Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Somatic mutation rates scale with lifespan across mammals.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1-7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined-including variation of around 30-fold in lifespan and around 40,000-fold in body mass-the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.

Phylogeny and diversity of alkane-degrading enzyme gene variants in the laurentian great lakes and western atlantic.

Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Two cryptic species of California mustard within Caulanthus lasiophyllus.

PREMISE: Cryptic species are evolutionarily distinct lineages lacking distinguishing morphological traits. Hidden diversity may be lurking in widespread species whose distributions cross phylogeographic barriers. This study investigates molecular and morphological variation in the widely distributed Caulanthus lasiophyllus (Brassicaceae) in comparison to its closest relatives. METHODS: Fifty-two individuals of C. lasiophyllus from across the species' range were sequenced for the nuclear ribosomal internal transcribed spacer region (ITS) and the chloroplast trnL-F region. A subset of these samples were examined for the chloroplast ndhF gene. All 52 individuals were scored for 13 morphological traits, as well as monthly and annual climate conditions at the collection locality. Morphological and molecular results are compared with the closest relatives-C. anceps and C. flavescens-in the "Guillenia Clade." To test for polyploidy, genome size estimates were made for four populations. RESULTS: Caulanthus lasiophyllus consists of two distinct lineages separated by eight ITS differences-eight times more variation than what distinguishes C. anceps and C. flavescens. Fewer variable sites were detected in trnL-F and ndhF regions, yet these data are consistent with the ITS results. The two lineages of C. lasiophyllus are geographically and climatically distinct; yet morphologically overlapping. Their genome sizes are not consistently different. CONCLUSIONS: Two cryptic species within C. lasiophyllus are distinguished at the molecular, geographic, and climatic scales. They have similar genome sizes and are morphologically broadly overlapping, but an ephemeral basal leaf character may help distinguish the species.

Transient commensal clonal interactions can drive tumor metastasis.

The extent and importance of functional heterogeneity and crosstalk between tumor cells is poorly understood. Here, we describe the generation of clonal populations from a patient-derived ovarian clear cell carcinoma model which forms malignant ascites and solid peritoneal tumors upon intraperitoneal transplantation in mice. The clonal populations are engineered with secreted Gaussia luciferase to monitor tumor growth dynamics and tagged with a unique DNA barcode to track their fate in multiclonal mixtures during tumor progression. Only one clone, CL31, grows robustly, generating exclusively malignant ascites. However, multiclonal mixtures form large solid peritoneal metastases, populated almost entirely by CL31, suggesting that transient cooperative interclonal interactions are sufficient to promote metastasis of CL31. CL31 uniquely harbors ERBB2 amplification, and its acquired metastatic activity in clonal mixtures is dependent on transient exposure to amphiregulin, which is exclusively secreted by non-tumorigenic clones. Amphiregulin enhances CL31 mesothelial clearance, a prerequisite for metastasis. These findings demonstrate that transient, ostensibly innocuous tumor subpopulations can promote metastases via "hit-and-run" commensal interactions.

Extensive heterogeneity in somatic mutation and selection in the human bladder.

The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted (n = 1914 microbiopsies), whole-exome (n = 655), and whole-genome (n = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.

Direct-from-sputum rapid phenotypic drug susceptibility test for mycobacteria.

BACKGROUND: The spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof. METHODS AND FINDINGS: We present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10µL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in < 3 days for all samples, allowing us to perform DST in < 3 days. CONCLUSION: We demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS.

Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

Biogeographic Patterns in Members of Globally Distributed and Dominant Taxa Found in Port Microbial Communities.

We conducted a global characterization of the microbial communities of shipping ports to serve as a novel system to investigate microbial biogeography. The community structures of port microbes from marine and freshwater habitats house relatively similar phyla, despite spanning large spatial scales. As part of this project, we collected 1,218 surface water samples from 604 locations across eight countries and three continents to catalogue a total of 20 shipping ports distributed across the East and West Coast of the United States, Europe, and Asia to represent the largest study of port-associated microbial communities to date. Here, we demonstrated the utility of machine learning to leverage this robust system to characterize microbial biogeography by identifying trends in biodiversity across broad spatial scales. We found that for geographic locations sharing similar environmental conditions, subpopulations from the dominant phyla of these habitats (Actinobacteria, Bacteroidetes, Cyanobacteria, and Proteobacteria) can be used to differentiate 20 geographic locations distributed globally. These results suggest that despite the overwhelming diversity within microbial communities, members of the most abundant and ubiquitous microbial groups in the system can be used to differentiate a geospatial location across global spatial scales. Our study provides insight into how microbes are dispersed spatially and robust methods whereby we can interrogate microbial biogeography.IMPORTANCE Microbes are ubiquitous throughout the world and are highly diverse. Characterizing the extent of variation in the microbial diversity across large geographic spatial scales is a challenge yet can reveal a lot about what biogeography can tell us about microbial populations and their behavior. Machine learning approaches have been used mostly to examine the human microbiome and, to some extent, microbial communities from the environment. Here, we display how supervised machine learning approaches can be useful to understand microbial biodiversity and biogeography using microbes from globally distributed shipping ports. Our findings indicate that the members of globally dominant phyla are important for differentiating locations, which reduces the reliance on rare taxa to probe geography. Further, this study displays how global biogeographic patterning of aquatic microbial communities (and other systems) can be assessed through populations of the highly abundant and ubiquitous taxa that dominant the system.

Global Comparison of the Bacterial Communities of Bilge Water, Boat Surfaces, and External Port Water.

In the past, ballast water has been a key vector in the ship-mediated dispersal of invasive species. Here, we evaluate the potential for port microorganisms to enter and colonize the hull and bilge water of ships. Due to the small size and ubiquitous nature of bacteria, they also have the potential to be spread through hull fouling and bilge water discharge. The goal of this study was to identify the extent to which the boat microbial community is shaped by the microbial community in the port water where the boat spends most of its time. Here, we compared the microbial communities of the hull and bilge compartments of 20 boats to those of the port water in 20 different ports in five regions around the world. We found that there was a significant difference in microbial diversity between boat and port microbial communities. Despite these differences, we found that Cyanobacteria were present at high abundances in the bilge water of most vessels. Due to the limited light in the bilge, the presence of Cyanobacteria suggests that port microorganisms can enter the bilge. Using source-tracking software, we found that, on average, 40% of the bilge and 52% of the hull microbial communities were derived from water. These findings suggest that the bilge of a vessel contains a diverse microbial community that is influenced by the port microbial community and has the potential to serve as an underappreciated vector for dispersal of life.IMPORTANCE Invasive species have been a worldwide problem for many years. However, the potential for microorganisms to become invasive is relatively underexplored. As the tools to study bacterial communities become more affordable, we are able to perform large-scale studies and examine bacterial communities in higher resolution than was previously practical. This study looked at the potential for bacteria to colonize both boat surfaces and bilge water. We describe the bacterial communities on boats in 20 shipping ports in five regions around the world, describing how these microorganisms were similar to microorganisms found in port water. This suggests that the water influences the bacterial community of a boat and that microorganisms living on a boat could be moved from place to place when the boat travels.

Microbial Community Dynamics During Lake Ice Freezing.

Many freshwater environments experience dramatic seasonal changes with some systems remaining ice-covered for most of the winter. Freshwater systems are also highly sensitive to environmental change. However, little is known about changes in microbial abundance and community composition during lake ice formation and times of persistent ice cover. The goal of this study is to characterize temporal dynamics of microbial communities during ice formation and persistent ice cover. Samples were collected in triplicate, five days per week from surface water in the Keweenaw Waterway between November and April. Environmental conditions along with microbial abundance and microbial community composition was determined. Distinct community composition was found between ice-free and ice-covered time periods with significantly different community composition between months. The microbial community underwent dramatic shifts in microbial abundance and diversity during the transitions into and out of ice cover. The richness of the microbial community increased during times of ice cover. Relatives of microbes involved in nitrogen cycling bloomed during times of ice cover as sequences related to known nitrifying taxa were significantly enriched during ice cover. These results help to elucidate how microbial abundance and diversity change over drastic seasonal transitions and how ice cover may affect microbial abundance and diversity.

Circulating tumor DNA dynamics using patient-customized assays are associated with outcome in neoadjuvantly treated breast cancer.

Pathological complete response (pCR) is an accurate predictor of good outcome following neoadjuvant chemotherapy (NAC) for locally advanced breast cancer. The presence of circulating-tumor DNA (ctDNA) has recently been reported to be strongly predictive of poor outcome in similar patient groups. We monitored ctDNA levels from 10 women undergoing NAC for locally advanced breast cancer using a patient-specific, hybrid-capture sequencing technique sensitive to the level of one altered allele in 10,000. Plasma was collected prior to the start of NAC, prior to each infusion of NAC, and during follow-up for between 350 and 1150 d after the start of NAC. Prior to the start of NAC, ctDNA was detectable in 3/3 triple negative, 3/3 HER2+, and 2/4 HER2-, ER+ breast cancer patients. Total cell-free DNA levels were considerably higher when patients were on NAC than at other times. ctDNA dynamics during NAC showed that patients with pCR experienced rapid declines in ctDNA levels, whereas patients without pCR typically showed evidence of residual ctDNA after initiation of treatment. Intriguingly, two of three patients that showed marked increases in ctDNA while on NAC experienced rapid recurrences (<2 yr following start of NAC). The third patient that had increases in ctDNA levels while on NAC had low-grade ER+ disease and showed residual ctDNA after surgery, which became undetectable after local radiation. Taken together, these results demonstrate the ability of our approach to sensitively serially monitor ctDNA during NAC, and identifies a need to further investigate the possibility of stratifying patients who need additional treatment or identify therapies that are ineffective.

Optical bandgap modelling from the structural arrangement of carbon nanotubes.

The optical bandgap properties of vertically-aligned carbon nanotube (VACNT) arrays were probed through their interaction with white light, with the light reflected from the rotating arrays measured with a spectrometer. The precise deterministic control over the structure of vertically-aligned carbon nanotube arrays through electron beam lithography and well-controlled growth conditions brings with it the ability to produce exotic photonic crystals over a relatively large area. The characterisation of the behaviour of these materials in the presence of light is a necessary first step toward application. Relatively large area array structures of high-quality VACNTs were fabricated in square, hexagonal, circular and pseudorandom patterned arrays with length scales on the order of those of visible light for the purpose of investigating how they may be used to manipulate an impinging light beam. In order to investigate the optical properties of these arrays a set of measurement apparatus was designed which allowed the accurate measurement of their optical bandgap characteristics. The patterned samples were rotated under the illuminating white light beam, revealing interesting optical bandgap results caused by the changing patterns and relative positions of the scattering elements (VACNTs).

Cross-ethnic meta-analysis identifies association of the GPX3-TNIP1 locus with amyotrophic lateral sclerosis.

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10-8), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10-3). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS.Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease. Here, Wray and colleagues identify association of the GPX3-TNIP1 locus with ALS using cross-ethnic meta-analyses.

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS).

BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.