p53 mutation in normal esophagus promotes multiple stages of carcinogenesis but is constrained by clonal competition.

Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.

Simulations reveal that different responses to cell crowding determine the expansion of p53 and Notch mutant clones in squamous epithelia.

During ageing, normal epithelial tissues progressively accumulate clones carrying mutations that increase mutant cell fitness above that of wild-type cells. Such mutants spread widely through the tissues, yet despite this cellular homeostasis and functional integrity of the epithelia are maintained. Two of the genes most commonly mutated in human skin and oesophagus are p53 and Notch1, both of which are also recurrently mutated in cancers of these tissues. From observations taken in human and mouse epithelia, we find that clones carrying p53 and Notch pathway mutations have different clone dynamics which can be explained by their different responses to local cell crowding. p53 mutant clone growth in mouse epidermis approximates a logistic curve, but feedbacks responding to local crowding are required to maintain tissue homeostasis. We go on to show that the observed ability of Notch pathway mutant cells to displace the wild-type population in the mouse oesophageal epithelium reflects a local density feedback that affects both mutant and wild-type cells equally. We then show how these distinct feedbacks are consistent with the distribution of mutations observed in human datasets and are suggestive of a putative mechanism to constrain these cancer-associated mutants.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Methods for analysing lineage tracing datasets.

A single population of progenitor cells maintains many epithelial tissues. Transgenic mouse cell tracking has frequently been used to study the growth dynamics of competing clones in these tissues. A mathematical model (the 'single-progenitor model') has been argued to reproduce the observed progenitor dynamics accurately. This requires three parameters to describe the growth dynamics observed in transgenic mouse cell tracking-a division rate, a stratification rate and the probability of dividing symmetrically. Deriving these parameters is a time intensive and complex process. We compare the alternative strategies for analysing this source of experimental data, identifying an approximate Bayesian computation-based approach as the best in terms of efficiency and appropriate error estimation. We support our findings by explicitly modelling biological variation and consider the impact of different sampling regimes. All tested solutions are made available to allow new datasets to be analysed following our workflows. Based on our findings, we make recommendations for future experimental design.

A single-progenitor model as the unifying paradigm of epidermal and esophageal epithelial maintenance in mice.

In adult skin epidermis and the epithelium lining the esophagus cells are constantly shed from the tissue surface and replaced by cell division. Tracking genetically labelled cells in transgenic mice has given insight into cell behavior, but conflicting models appear consistent with the results. Here, we use an additional transgenic assay to follow cell division in mouse esophagus and the epidermis at multiple body sites. We find that proliferating cells divide at a similar rate, and place bounds on the distribution cell cycle times. By including these results in a common analytic approach, we show that data from eight lineage tracing experiments is consistent with tissue maintenance by a single population of proliferating cells. The outcome of a given cell division is unpredictable but, on average, the likelihood of producing proliferating and differentiating cells is equal, ensuring cellular homeostasis. These findings are key to understanding squamous epithelial homeostasis and carcinogenesis.

Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations.

Aging human tissues, such as sun-exposed epidermis, accumulate a high burden of progenitor cells that carry oncogenic mutations. However, most progenitors carrying such mutations colonize and persist in normal tissue without forming tumors. Here, we investigated tissue-level constraints on clonal progenitor behavior by inducing a single-allele p53 mutation (Trp53R245W; p53∗/wt), prevalent in normal human epidermis and squamous cell carcinoma, in transgenic mouse epidermis. p53∗/wt progenitors initially outcompeted wild-type cells due to enhanced proliferation, but subsequently reverted toward normal dynamics and homeostasis. Physiological doses of UV light accelerated short-term expansion of p53∗/wt clones, but their frequency decreased with protracted irradiation, possibly due to displacement by UV-induced mutant clones with higher competitive fitness. These results suggest multiple mechanisms restrain the proliferation of p53∗/wt progenitors, thereby maintaining epidermal integrity.

Transmission of Staphylococcus aureus between health-care workers, the environment, and patients in an intensive care unit: a longitudinal cohort study based on whole-genome sequencing.

BACKGROUND: Health-care workers have been implicated in nosocomial outbreaks of Staphylococcus aureus, but the dearth of evidence from non-outbreak situations means that routine health-care worker screening and S aureus eradication are controversial. We aimed to determine how often S aureus is transmitted from health-care workers or the environment to patients in an intensive care unit (ICU) and a high-dependency unit (HDU) where standard infection control measures were in place. METHODS: In this longitudinal cohort study, we systematically sampled health-care workers, the environment, and patients over 14 months at the ICU and HDU of the Royal Sussex County Hospital, Brighton, England. Nasal swabs were taken from health-care workers every 4 weeks, bed spaces were sampled monthly, and screening swabs were obtained from patients at admission to the ICU or HDU, weekly thereafter, and at discharge. Isolates were cultured and their whole genome sequenced, and we used the threshold of 40 single-nucleotide variants (SNVs) or fewer to define subtypes and infer recent transmission. FINDINGS: Between Oct 31, 2011, and Dec 23, 2012, we sampled 198 health-care workers, 40 environmental locations, and 1854 patients; 1819 isolates were sequenced. Median nasal carriage rate of S aureus in health-care workers at 4-weekly timepoints was 36·9% (IQR 35·7-37·3), and 115 (58%) health-care workers had S aureus detected at least once during the study. S aureus was identified in 8-50% of environmental samples. 605 genetically distinct subtypes were identified (median SNV difference 273, IQR 162-399) at a rate of 38 (IQR 34-42) per 4-weekly cycle. Only 25 instances of transmission to patients (seven from health-care workers, two from the environment, and 16 from other patients) were detected. INTERPRETATION: In the presence of standard infection control measures, health-care workers were infrequently sources of transmission to patients. S aureus epidemiology in the ICU and HDU is characterised by continuous ingress of distinct subtypes rather than transmission of genetically related strains. FUNDING: UK Medical Research Council, Wellcome Trust, Biotechnology and Biological Sciences Research Council, UK National Institute for Health Research, and Public Health England.

GprotPRED: Annotation of Gα, Gß and Gγ subunits of G-proteins using profile Hidden Markov Models (pHMMs) and application to proteomes.

Heterotrimeric G-proteins form a major protein family, which participates in signal transduction. They are composed of three subunits, Gα, Gß and Gγ. The Gα subunit is further divided in four distinct families Gs, Gi/o, Gq/11 and G12/13. The goal of this work was to detect and classify members of the four distinct families, plus the Gß and the Gγ subunits of G-proteins from sequence alone. To achieve this purpose, six specific profile Hidden Markov Models (pHMMs) were built and checked for their credibility. These models were then applied to ten (10) proteomes and were able to identify all known G-protein and classify them into the distinct families. In a separate case study, the models were applied to twenty seven (27) arthropod proteomes and were able to give more credible classification in proteins with uncertain annotation and in some cases to detect novel proteins. An online tool, GprotPRED, was developed that uses these six pHMMs. The sensitivity and specificity for all pHMMs were equal to 100% with the exception of the Gß case, where sensitivity equals to 100%, while specificity is 99.993%. In contrast to Pfam's pHMM which detects Gα subunits in general, our method not only detects Gα subunits but also classifies them into the appropriate Gα-protein family and thus could become a useful tool for the annotation of G-proteins in newly discovered proteomes. GprotPRED online tool is publicly available for non-commercial use at http://bioinformatics.biol.uoa.gr/GprotPRED and, also, a standalone version of the tool at https://github.com/vkostiou/GprotPRED.