Search

Clopidogrel, a prescription drug to reduce ischemic events in cardiovascular patients, has been extensively studied in mostly European individuals but not among Caribbean Hispanics. This study evaluated the low abundance and reduced activity of paraoxonase-1 (PON1) in clopidogrel-resistant patients as a predictive risk biomarker of poor responders and disease severity in this population. Thirty-six patients on clopidogrel (cases divided into poor and normal responders) were enrolled, along with 11 cardiovascular patients with no clopidogrel indications (positive control) and 13 healthy volunteers (negative control). Residual on-treatment platelet reactivity unit (PRU), PON1 abundance by Western blotting, and PON1 activity by enzymatic assays were measured. PON1 genotyping and computational haplotype phasing were performed on 512 DNA specimens for two genetic loci (rs662 and rs854560). No statistical differences in mean relative PON1 abundance were found among the groups (p > 0.05). However, a significantly lower enzymatic activity was found in poor responders (10.57 ± 6.79 µU/mL) when compared to controls (22.66 ± 8.30 µU/mL and 22.21 ± 9.66 µU/mL; p = 0.004). PON1 activity among carriers of the most prevalent PON1 haplotype (AA|AA) was significantly lower than in wild types (7.90 µU/mL vs. 22.03 µU/mL; p = 0.005). Our findings suggested that PON1 is a potential biomarker of cardiovascular disease severity in Caribbean Hispanics.

Abstract Background Accessory proteins have diverse roles in coronavirus pathobiology. One of them in SARS-CoV (the causative agent of the severe acute respiratory syndrome outbreak in 2002–2003) is encoded by the open reading frame 8 (ORF8). Among the most dramatic genomic changes observed in SARS-CoV isolated from patients during the peak of the pandemic in 2003 was the acquisition of a characteristic 29-nucleotide deletion in ORF8. This deletion cause splitting of ORF8 into two smaller ORFs, namely ORF8a and ORF8b. Functional consequences of this event are not entirely clear. Results Here, we performed evolutionary analyses of ORF8a and ORF8b genes and documented that in both cases the frequency of synonymous mutations was greater than that of nonsynonymous ones. These results suggest that ORF8a and ORF8b are under purifying selection, thus proteins translated from these ORFs are likely to be functionally important. Comparisons with several other SARS-CoV genes revealed that another accessory gene, ORF7a, has a similar ratio of nonsynonymous to synonymous mutations suggesting that ORF8a, ORF8b, and ORF7a are under similar selection pressure. Conclusions Our results for SARS-CoV echo the known excess of deletions in the ORF7a-ORF7b-ORF8 complex of accessory genes in SARS-CoV-2. A high frequency of deletions in this gene complex might reflect recurrent searches in “functional space” of various accessory protein combinations that may eventually produce more advantageous configurations of accessory proteins similar to the fixed deletion in the SARS-CoV ORF8 gene.

Summary: Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus’s macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

Sofya Garushyants et al. survey publicly available alignments of SARS-CoV-2 genomes to identify and characterize potential insertion variants throughout the COVID-19 pandemic. They altogether identify 347 inserts, some of which might affect SARS-CoV-2 pathogenicity.

Nucleotide substitutions in protein-coding genes can be divided into synonymous (S) and non-synonymous (N) ones that alter amino acids (including nonsense mutations causing stop codons). The S substitutions are expected to have little effect on function. The N substitutions almost always are affected by strong purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can modulate the deleterious effect of single N substitutions and, thus, could be subjected to the positive selection. This effect has been demonstrated for mutations in the serine codons, stop codons and double N substitutions in prokaryotes. In all abovementioned cases, a novel technique was applied that allows elucidating the effects of selection on double substitutions considering mutational biases. Here, we applied the same technique to study double N substitutions in eukaryotic lineages of primates and yeast. We identified markedly fewer cases of purifying selection relative to prokaryotes and no evidence of codon double substitutions under positive selection. This is consistent with previous studies of serine codons in primates and yeast. In general, the obtained results strongly suggest that there are major differences between studied pro- and eukaryotes; double substitutions in primates and yeasts largely reflect mutational biases and are not hallmarks of selection. This is especially important in the context of detection of positive selection in codons because it has been suggested that multiple mutations in codons cause false inferences of lineage-specific site positive selection. It is likely that this concern is applicable to previously studied prokaryotes but not to primates and yeasts where markedly fewer double substitutions are affected by positive selection.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

ABSTRACT Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

Abstract Background Medium spiny neurons (MSNs) comprise the main body (95% in mouse) of the dorsal striatum neurons and represent dopaminoceptive GABAergic neurons. The cAMP (cyclic Adenosine MonoPhosphate)—mediated cascade of excitation and inhibition responses observed in MSN intracellular signal transduction is crucial for neuroscience research due to its involvement in the motor and behavioral functions. In particular, all types of addictions are related to MSNs. Shedding the light on the mechanics of the above-mentioned cascade is of primary importance for this research domain. Results A mouse model of chronic social conflicts in daily agonistic interactions was used to analyze dorsal striatum neurons genes implicated in cAMP-mediated phosphorylation activation pathways specific for MSNs. Based on expression correlation analysis, we succeeded in dissecting Drd1- and Drd2-dopaminoceptive neurons (D1 and D2, correspondingly) gene pathways. We also found that D1 neurons genes clustering are split into two oppositely correlated states, passive and active ones, the latter apparently corresponding to D1 firing stage upon protein kinase A (PKA) activation. We observed that under defeat stress in chronic social conflicts the loser mice manifest overall depression of dopamine-mediated MSNs activity resulting in previously reported reduced motor activity, while the aggressive mice with positive fighting experience (aggressive mice) feature an increase in both D1-active phase and D2 MSNs genes expression leading to hyperactive behavior pattern corresponded by us before. Based on the alternative transcript isoforms expression analysis, it was assumed that many genes (Drd1, Adora1, Pde10, Ppp1r1b, Gnal), specifically those in D1 neurons, apparently remain transcriptionally repressed via the reversible mechanism of promoter CpG island silencing, resulting in alternative promoter usage following profound reduction in their expression rate. Conclusion Based on the animal stress model dorsal striatum pooled tissue RNA-Seq data restricted to cAMP related genes subset we elucidated MSNs steady states exhaustive projection for the first time. We correspond the existence of D1 active state not explicitly outlined before, and connected with dynamic dopamine neurotransmission cycles. Consequently, we were also able to indicate an oscillated postsynaptic dopamine vs glutamate action pattern in the course of the neurotransmission cycles.

The analysis of deletions may reveal evolutionary trends and provide new insight into the surprising variability and rapidly spreading capability that SARS-CoV-2 has shown since its emergence. To understand the factors governing genomic stability, it is important to define the molecular mechanisms of deletions in the viral genome. In this work, we performed a statistical analysis of deletions. Specifically, we analyzed correlations between deletions in the SARS-CoV-2 genome and repetitive elements and documented a significant association of deletions with runs of identical (poly-) nucleotides and direct repeats. Our analyses of deletions in the accessory genes of SARS-CoV-2 suggested that there may be a hypervariability in ORF7A and ORF8 that is not associated with repetitive elements. Such recurrent search in a “sequence space” of accessory genes (that might be driven by natural selection) did not yet cause increased viability of the SARS-CoV-2 variants. However, deletions in the accessory genes may ultimately produce new variants that are more successful compared to the viral strains with the conventional architecture of the SARS-CoV-2 accessory genes.

Abstract Background Single nucleotide substitutions in protein-coding genes can be divided into synonymous (S), with little fitness effect, and non-synonymous (N) ones that alter amino acids and thus generally have a greater effect. Most of the N substitutions are affected by purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases potentially could alleviate the deleterious effect of single substitutions, making them subject to positive selection. To elucidate the effects of selection on double substitutions in all codons, it is critical to differentiate selection from mutational biases. Results We addressed the evolutionary regimes of within-codon double substitutions in 37 groups of closely related prokaryotic genomes from diverse phyla by comparing the fractions of double substitutions within codons to those of the equivalent double S substitutions in adjacent codons. Under the assumption that substitutions occur one at a time, all within-codon double substitutions can be represented as “ancestral-intermediate-final” sequences (where “intermediate” refers to the first single substitution and “final” refers to the second substitution) and can be partitioned into four classes: (1) SS, S intermediate–S final; (2) SN, S intermediate–N final; (3) NS, N intermediate–S final; and (4) NN, N intermediate–N final. We found that the selective pressure on the second substitution markedly differs among these classes of double substitutions. Analogous to single S (synonymous) substitutions, SS double substitutions evolve neutrally, whereas analogous to single N (non-synonymous) substitutions, SN double substitutions are subject to purifying selection. In contrast, NS show positive selection on the second step because the original amino acid is recovered. The NN double substitutions are heterogeneous and can be subject to either purifying or positive selection, or evolve neutrally, depending on the amino acid similarity between the final or intermediate and the ancestral states. Conclusions The results of the present, comprehensive analysis of the evolutionary landscape of within-codon double substitutions reaffirm the largely conservative regime of protein evolution. However, the second step of a double substitution can be subject to positive selection when the first step is deleterious. Such positive selection can result in frequent crossing of valleys on the fitness landscape.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

Cancer genomes harbor numerous genomic alterations and many cancers accumulate thousands of nucleotide sequence variations. A prominent fraction of these mutations arises as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases followed by the replication/repair of edited sites by DNA polymerases (pol), as deduced from the analysis of the DNA sequence context of mutations in different tumor tissues. We have used the weight matrix (sequence profile) approach to analyze mutagenesis due to Activation Induced Deaminase (AID) and two error-prone DNA polymerases. Control experiments using shuffled weight matrices and somatic mutations in immunoglobulin genes confirmed the power of this method. Analysis of somatic mutations in various cancers suggested that AID and DNA polymerases η and θ contribute to mutagenesis in contexts that almost universally correlate with the context of mutations in A:T and G:C sites during the affinity maturation of immunoglobulin genes. Previously, we demonstrated that AID contributes to mutagenesis in (de)methylated genomic DNA in various cancers. Our current analysis of methylation data from malignant lymphomas suggests that driver genes are subject to different (de)methylation processes than non-driver genes and, in addition to AID, the activity of pols η and θ contributes to the establishment of methylation-dependent mutation profiles. This may reflect the functional importance of interplay between mutagenesis in cancer and (de)methylation processes in different groups of genes. The resulting changes in CpG methylation levels and chromatin modifications are likely to cause changes in the expression levels of driver genes that may affect cancer initiation and/or progression.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Abstract Here we describe a new family of carotenoid cleavage oxygenases (CCOs) in metazoans, the BCO2-like (BCOL) clade, which contains lancelet, nematode, and molluscan carotenoid oxygenase sequences. Phylogenetic analysis of CCOs in all kingdoms of life confirmed that the BCOL enzymes are an independent clade of ancient origin. One of the predicted lancelet BCOL proteins, cloned and analyzed for carotenoid cleavage activity in a bacterial carotenoid expression system, had activity similar to lancelet BCO2 proteins, although with a preference for cis isomers. Our docking predictions correlated well with the cis-favored activity. The extensive expansions of the new animal BCOL family in some species (e.g., lancelet) suggests that the carotenoid cleavage oxygenase superfamily has evolved in the “extremely high turnover” fashion: numerous losses and duplications of this family are likely to reflect complex regulation processes during development, and interactions with the environment. These findings also serve to provide a rationale for the evolution of the BCO-related outlier RPE65 retinol isomerase, an enzyme that does not utilize carotenoids as substrate or perform double-bond cleavage.

Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE , consistent with lack of translation of RPE65 mRNA in cell cultures.

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons—where an intermediate step is a nonsense substitution—show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.

The Leishmania donovani species complex consists of all L. donovani and L. infantum strains mainly responsible for visceral leishmaniasis (VL). It was suggested that genome rearrangements in Leishmania spp. occur very often, thus enabling parasites to adapt to the different environmental conditions. Some of these rearrangements may be directly linked to the virulence or explain the reduced efficacy of antimonial drugs in some isolates. In the current study, we focused on a large-scale analysis of putative gene conversion events using publicly available datasets. Previous population study of L. donovani suggested that population variability of L. donovani is relatively low, however the authors used masking procedures and strict read selection criteria. We decided to re-analyze DNA-seq data without masking sequences, because we were interested in the most dynamic fraction of the genome. The majority of samples have an excess of putative gene conversion/recombination events in the noncoding regions, however we found an overall excess of putative intrachromosomal gene conversion/recombination in the protein coding genes, compared to putative interchromosomal gene conversion/recombination events.

Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate β-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate β-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.

Background: The human gut microbiota is a dynamic community of microorganisms that mediate important biochemical processes. Differences in the gut microbial composition have been associated with inflammatory bowel diseases (IBD) and other intestinal disorders. In this study, we quantified and compared the frequencies of eight genotoxic and/or pro-inflammatory bacterial genes found in metagenomic Whole Genome Sequences (mWGSs) of samples from individuals with IBD vs. a cohort of healthy human subjects.Methods: The eight selected gene sequences were clbN, clbB, cif, cnf-1, usp, tcpC from Escherichia coli, gelE from Enterococcus faecalis and murB from Akkermansia muciniphila. We also included the sequences for the conserved murB genes from E. coli and E. faecalis as markers for the presence of Enterobacteriaceae or Enterococci in the samples. The gene sequences were chosen based on their previously reported ability to disrupt normal cellular processes to either promote inflammation or to cause DNA damage in cultured cells or animal models, which could be linked to a role in IBD. The selected sequences were searched in three different mWGS datasets accessed through the Human Microbiome Project (HMP): a healthy cohort (N = 251), a Crohn’s disease cohort (N = 60) and an ulcerative colitis cohort (N = 17).Results: Firstly, the sequences for the murB housekeeping genes from Enterobacteriaceae and Enterococci were more frequently found in the IBD cohorts (32% E. coli in IBD vs. 12% in healthy; 13% E. faecalis in IBD vs. 3% in healthy) than in the healthy cohort, confirming earlier reports of a higher presence of both of these taxa in IBD. For some of the sequences in our study, especially usp and gelE, their frequency was even more sharply increased in the IBD cohorts than in the healthy cohort, suggesting an association with IBD that is not easily explained by the increased presence of E. coli or E. faecalis in those samples.Conclusion: Our results suggest a significant association between the presence of some of these genotoxic or pro-inflammatory gene sequences and IBDs. In addition, these results illustrate the power and limitations of the HMP database in the detection of possible clinical correlations for individual bacterial genes.

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell’s history and to acquisition of new mutations near original break.

Transposable elements (TE) are abundant in mammalian genomes and appear to have contributed to the evolution of their hosts by providing novel regulatory or coding sequences. We analyzed different regions of long intergenic non-coding RNA (lincRNA) genes in human and mouse genomes to systematically assess the potential contribution of TEs to the evolution of the structure and regulation of expression of lincRNA genes. Introns of lincRNA genes contain the highest percentage of TE-derived sequences, followed by exons and then promoter regions although the density of TEs is not significantly different between exons and promoters. Higher frequencies of ancient TEs in promoters and exons compared to introns implies that many lincRNA genes emerged before the split of primates and rodents. The content of TE-derived sequences in lincRNA genes is substantially higher than that in protein-coding genes, especially in exons and promoter regions. A significant positive correlation was detected between the content of TEs and evolutionary rate of lincRNAs indicating that inserted TEs are preferentially fixed in fast-evolving lincRNA genes. These results are consistent with the RIDL (Repeat Insertion Domains of LncRNAs) hypothesis under which TEs have substantially contributed to the origin, evolution, and in particular functional diversification, of lincRNA genes.

The abundance of mammalian long intergenic non-coding RNA (lincRNA) genes is high, yet their functions remain largely unknown. One possible way to study this important question is to use large-scale comparisons of various characteristics of lincRNA with those of protein-coding genes for which a large body of functional information is available. A prominent feature of mammalian protein-coding genes is the high evolutionary conservation of the exon-intron structure. Comparative analysis of putative intron positions in lincRNA genes from various mammalian genomes suggests that some lincRNA introns have been conserved for over 100 million years, thus the primary and/or secondary structure of these molecules is likely to be functionally important.