Tobacco smoking and somatic mutations in human bronchial epithelium

Tobacco smoking causes lung cancer, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA. The profound effects of tobacco on the genome of lung cancer cells are well-documented, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4–14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0–6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts

Single cell derived mRNA signals across human kidney tumors.

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre

Abstract: Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development

The potential for immunoglobulins and host defense peptides (HDPs) to reduce the use of antibiotics in animal production

Abstract Innate defense mechanisms are aimed at quickly containing and removing infectious microorganisms and involve local stromal and immune cell activation, neutrophil recruitment and activation and the induction of host defense peptides (defensins and cathelicidins), acute phase proteins and complement activation. As an alternative to antibiotics, innate immune mechanisms are highly relevant as they offer rapid general ways to, at least partially, protect against infections and enable the build-up of a sufficient adaptive immune response. This review describes two classes of promising alternatives to antibiotics based on components of the innate host defense. First we describe immunoglobulins applied to mimic the way in which they work in the newborn as locally acting broadly active defense molecules enforcing innate immunity barriers. Secondly, the potential of host defense peptides with different modes of action, used directly, induced in situ or used as vaccine adjuvants is described

Three-dimensional Surface Imaging for Clinical Decision Making in Pectus Excavatum

To evaluate pectus excavatum, 3-dimensional surface imaging is a promising radiation-free alternative to computed tomography and plain radiographs. Given that 3-dimensional images concern the external surface, the conventional Haller index, and correction index are not applicable as these are based on internal diameters. Therefore, external equivalents have been introduced for 3-dimensional images. However, cut-off values to help determine surgical candidacy using external indices are lacking. A prospective cohort study was conducted. Consecutive patients referred for suspected pectus excavatum received a computed tomography (≥18 years) or plain radiographs (<18 years). The external Haller index and external correction index were calculated from additionally acquired 3-dimensional images. Cut-off values for the 3-dimensional image derived indices were obtained by receiver-operating characteristic curve analyses, using a conventional Haller index ≥3.25, and computed tomography derived correction index ≥28.0% as indicative for surgery. Sixty-one and 63 patients were included in the computed tomography and radiograph group, respectively. To determine potential surgical candidacy, receiver-operating characteristic analyses found an optimum cut-off of ≥1.83 for the external Haller index in both the computed tomography and radiograph group with a positive predictive value between 0.90 and 0.97 and a negative predictive value between 0.72 and 0.81. The optimal cut-off for the external correction index was ≥15.2% with a positive predictive value of 0.86 and negative predictive value of 0.93. The 3-dimensional image derived external Haller index and external correction index are accurate radiation-free alternatives to facilitate surgical decision-making among patients suspected of pectus excavatum with values of ≥1.83 and ≥15.2% indicative for surgery

Predicting Aesthetic Outcome of the Nuss Procedure in Patients with Pectus Excavatum

Patients suffering from pectus excavatum often experience psychosocial distress due to perceived anomalies in their physical appearance. The ability to visually inform patients about their expected aesthetic outcome after surgical correction is still lacking. This study aims to develop an automatic, patient-specific model to predict aesthetic outcome after the Nuss procedure. Patients prospectively received preoperative and postoperative 3-dimensional optical surface scanning of their chest during the Nuss procedure. A prediction model was composed based on nonlinear least squares data-fitting, regression methods and a 2-dimensional Gaussian function with adjustable amplitude, variance, rotation, skewness, and kurtosis components. Morphological features of pectus excavatum were extracted from preoperative images using a previously developed surface analysis tool to generate a patient-specific model. Prediction accuracy was evaluated through cross-validation, utilizing the mean root squared deviation and maximum positive and negative deviations as performance measures. The prediction model was evaluated on 30 (90% male) prospectively imaged patients. The model achieved an average root mean squared deviation of 6.3 ± 2.0 mm, with average maximum positive and negative deviations of 12.7 ± 6.1 and -10.2 ± 5.7 mm, respectively, between the predicted and actual postoperative aesthetic result. Our developed 2-dimensional Gaussian model based on 3-dimensional optical surface images is a clinically promising tool to predict postsurgical aesthetic outcome in patients with pectus excavatum. Prediction of the aesthetic outcome after the Nuss procedure potentially improves information provision and expectation management among patients. Further research should assess whether increasing the sample size may reduce deviations and improve performance