Instituto de Biomedicina y Biotecnología de Cantabria

Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.

Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.

Abstract Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations. Here, we analyze genomic composition and pairwise sequence identity for over 10,000 reference plasmids to obtain a global map of the prokaryotic plasmidome. Plasmids in this map organize into discrete clusters, which we call plasmid taxonomic units (PTUs), with high average nucleotide identity between its members. We identify 83 PTUs in the order Enterobacterales , 28 of them corresponding to previously described archetypes. Furthermore, we develop an automated algorithm for PTU identification, and validate its performance using stochastic blockmodeling. The algorithm reveals a total of 276 PTUs in the bacterial domain. Each PTU exhibits a characteristic host distribution, organized into a six-grade scale (I–VI), ranging from plasmids restricted to a single host species (grade I) to plasmids able to colonize species from different phyla (grade VI). More than 60% of the plasmids in the global map are in groups with host ranges beyond the species barrier.

BACKGROUND: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer.

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.

Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.

Hematopoiesis is a process capable of generating millions of cells every second, as distributed in many cell types. The process is regulated by a number of transcription factors that regulate the differentiation along the distinct lineages and dictate the genetic program that defines each mature phenotype. Myc was first discovered as the oncogene of avian leukemogenic retroviruses; it was later found translocated in human lymphoma. From then on, evidence accumulated showing that c-Myc is one of the transcription factors playing a major role in hematopoiesis. The study of genetically modified mice with overexpression or deletion of Myc has shown that c-Myc is required for the correct balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Enforced Myc expression in mice leads to reduced HSC pools owing to loss of self-renewal activity at the expense of increased proliferation of progenitor cells and differentiation. c-Myc deficiency consistently results in the accumulation of HSCs. Other models with conditional Myc deletion have demonstrated that different lineages of hematopoietic cells differ in their requirement for c-Myc to regulate their proliferation and differentiation. When transgenic mice overexpress c-Myc or N-Myc in mature cells from the lymphoid or myeloid lineages, the result is lymphoma or leukemia. In agreement, enforced expression of c-Myc blocks the differentiation in several leukemia-derived cell lines capable of differentiating in culture. Not surprising, MYC deregulation is recurrently found in many types of human lymphoma and leukemia. Whereas MYC is deregulated by translocation in Burkitt lymphoma and, less frequently, other types of lymphoma, MYC is frequently overexpressed in acute lymphoblastic and myeloid leukemia, through mechanisms unrelated to chromosomal translocation, and is often associated with disease progression.

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.

Promotion of the cell cycle is a major oncogenic mechanism of the oncogene c-MYC (MYC). MYC promotes the cell cycle by not only activating or inducing cyclins and CDKs but also through the downregulation or the impairment of the activity of a set of proteins that act as cell-cycle brakes. This review is focused on the role of MYC as a cell-cycle brake releaser i.e., how MYC stimulates the cell cycle mainly through the functional inactivation of cell cycle inhibitors. MYC antagonizes the activities and/or the expression levels of p15, ARF, p21, and p27. The mechanism involved differs for each protein. p15 (encoded by CDKN2B) and p21 (CDKN1A) are repressed by MYC at the transcriptional level. In contrast, MYC activates ARF, which contributes to the apoptosis induced by high MYC levels. At least in some cells types, MYC inhibits the transcription of the p27 gene (CDKN1B) but also enhances p27's degradation through the upregulation of components of ubiquitin ligases complexes. The effect of MYC on cell-cycle brakes also opens the possibility of antitumoral therapies based on synthetic lethal interactions involving MYC and CDKs, for which a series of inhibitors are being developed and tested in clinical trials.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

Conjugation of DNA through a type IV secretion system (T4SS) drives horizontal gene transfer. Yet little is known on the diversity of these nanomachines. We previously found that T4SS can be divided in eight classes based on the phylogeny of the only ubiquitous protein of T4SS (VirB4). Here, we use an ab initio approach to identify protein families systematically and specifically associated with VirB4 in each class. We built profiles for these proteins and used them to scan 2262 genomes for the presence of T4SS. Our analysis led to the identification of thousands of occurrences of 116 protein families for a total of 1623 T4SS. Importantly, we could identify almost always in our profiles the essential genes of well-studied T4SS. This allowed us to build a database with the largest number of T4SS described to date. Using profile-profile alignments, we reveal many new cases of homology between components of distant classes of T4SS. We mapped these similarities on the T4SS phylogenetic tree and thus obtained the patterns of acquisition and loss of these protein families in the history of T4SS. The identification of the key VirB4-associated proteins paves the way toward experimental analysis of poorly characterized T4SS classes.

Genetic exchange by conjugation is responsible for the spread of resistance, virulence, and social traits among prokaryotes. Recent works unraveled the functioning of the underlying type IV secretion systems (T4SS) and its distribution and recruitment for other biological processes (exaptation), notably pathogenesis. We analyzed the phylogeny of key conjugation proteins to infer the evolutionary history of conjugation and T4SS. We show that single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) conjugation, while both based on a key AAA(+) ATPase, diverged before the last common ancestor of bacteria. The two key ATPases of ssDNA conjugation are monophyletic, having diverged at an early stage from dsDNA translocases. Our data suggest that ssDNA conjugation arose first in diderm bacteria, possibly Proteobacteria, and then spread to other bacterial phyla, including bacterial monoderms and Archaea. Identifiable T4SS fall within the eight monophyletic groups, determined by both taxonomy and structure of the cell envelope. Transfer to monoderms might have occurred only once, but followed diverse adaptive paths. Remarkably, some Firmicutes developed a new conjugation system based on an atypical relaxase and an ATPase derived from a dsDNA translocase. The observed evolutionary rates and patterns of presence/absence of specific T4SS proteins show that conjugation systems are often and independently exapted for other functions. This work brings a natural basis for the classification of all kinds of conjugative systems, thus tackling a problem that is growing as fast as genomic databases. Our analysis provides the first global picture of the evolution of conjugation and shows how a self-transferrable complex multiprotein system has adapted to different taxa and often been recruited by the host. As conjugation systems became specific to certain clades and cell envelopes, they may have biased the rate and direction of gene transfer by conjugation within prokaryotes.

We show that BRAF(V600E) initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf(V600E) expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.

N-methyl-D-aspartate (NMDA) receptor antagonists such as phencyclidine (PCP), dizocilpine (MK-801) and ketamine have long been considered a model of schizophrenia, both in animals and humans. However, ketamine has been recently approved for treatment-resistant depression, although with severe restrictions. Interestingly, the dosage in both conditions is similar, and positive symptoms of schizophrenia appear before antidepressant effects emerge. Here, we describe the temporal mechanisms implicated in schizophrenia-like and antidepressant-like effects of NMDA blockade in rats, and postulate that such effects may indicate that NMDA receptor antagonists induce similar mechanistic effects, and only the basal pre-drug state of the organism delimitates the overall outcome. Hence, blockade of NMDA receptors in depressive-like status can lead to amelioration or remission of symptoms, whereas healthy individuals develop psychotic symptoms and schizophrenia patients show an exacerbation of these symptoms after the administration of NMDA receptor antagonists.

Plasmids contain a backbone of core genes that remains relatively stable for long evolutionary periods, making sense to speak about plasmid species. The identification and characterization of the core genes of a plasmid species has a special relevance in the study of its epidemiology and modes of transmission. Besides, this knowledge will help to unveil the main routes that genes, for example antibiotic resistance (AbR) genes, use to travel from environmental reservoirs to human pathogens. Global dissemination of multiple antibiotic resistances and virulence traits by plasmids is an increasing threat for the treatment of many bacterial infectious diseases. To follow the dissemination of virulence and AbR genes, we need to identify the causative plasmids and follow their path from reservoirs to pathogens. In this review, we discuss how the existing diversity in plasmid genetic structures gives rise to a large diversity in propagation strategies. We would like to propose that, using an identification methodology based on plasmid mobility types, we can follow the propagation routes of most plasmids in Gammaproteobacteria, as well as their cargo genes, in complex ecosystems. Once the dissemination routes are known, designing antidissemination drugs and testing their efficacy will become feasible. We discuss in this review how the existing diversity in plasmid genetic structures gives rise to a large diversity in propagation strategies. We would like to propose that, by using an identification methodology based on plasmid mobility types, we can follow the propagation routes of most plasmids in ?-proteobacteria, as well as their cargo genes, in complex ecosystems.