Lamin B1 Depletion in Senescent Cells Triggers Large-Scale Changes in Gene Expression and the Chromatin Landscape

Senescence is a stable proliferation arrest, associated with an altered secretory pathway, thought to promote tumor suppression and tissue aging. While chromatin regulation and lamin B1 down-regulation have been implicated as senescence effectors, functional interactions between them are poorly understood. We compared genome-wide Lys4 trimethylation on histone H3 (H3K4me3) and H3K27me3 distributions between proliferating and senescent human cells and found dramatic differences in senescence, including large-scale domains of H3K4me3- and H3K27me3-enriched “mesas” and H3K27me3-depleted “canyons.” Mesas form at lamin B1-associated domains (LADs) in replicative senescence and oncogene-induced senescence and overlap DNA hypomethylation regions in cancer, suggesting that pre-malignant senescent chromatin changes foreshadow epigenetic cancer changes. Hutchinson-Gilford progeria syndrome fibroblasts (mutant lamin A) also show evidence of H3K4me3 mesas, suggesting a link between premature chromatin changes and accelerated cell senescence. Canyons mostly form between LADs and are enriched in genes and enhancers. H3K27me3 loss is correlated with up-regulation of key senescence genes, indicating a link between global chromatin changes and local gene expression regulation. Lamin B1 reduction in proliferating cells triggers senescence and formation of mesas and canyons. Our data illustrate profound chromatin reorganization during senescence and suggest that lamin B1 down-regulation in senescence is a key trigger of global and local chromatin changes that impact gene expression, aging, and cancer

Human mutation/substitution rate: Variability, modeling and applications

Mutation generates genetic variation, and in turn selection purges deleterious variants from the population. Understanding both is critical for discovering causal genes and variants behind diseases or making inferences about evolutionary processes. Human mutation rate varies significantly across the genome although most studies have only considered the immediate flanking nucleotides around the polymorphic site to model and study patterns of variability. The impact of larger sequence context has not been fully clarified, even though it substantially influences rates of mutation. In the first part of this thesis, I develop a novel statistical framework and using data from the 1000 Genomes project, demonstrate that a larger heptanucleotide sequence context explains \u3e81% variability in substitution probabilities, discovering novel mutation promoting motifs at ApT dinucleotides, CAAT, and TACG sequences. My approach also reveals previously undocumented variability in C-to-T substitutions at CpG sites, not immediately explained by differential methylation intensity. Building on this framework, I model the selective forces acting on the coding genome and develop statistical scores that measures the intolerance at the gene or amino-acid level for functional variants. I demonstrate clinical utility of such intolerance scores in identifying genes associated with multiple human diseases including Autism. Next, I apply these lessons of mutation rate variability to develop an algorithm to detect sub-genic enrichment of de novo germline mutations in RB1 gene of bilateral Retinoblastoma (RB) probands to further elucidate disease biology. I demonstrate that previously noted ‘hotspots’ of nonsense mutations in RB1 are compatible with the elevated mutation rates expected at CpG sites, refuting a specific mechanism in RB pathogenesis. I also find enrichment of splice-site donor mutations of exon 6 and 12 but depletion at exon 5, indicative of previously unappreciated heterogeneity in penetrance within this class of substitution. Finally, I generate more accurate and informative estimates of de novo germline mutation rate in humans, and develop a toolkit to simulate, distribute and interpret mutations in human diseases. Overall, my research uncovers novel variability in human mutation rate and provides a systematic framework for analyzing mutational data, which can be used from causal gene discovery to elucidating specific disease mechanisms

Human mutation/substitution rate: Variability, modeling and applications

Mutation generates genetic variation, and in turn selection purges deleterious variants from the population. Understanding both is critical for discovering causal genes and variants behind diseases or making inferences about evolutionary processes. Human mutation rate varies significantly across the genome although most studies have only considered the immediate flanking nucleotides around the polymorphic site to model and study patterns of variability. The impact of larger sequence context has not been fully clarified, even though it substantially influences rates of mutation. In the first part of this thesis, I develop a novel statistical framework and using data from the 1000 Genomes project, demonstrate that a larger heptanucleotide sequence context explains \u3e81% variability in substitution probabilities, discovering novel mutation promoting motifs at ApT dinucleotides, CAAT, and TACG sequences. My approach also reveals previously undocumented variability in C-to-T substitutions at CpG sites, not immediately explained by differential methylation intensity. Building on this framework, I model the selective forces acting on the coding genome and develop statistical scores that measures the intolerance at the gene or amino-acid level for functional variants. I demonstrate clinical utility of such intolerance scores in identifying genes associated with multiple human diseases including Autism. Next, I apply these lessons of mutation rate variability to develop an algorithm to detect sub-genic enrichment of de novo germline mutations in RB1 gene of bilateral Retinoblastoma (RB) probands to further elucidate disease biology. I demonstrate that previously noted ‘hotspots’ of nonsense mutations in RB1 are compatible with the elevated mutation rates expected at CpG sites, refuting a specific mechanism in RB pathogenesis. I also find enrichment of splice-site donor mutations of exon 6 and 12 but depletion at exon 5, indicative of previously unappreciated heterogeneity in penetrance within this class of substitution. Finally, I generate more accurate and informative estimates of de novo germline mutation rate in humans, and develop a toolkit to simulate, distribute and interpret mutations in human diseases. Overall, my research uncovers novel variability in human mutation rate and provides a systematic framework for analyzing mutational data, which can be used from causal gene discovery to elucidating specific disease mechanisms

Viral communities of the human gut: metagenomic analysis of composition and dynamics

Abstract Background The numerically most abundant biological entities on Earth are viruses. Vast populations prey on the cellular microbiota in all habitats, including the human gut. Main body Here we review approaches for studying the human virome, and some recent results on movement of viral sequences between bacterial cells and eukaryotic hosts. We first overview biochemical and bioinformatic methods, emphasizing that specific choices in the methods used can have strong effects on the results obtained. We then review studies characterizing the virome of the healthy human gut, which reveal that most of the viruses detected are typically uncharacterized phage - the viral dark matter - and that viruses that infect human cells are encountered only rarely. We then review movement of phage between bacterial cells during antibiotic treatment. Here a radical proposal for extensive movement of antibiotic genes on phage has been challenged by a careful reanalysis of the metagenomic annotation methods used. We then review two recent studies of movement of whole phage communities between human individuals during fecal microbial transplantation, which emphasize the possible role of lysogeny in dispersal. Short conclusion Methods for studying the human gut virome are improving, yielding interesting data on movement of phage genes between cells and mammalian host organisms. However, viral populations are vast, and studies of their composition and function are just beginning

Additional file 2: Figure S1. of De novo mutational profile in RB1 clarified using a mutation rate modeling algorithm

Comparison of observed mutations and the simulated frequency of essential splice acceptor mutations in RB (99% CI) to find exon specific differential pathogenicity within essential splice mutations. Exons where the observed mutations are higher or lower than the 99% confidence interval of simulations are denoted by an asterisk (*). (PDF 143 kb

Additional file 3: Figure S2. of De novo mutational profile in RB1 clarified using a mutation rate modeling algorithm

Donor splice mutations in Exons 5, 6 and 12, and their effect on codon structure. The codon structures are shown prior and after the donor splice mutation. The donor splice mutation results in exon skipping or deletion, but can also cause a frameshift mutation in certain cases. (PDF 84 kb

Additional file 1: Table S1. of De novo mutational profile in RB1 clarified using a mutation rate modeling algorithm

All de novo germline variants in RB1 gene of patients with RB. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S2. All ExAC variants in RB1 gene that were considered in our analysis. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S3. All Nonsense variants in RB1 gene from Onadim and Houdayer groups. “gDNA position” is the nucleotide position in the GENBANK accession number L11910 of the gene. Table S4. Comparison of observed mutations and the simulated frequency of nonsense changes per exon, to find differential pathogenicity within nonsense mutations. Analysis was performed on data from Onadim and Houdayer groups. Table S5. Comparison of observed mutations and the simulated frequency of nonsense changes to find differential pathogenicity within nonsense mutations. Data shown for all amino acids and two arginine codons (99% CI) which can change to a stop codon. Analysis was performed on data from Onadim and Houdayer groups. Table S6. Polyphen predictions on the de novo germline missense mutations or some potential variants near codon 661 in RB1 gene. “Polyphen2_format” is the variant format accepted by the Polyphen2 tool. “Polyphen_prediction” is the result of Polyphen2 on the missense variant. Table S7. Comparison between observed mutations and the simulated frequency of missense changes at amino acids and codons in exon 20, to find localized pathogenicity within missense mutations. Only the significant results are reported here. Table S8. Genomic territory of RB1 gene analyzed in our study. “Position Start” is the start position of the entry as per GENBANK database. “Position End” is the end positon of the entry as per GENBANK database. “Annotation” is the description of the entry. Possible keywords are exon or donor/acceptor region in essential splice or nonessential intronic region. “Exon” corresponds to exon number of the entry. (XLSX 30 kb

Treatment of Partial Thickness Burns: A Prospective, Randomized Controlled Trial Comparing Four Routinely Used Burns Dressings in an Ambulatory Care Setting

Abstract This prospective, randomized controlled trial study compared the effects of four dressings for adult partial thickness burns, focusing on re-epithelialization time and cost effectiveness. Adults with partial thickness burns meeting inclusion criteria were randomized to either Biobrane™, Acticoat™, Mepilex® Ag, or Aquacel® Ag. Primary endpoint for analysis was &amp;gt;95% re-epithelialization. Incremental cost-effectiveness ratios were calculated based on dressing costs. Dominance probabilities between treatment arms were calculated from bootstrap resampling trial data. One hunderd thirty-one partial thickness burn wounds in 119 patients were randomized. Adjusting for sex, age, smoking status, burn mechanism, TBSA, and first aid adequacy, Mepilex® Ag had a reduced time to re-epithelialization compared to Biobrane™ (IRR: 1.26; 95% CI: 1.07–1.48, P &amp;lt; .01). Economic analysis showed that there was a 99%, 71%, and 53% probability that Mepilex® Ag dominated (cheaper and more effective) Biobrane™, Acticoat™, and Aquacel® Ag, respectively. Mepilex® Ag achieved faster re-epithelialization and better cost effectiveness. Patient satisfaction and comfort seems better with Biobrane™ although not reflected within the end outcome of the healed wound. It is the patients’ (after extensive education) and clinicians’ choice, level of experience, and availability of products in praxis that will guide the decision as to which the product is used individually on which patient.</jats:p

Strain population structure varies widely across bacterial species and predicts strain colonization in unrelated individuals

SummaryThe population structure of strains within a bacterial species is poorly defined, despite the functional importance of strain variation in the human gut microbiota on health. Here we analyzed &gt;1000 sequenced bacterial strains from the fecal microbiota of 47 individuals from two countries and combined them with &gt;150,000 bacterial genomes from NCBI to quantify the strain population size of different bacterial species, as well as the frequency of finding the same strain colonized in unrelated individuals who had no opportunities for direct microbial strain transmission. Strain population sizes ranged from tens to over one-hundred thousand per species. Prevalent strains in common gut microbiota species with small population sizes were the most likely to be harbored in two or more unrelated individuals. The finite strain population size of certain species creates the opportunity to comprehensively sequence the entirety of these species’ prevalent strains and associate their presence in different individuals with health outcomes.</jats:p

Precise quantification of bacterial strains after fecal microbiota transplantation delineates long-term engraftment and explains outcomes

AbstractFecal microbiota transplantation (FMT) has been successfully applied to treat recurrent Clostridium difficile infection in humans, but a precise method to measure which bacterial strains stably engraft in recipients and evaluate their association with clinical outcomes is lacking. We assembled a collection of &gt;1,000 different bacterial strains that were cultured from the fecal samples of 22 FMT donors and recipients. Using our strain collection combined with metagenomic sequencing data from the same samples, we developed a statistical approach named Strainer for the detection and tracking of bacterial strains from metagenomic sequencing data. We applied Strainer to evaluate a cohort of 13 FMT longitudinal clinical interventions and detected stable engraftment of 71% of donor microbiota strains in recipients up to 5 years post-FMT. We found that 80% of recipient gut bacterial strains pre-FMT were eliminated by FMT and that post-FMT the strains present persisted up to 5 years later, together with environmentally acquired strains. Quantification of donor bacterial strain engraftment in recipients independently explained (precision 100%, recall 95%) the clinical outcomes (relapse or success) after initial and repeat FMT. We report a compendium of bacterial species and strains that consistently engraft in recipients over time that could be used in defined live biotherapeutic products as an alternative to FMT. Our analytical framework and Strainer can be applied to systematically evaluate either FMT or defined live bacterial therapeutic studies by quantification of strain engraftment in recipients.</jats:p