‐entry system. A detailed flow diagram of study participants has been presented elsewhere.2.3Quantitative data on experience and expectations of patients were analysed using chi‐square tests to compare categorical variables across BMI category groups and analysis of variance to compare continuous variables across groups. A single multiple linear regression model using the respondents' self‐reported belief in their ability to lose weight as dependent variable was used to analyse data for potentially associated characteristics.Qualitative data from patients' responses on weight loss measures that had been used in the past were grouped into major themes.3Among the 2,263 participants who completed the survey section on ‘Patients’ Experience with Weight Management’, 648 (29%) had overweight, 619 (27%) had class I obesity, 474 (21%) had class II obesity and 522 (23%) had class III obesity. Demographic characteristics differed by weight category; persons in the class III obesity category were younger, were more likely to be female, were more likely to be non‐Caucasian, and have some college education or more (Table N = 489). Weight loss programmes, medications and surgery were utilized more by those in the higher BMI categories; 23 of 37 participants who had bariatric surgery and 15 of 31 participants who took weight loss medications had a BMI of 35 and above , exercise, change in eating/physical activity patterns and surgery, with the largest percentage of responders having attempted dietary management . Lower BMI categories were associated with perceived need for lesser weight loss.Table p value < 0.001). However, the same group that had the greatest desire to lose weight had the lowest belief in ability to lose weight .To the survey questions that asked the responders to rate, on a scale of 1–10, their desire to lose weight and belief on their ability to lose weight, those with BMI of 40 and higher reported significantly higher desire to lose weight than those with BMI in overweight category  result in 5–7% weight loss; a slightly higher percentage of weight loss (8–10%) had been achieved in more intensive, comprehensive programmes.Interestingly, the same group of patients who would benefit most from weight loss interventions, i.e. those with higher BMI and those who felt the need to lose >10 lb, had the least confidence in their ability to lose weight. While an independent correlation between greater desire to lose weight and increased belief in ability to lose weight was seen in a multiple regression analysis, the perceived confidence in being able to lose weight decreased with higher BMI. One plausible explanation would be the increased likelihood that this group of patients may have already tried several weight loss measures but were unsuccessful in their attempts at losing weight, which consequently decreased their perceived confidence in their ability to lose weight. This had been observed even among well‐motivated individuals who have had several unsuccessful quit attempts at smoking cessation.This inverse relationship between desire and confidence to lose weight may also partly be due to ambivalence to come forward and seek help due to stigma‐related cognitions commonly experienced by those with higher BMI. In a semi‐structured interview conducted among a small group of individuals with diverse backgrounds and levels of obesity, reluctance in presenting their concerns to their primary care physicians stemmed from self‐consciousness to their features and uncertainty as to how they would be perceived.In his social cognitive theory, Albert Bandura defined self‐efficacy as confidence in one's ability to perform a task that affects whether thought is translated into action.An association between being female and decreased confidence in ability to lose weight was also seen in a multiple regression analysis. Several factors may have contributed to this observation. Women not only tend to experience transitional events in life that trigger weight gain such as pregnancy and menopause but also are more vulnerable than men to weight stigmatization.The major strength of this study is the inclusion of five diverse sites and large sample groups. The surveys were also anonymously completed, which helped reduce potential barriers in patients' participation at reporting personal perceptions and experiences. This anonymity, however, prevented linking the participants' data to health records, which is one limitation of this study. The survey asked the participants if they had previously lost weight and what measures helped them to lose weight. It did not ask how much weight loss was achieved or any behavioural change made as well as the number of attempts at weight loss and measures used for weight loss maintenance, which would have provided additional relevant information regarding patients' experience. The 12% response rate may have resulted in a study sample not being representative of the general population of patients with overweight and obesity receiving health care at study sites; however, it should be noted that response rates of mailed surveys have steadily declined in recent years.5Participants in this study had varying expectations regarding weight loss, both on perceived need and on belief in ability to lose weight. Those with higher BMI had perceived need for greater, potentially unrealistic weight loss but also reported being least confident at their ability to lose weight. In a multiple regression analysis, being female, having higher BMI and needing to lose 11 lb or more were independently associated with lesser belief in being able to lose weight. Results from this study affirmed the importance of incorporating goal‐setting, problem‐solving and self‐efficacy skills through motivational interviewing, coaching or other strategies in weight management programmes.The authors report no competing interests.This research was supported in part by the Patient‐Centered Outcomes Research Institute (PCORI) CDRN‐1501‐26638  and by the Mayo Clinic Robert D. and Patricia E. Kern Center for the Science of Health Care Delivery.


P < .01). The presence of Ioversol in the IVD combined with high‐resolution microCT allow for nondestructive visualization of structural features of the IVD. These results demonstrate CEμCT with Ioversol as a viable strategy for the in vivo monitoring of multiple mouse IVDs during degeneration, disease, and injury.Mouse models are often used for studies of intervertebral disc (IVD) homeostasis and degeneration, yet the relatively small size of the IVD poses challenges for noninvasive, longitudinal imaging modalities. The recently developed contrast‐enhanced microCT (CEμCT) using Ioversol has been successful in detecting degenerative changes in the murine IVD ex vivo at the micrometer scale. Further leveraging the superior biocompatibility of Ioversol as a contrast agent, we demonstrate the in vivo use of this CEμCT technique to examine IVDs at multiple spinal sites. Ioversol was administered via tail vein injection (TVI) in growing and adult male FVB/NJ mice (n = 5 /group). The animals were anesthetized and underwent in vivo micro‐computed tomographic (microCT) at the coccygeal (CC5/CC6), lumbar (L5/6), and thoracic (T12/T13) IVDs. TVI of Ioversol was well‐tolerated by all animals. As Ioversol filtered through the kidneys and accumulated in the bladder, the attenuations of the mouse bladder and kidneys increased due to the high molecular weight of Ioversol, confirming that the Ioversol is biological available. Average IVD attenuations increased 3%‐15% following TVI (ANOVA;  Mice were injected with 8 mL/kg of 350 mg/mL of Ioversol  which reflects veterinary recommendations for a mouse at the highest available concentration of Ioversol. Mice were placed in a restrainer on a heat pad and lidocaine was applied to the tail. A 29G syringe was used to inject Ioversol into the lateral tail vein of the mouse. Mice were then anesthetized by isoflurane prior to microCT imaging.All procedures were performed with the approval of Washington University School of Medicine Institutional Animal Care and Use Committee. Male FVBN/J mice at two age groups, 3‐ (growing) and 8‐ (adult) month old, were used in this study (n = 5 / group). Wild‐type mice were selected for this study to examine the diffusion of Ioversol into the IVD under healthy conditions. Mice were kept in standard husbandry conditions with 12‐hour light/dark cycles as described by Jackson Labs  at 10 μm voxel size . Our preliminary studies showed that at least 20 minutes is required for TVI‐administration of Ioversol to diffuse into the intradiscal space to increase the attenuation of the tissue. We observed that the bioavailability of Ioversol diminish significantly 2‐hours post‐TVI.To determine the optimum time to achieve the maximum attenuation, each site is scanned at several timepoints following the injection. During the 110 minutes scanning period, three intervertebral levels . Following an initial median filter , vertebral bodies were used as landmarks to remove the surrounding soft tissue. Contours were drawn around the outer edge of the vertebral bodies and morphed using linear interpolation. The bone was excluded to generate a mask of the IVD. The threshold was verified by visual inspection to ensure that all voxels containing bone were removed from the mask. The mask undergoes a morphological open followed by a morphological close to smooth edges and fill voids. From the mask of the whole disc, volume and average attenuation were calculated. The volume was determined from the total number of voxels included in the mask. The mean attenuation was taken as the average 16‐bit grayscale value of the voxels contained within the mask. The mean attenuation of scans following TVI of Ioversol was normalized to their respective baseline scans. The ratio of the mean attenuation of the post‐TVI scan (DI) to the pre‐TVI (DI0) was calculated for both groups at all levels. Disc heights and visualizations were obtained using OsiriX . Disc height represents the average disc height across five evenly spaced points along the mid‐sagittal plane of the IVD.The raw data was exported to DICOM for further analysis using a custom MATLAB program .3Tail vein injection (TVI) of Ioversol was well‐tolerated by the mice with no adverse events observed up to 4 weeks post injection. Shortly following TVI, the bladder and kidney became attenuating features in the microCT‐scout view, confirming the metabolism and bioavailability of Ioversol Figure B.P < .01; Figure The post‐TVI scans showed increased attenuations of the IVD regions compared with the pre‐TVI scans. Qualitative inspection also showed that the IVD attenuation increased compared to other soft tissues in the IVD's vicinity. Quantitative comparisons of the voxel densities versus attenuations showed that the entire intradiscal region shifts towards elevated values Figure C, with tIn order to explore the time‐dependence of Ioversol retention in the IVD, we monitored each intervertebral disc at three timepoints as technically allowed by the microCT system. Mean attenuation varied by less than 5% between the three post‐TVI scans (data not shown). Increased attenuation following injection with Ioversol was observed across all levels and ages Figure A, D. At 4To date, this is the first study to describe the in vivo use of CEμCT for the imaging of the mouse intervertebral disc. This approach was previously demonstrated in several ex vivo rodent models of IVD degeneration, and it achieved superior spatial resolution compared with microMRI.The TVIs were well‐tolerated by all mice with no adverse consequences, suggesting that the technique would be suitable for longitudinal monitoring. However, it is worth noting that Ioversol is metabolized through the mouse quickly, and we observe declines in IVD and kidney signal as early as 120 minutes post‐TVI. As the natural course of urinary homeostasis, the bladder attenuation increases over time until the Ioversol is expelled. This accumulation of contrast‐agent in the bladder could occasionally introduce beam‐hardening artifacts if the IVD of interest is localized near the bladder, and we would suggest users of this approach to be cognizant of the anatomical variations and metabolic rate of the mouse model. We observed that time required to achieve maximum attenuation depended on age of the animal and spinal level, and it may be important to optimize injection and imaging parameters for animals of different sex, strain, and backgrounds. Here, we were limited by the scan time and time for detector repositioning for optimizing post‐TVI scan time; future work focused on a single level would allow additional timepoints to be investigated for further refinement of the optimal time between injection and post‐TVI scan. The attenuation signals are influenced by motion and partial volume effects, and this is particularly evident in the thoracic IVD due to the narrow intradiscal space (approximately 100‐200 μm disc height) and proximity to the lungs during breathing. Motion artifacts and partial volume effects may have contributed to the variation in the attenuation response post‐TVI. As a result, thoracic IVDs may not be suited for this technique. Yet the lumbar and coccygeal vertebrae did not exhibit motion artifacts, suggesting that motion was not a factor at these sites. It is worth noting that alterations in endplate porosity and hydration levels may also influence the infiltration of Ioversol and attenuation of the IVD following TVI. Our approach here accounts for these variations by scanning each spinal site at multiple timepoints to determine the time between injection and imaging to maximize the diffusion of Ioversol into the IVD. If only one or two adjacent IVDs were monitored, the signal‐to‐noise can be improved through increasing integration and/or exposure times. Variations due to TVI contrast‐agent delivery may be mitigated with blood vessel cannulation in the future.The current CEμCT technique allows for the high‐resolution imaging of the mouse IVD in vivo at spatial resolutions at tens of microns,All authors state they have no conflicts of interest.Performed experimentsAnalysis and interpretation of the dataDrafted manuscriptApproved final version of manuscriptRemy WalkConceived and designed researchAnalysis and interpretation results of experimentsEdited and revised manuscriptApproved final version of manuscriptSimon Tang


Gastrointestinal (GI) adenocarcinoma, especially colorectal cancer (CRC), is a devastating complication of inflammatory bowel disease (IBD). We sought to examine the role of chronic inflammation and other possible predictors of the development of CRC, as well as assess as yet unexamined factors such as psychological comorbidity and engagement in care.This study included all patients involved in a tertiary hospital IBD service diagnosed with CRC between 2007 and 2017. Reports from histological specimens were assessed, and all those with adenocarcinoma, high‐grade dysplasia (HGD), or multifocal low‐grade dysplasia (LGD) originating within IBD‐affected mucosa were included in the study.A total of 32 patients were included in the study . The majority had a duration of disease >20 years. Eleven patients  had previous disease‐related surgery, and 16  had multiple previous disease‐related admissions. Thirteen patients  had >50% of CRP results higher than 8 mg/L. Psychiatric comorbidities were common, with 19 patients  having a psychiatric comorbidity or poor engagement in treatment.In this cohort, we have highlighted poor engagement, hesitation to up‐titrate therapy when indicated, and psychological comorbidities as likely contributors to poor disease control and development of GI adenocarcinoma. Based on our data, these easily identifiable clinical care factors should not be overlooked when addressing IBD‐related GI malignancy prevention. Additional research is required to assess a direct causal relationship, but this study would support the incorporation of psychology services into IBD clinics. Gastrointestinal adenocarcinoma, especially colorectal cancer (CRC), is a devastating complication of inflammatory bowel disease (IBD). This study highlights poor engagement, hesitation to up‐titrate therapy when indicated, and psychological comorbidities as new risk factors of which clinicians should be aware. Gastrointestinal (GI) adenocarcinoma, particularly colorectal cancer (CRC), is a devastating complication of inflammatory bowel disease (IBD), accounting for between 15 and 20% of IBD‐related mortality.Many larger‐scale studies have assessed risk factors of CRC in IBD, as well as prevention and detection strategies—often at a population level. The aim of our study was to analyze our cohort of patients who developed GI adenocarcinoma in the context of IBD in greater clinical detail. We aimed to assess known risk factors for GI adenocarcinoma in addition to evaluating other unexamined factors, such as psychological comorbidity and engagement in care, given the large psychological burden of IBD.Our study included all patients known to the Royal Adelaide Hospital IBD service between 2007 and 2017. Retrospective analysis of prospectively collected data was undertaken. We reviewed our cohort to identify and examine clinical data from all cases of GI adenocarcinoma and high‐grade dysplasia (HGD) or multifocal low‐grade dysplasia (LGD) originating within IBD‐affected areas.n = 8, CI 26–69%) had UC, and 53%  had CD. There was no significant difference between males and females. Median age at diagnosis was 55 years for adenocarcinoma and 54 years for HGD/multifocal LGD. Ileocolonic disease was the most common phenotype in patients with CD and pancolitis the most common in UC  of those with CD.A total of 17 patients with adenocarcinoma and 15 patients with HGD/multifocal LGD were included  had used long‐term prednisolone (defined as >12 months) at some stage in their disease course.There were multiple markers for long‐term active inflammation in this population Tables  and 4. TP = 0.01). Of those with 10 or more available C‐reactive protein (CRP) measurements, 13 patients  had >50% of these measurements above 8 mg/L , with 10 patients  having 3 or more measurements above 50 mg/L.Of the patients with CD, 11  had received treatment with biologic agents. In the overall IBD cohort, 11  had required previous disease‐related surgery, and 3  had more than two previous operations. Sixteen patients  had multiple previous IBD‐related hospital admissions, with five  having more than 5 and a further two  having more than 10 prior admissions. Patients with CD were more likely to have been hospitalized than those with UC  had exposure to biologics, mostly infliximab; however, three received infliximab for less than 6 months prior to the adenocarcinoma/dysplasia diagnosis  had severe inflammation as defined by the presence of deep ulceration, and only one patient had no visible inflammation endoscopically Table . Only ni–69% had Our data are novel in that they indicate new potential contributors to the development of GI adenocarcinoma and dysplasia in IBD. Psychiatric comorbidity and/or poor engagement in medical care were highly prevalent in patients presenting to a tertiary IBD service late in their disease course, and a large proportion  of the service's total diagnoses of neoplasia over the last decade came from this group. Those at risk presented to the referral service after many years of poorly controlled inflammation without exposure to maximal available therapy. These data suggest that, from a community perspective, efforts should be made to better educate patients and nonspecialist IBD clinicians about the cancer risks of poor disease control. Specific attention to mental health issues and exploration of the reasons for failure of up‐titration of therapy are also warranted. These novel findings warrant investigation in a larger case–control study to evaluate whether they are indeed independent risk factors for poor disease control and the development of IBD‐associated malignancy/dysplasia.It has been well established that chronic inflammation is the key component to the development of CRC in IBD.n = 8, CI 26–69%) only received a biologic for 6 months or less prior to the neoplasia diagnosis. While there are few long‐term data regarding CRC risk reduction with anti‐TNF agents, a 2011 Dutch case–control study of 173 cases of IBD‐associated CRC found a significant reduction in CRC risk with the use of infliximab, with an odds ratio of 0.09.Despite the severity of inflammation, many of these patients did not have more aggressive therapy initiated. In those with UC, only nine  had been up‐titrated to a thiopurine. A 2018 meta‐analysis by Zhu et al. reinforced the antineoplastic activity of thiopurine‐based immunomodulators, with an odds ratio of 0.51 for HGD and 0.55 for CRC.The failure to deliver appropriate  treatment may be partly a result of patient engagement factors. The high rates of anxiety and depression in our cohort likely contributed significantly to the rates of difficult engagement in treatment. Engagement issues included self‐cessation of medications, hesitancy to up‐titrate therapies, and—in one instance—declining colorectal surgical input for severe perianal disease and thus rendering the patient ineligible for anti‐TNF therapy (due to the sepsis risk). Given the chronic nature of IBD and the requirement for long‐term adherence to therapy to maintain disease control, psychological comanagement is imperative to optimize outcomes. Psychologist involvement in specialist IBD clinics should be considered as it has been shown to improve medication adherence and reduce hospital admissions.Those at greatest risk of GI adenocarcinoma in IBD should be recognized in clinical care. They have extensive disease, poor disease control, prolonged steroid use, recurrent admissions, and a history of IBD surgery. In this cohort, we have, for the first time, highlighted poor engagement in care, hesitation to up‐titrate therapy when indicated, and psychological comorbidities as potential additional contributors of which clinicians should be aware.Based on our data, the primary focus of IBD‐related GI malignancy prevention should be on these identifiable clinical care factors to reduce inflammation in the first instance and subsequently reduce the eventual development of dysplasia requiring detection. One such method for addressing these factors would be the incorporation of psychology services into IBD centers, with the aim of improving patient engagement and addressing psychiatric comorbidities.