Georgiy Golovko

Mycoplasma CG- and GATC-specific DNA methyltransferases selectively and efficiently methylate the host genome and alter the epigenetic landscape in human cells

Aberrant DNA methylation is frequently observed in disease, including many cancer types, yet the underlying mechanisms remain unclear. Because germline and somatic mutations in the genes that are responsible for DNA methylation are infrequent in malignancies, additional mechanisms must be considered. Mycoplasmas spp., including Mycoplasma hyorhinis, efficiently colonize human cells and may serve as a vehicle for delivery of enzymatically active microbial proteins into the intracellular milieu. Here, we performed, for the first time, genome-wide and individual gene mapping of methylation marks generated by the M. hyorhinis CG- and GATC-specific DNA cytosine methyltransferases (MTases) in human cells. Our results demonstrated that, upon expression in human cells, MTases readily translocated to the cell nucleus. In the nucleus, MTases selectively and efficiently methylated the host genome at the DNA sequence sites free from pre-existing endogenous methylation, including those in a variety of cancer-associated genes. We also established that mycoplasma is widespread in colorectal cancers, suggesting that either the infection contributed to malignancy onset or, alternatively, that tumors provide a favorable environment for mycoplasma growth. In the human genome, ∼11% of GATC sites overlap with CGs (e.g., CGATmCG); therefore, the methylated status of these sites can be perpetuated by human DNMT1. Based on these results, we now suggest that the GATC-specific methylation represents a novel type of infection-specific epigenetic mark that originates in human cells with a previous exposure to infection. Overall, our findings unveil an entirely new panorama of interactions between the human microbiome and epigenome with a potential impact in disease etiology.

Slim-Filter: an interactive windows-based application for illumina genome analyzer data assessment and manipulation

BackgroundThe emergence of Next Generation Sequencing technologies has made it possible for individual investigators to generate gigabases of sequencing data per week. Effective analysis and manipulation of these data is limited due to large file sizes, so even simple tasks such as data filtration and quality assessment have to be performed in several steps. This requires (potentially problematic) interaction between the investigator and a bioinformatics/computational service provider. Furthermore, such services are often performed using specialized computational facilities.ResultsWe present a Windows-based application, Slim-Filter designed to interactively examine the statistical properties of sequencing reads produced by Illumina Genome Analyzer and to perform a broad spectrum of data manipulation tasks including: filtration of low quality and low complexity reads; filtration of reads containing undesired subsequences (such as parts of adapters and PCR primers used during the sample and sequencing libraries preparation steps); excluding duplicated reads (while keeping each read’s copy number information in a specialized data format); and sorting reads by copy numbers allowing for easy access and manual editing of the resulting files. Slim-Filter is organized as a sequence of windows summarizing the statistical properties of the reads. Each data manipulation step has roll-back abilities, allowing for return to previous steps of the data analysis process. Slim-Filter is written in C++ and is compatible with fasta, fastq, and specialized AS file formats presented in this manuscript. Setup files and a user’s manual are available for download at the supplementary web site (https://www.bioinfo.uh.edu/Slim_Filter/).ConclusionThe presented Windows-based application has been developed with the goal of providing individual investigators with integrated sequencing reads analysis, curation, and manipulation capabilities.

Nasopharyngeal microbiota in infants and changes during viral upper respiratory tract infection and acute otitis media

Background Interferences between pathogenic bacteria and specific commensals are known. We determined the interactions between nasopharyngeal microbial pathogens and commensals during viral upper respiratory tract infection (URI) and acute otitis media (AOM) in infants. Methods We analyzed 971 specimens collected monthly and during URI and AOM episodes from 139 infants. The 16S rRNA V4 gene regions were sequenced on the Illumina MiSeq platform. Results Among the high abundant genus-level nasopharyngeal microbiota were Moraxella, Haemophilus, and Streptococcus (3 otopathogen genera), Corynebacterium, Dolosigranulum, Staphylococcus, Acinetobacter, Pseudomonas, and Bifidobacterium. Bacterial diversity was lower in culture-positive samples for Streptococcus pneumoniae, and Haemophilus influenzae, compared to cultured-negative samples. URI frequencies were positively associated with increasing trend in otopathogen colonization. AOM frequencies were associated with decreasing trend in Micrococcus colonization. During URI and AOM, there were increases in abundance of otopathogen genera and decreases in Pseudomonas, Myroides, Yersinia, and Sphingomonas. Otopathogen abundance was increased during symptomatic viral infection, but not during asymptomatic infection. The risk for AOM complicating URI was reduced by increased abundance of Staphylococcus and Sphingobium. Conclusion Otopathogen genera played the key roles in URI and AOM occurrences. Staphylococcus counteracts otopathogens thus Staphylococcal colonization may be beneficial, rather than harmful. While Sphingobium may play a role in preventing AOM complicating URI, the commonly used probiotic Bifidobacterium did not play a significant role during URI or AOM. The role of less common commensals in counteracting the deleterious effects of otopathogens requires further studies.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells

BackgroundGlucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents.MethodsWe analyzed the effect of treatment with the cytosine analogue 5 aza-2’ deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status.ConclusionsTreatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

CoCo: An application to store High-Throughput Sequencing data in compact text and binary file formats

The storage, manipulation, and especially internet transfer of large amounts of data produced by High-Throughput Sequencing (HTS) instruments present major obstacles utilizing the full potential of this promising technology. The current standard is based on storing all data, which are produced in text (FASTQ and FASTA) and often stored in binary (SRA and BAM) formats. To date, significant effort has been devoted to efficiently compressing these cumbersome sequencing data sets in their existing formats. However, given the substantial improvements in the quality of HTS data, we believe that if one can afford to exclude low quality data and read headers, new much more compressed data formats can be used to reduce size of HTS data files by at least two orders of magnitude. Here we present several examples of file formats specifically designed to store only high quality sequencing reads in space efficient text and binary form. The basic principles used to decrease file size include storage of only one copy of a sequence when reads are present in multiple copies; alphabetical sorting of all reads and storage of only the differences (suffixes) between consecutive reads; and optimization of the number of bits/bytes required to store the information in binary formats. While file size reduction depends on properties of the sequencing data, the size of the resulting files can be as low as 0.1 %-5% of the original FASTQ, SRA, or BAM files. The greatest advantage of the proposed formats however, is based on its time and memory efficiency. The time required to convert reads from FASTQ/FAST A files into the proposed formats is up to 10 times faster than gzip and SRA. The conversion of files in the proposed formats back to FAST A is limited only by the time required to read the file from the hard drive. We present the source code of the C++ object (class) implemented to store, sort, and perform I/O operations with equal length subsequences; and two executable LINUX command line applications (CoCo and CoCo-PIus) able to work with all types of sequencing data including paired-end and flexible size reads. Source code, Linux executables, as well as user manual can be downloaded from http://bgl.utmb.edu/publications/34cocoplus.

Secondary Analysis of the NCI-60 Whole Exome Sequencing Data Indicates Significant Presence of Propionibacterium acnes Genomic Material in Leukemia (RPMI-8226) and Central Nervous System (SF-295, SF-539, and SNB-19) Cell Lines.

The NCI-60 human tumor cell line panel has been used in a broad range of cancer research over the last two decades. A landmark 2013 whole exome sequencing study of this panel added an exceptional new resource for cancer biologists. The complementary analysis of the sequencing data produced by this study suggests the presence of Propionibacterium acnes genomic sequences in almost half of the datasets, with the highest abundance in the leukemia (RPMI-8226) and central nervous system (SF-295, SF-539, and SNB-19) cell lines. While the origin of these contaminating bacterial sequences remains to be determined, observed results suggest that computational control for the presence of microbial genomic material is a necessary step in the analysis of the high throughput sequencing (HTS) data.

Mosquito bottlenecks alter viral mutant swarm in a tissue and time-dependent manner with contraction and expansion of variant positions and diversity

Abstract Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.

Draft genome sequence of Zobellella denitrificans ZD1 (JCM 13380), a salt-tolerant denitrifying bacterium capable of producing poly(3-hydroxybutyrate)

ABSTRACT Zobellella denitrificans ZD1, isolated from sediments of an estuarine mangrove ecosystem in Taiwan, exhibits growth-associated production of biopolymer poly(3-hydroxybutyrate) (PHB). This work reports the 4.05-Mbp draft genome sequence of Z. denitrificans ZD1, consisting of 217 contigs with a G+C content of 63.8% and 3,672 protein-coding sequences.