Gut Mycobiota Dysbiosis Is Associated with Melanoma and Response to Anti-PD-1 Therapy.

Recent research indicates that gut microbiota may be vital in the advancement of melanoma. In this study, we found that melanoma patients exhibited a distinct gut mycobiota structure compared with healthy participants. Candida albicans, Candida dubliniensis, and Neurospora crassa were more abundant in samples from patients with melanoma, whereas Saccharomyces cerevisiae and Debaryomyces hansenii were less abundant. During anti-PD-1 treatment, the relative amount of Malassezia restricta and C. albicans increased. A higher level of Saccharomyces paradoxus was associated with a positive response to anti-PD-1 treatment, whereas a higher level of Tetrapisispora blattae was associated with a lack of clinical benefits. High levels of M. restricta and C. albicans, elevated serum lactate dehydrogenase, and being overweight were linked to increased risk of melanoma progression and poorer response to anti-PD-1 treatment. Thus, this study has revealed melanoma-associated mycobiome dysbiosis, characterized by altered fungal composition and fungi species associated with a higher risk of melanoma progression, identifying a role for the gut mycobiome in melanoma progression.

CMC: Cancer miRNA Census - a list of cancer-related miRNA genes.

A growing body of evidence indicates an important role of miRNAs in cancer; however, there is no definitive, convenient-to-use list of cancer-related miRNAs or miRNA genes that may serve as a reference for analyses of miRNAs in cancer. To this end, we created a list of 165 cancer-related miRNA genes called the Cancer miRNA Census (CMC). The list is based on a score, built on various types of functional and genetic evidence for the role of particular miRNAs in cancer, e.g. miRNA-cancer associations reported in databases, associations of miRNAs with cancer hallmarks, or signals of positive selection of genetic alterations in cancer. The presence of well-recognized cancer-related miRNA genes, such as MIR21, MIR155, MIR15A, MIR17 or MIRLET7s, at the top of the CMC ranking directly confirms the accuracy and robustness of the list. Additionally, to verify and indicate the reliability of CMC, we performed a validation of criteria used to build CMC, comparison of CMC with various cancer data (publications and databases), and enrichment analyses of biological pathways and processes such as Gene Ontology or DisGeNET. All validation steps showed a strong association of CMC with cancer/cancer-related processes confirming its usefulness as a reference list of miRNA genes associated with cancer.

LINE-1 mRNA 3' end dynamics shape its biology and retrotransposition potential.

LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.

The emerging role of the gut mycobiome in liver diseases.

In recent years, it has become clear that gut microbiota plays a major role in the human body, both in health and disease. Because of that, the gut microbiome and its impact on human well-being are getting wider and wider attention. Studies focused on the liver are not an exception. However, the majority of the analyses are concentrated on the bacterial part of the gut microbiota, while the fungi living in the human intestines are often omitted or underappreciated. This review is focused on the gut mycobiome as an important factor that should be taken into consideration regarding liver homeostasis and its perturbations. We have collected the findings in this field and we discuss their importance. We aim to emphasize the fungal compositional changes related to liver diseases and, by that, provide novel insights into the directions of liver research and gut microbiota as a therapeutic target for liver diseases.

Host Factors Associated with Gut Mycobiome Structure.

Recent studies revealed a significant role of the gut fungal community in human health. Here, we investigated the content and variation of gut mycobiota among subjects from the European population. We explored the interplay between gut fungi and various host-related sociodemographic, lifestyle, health, and dietary factors. The study included 923 participants. Fecal DNA samples were analyzed by whole-metagenome high-throughput sequencing. Subsequently, fungi taxonomic profiles were determined and accompanied by computational and statistical analyses of the association with 53 host-related factors. Fungal communities were characterized by a high prevalence of Saccharomyces, Candida, and Sporisorium. Ten factors were found to correlate significantly with the overall mycobiota variation. Most were diet related, including the consumption of chips, meat, sodas, sweetening, processed food, and alcohol, followed by age and marital status. Differences in α- and/or ß-diversity were also reported for other factors such as body mass index (BMI), job type, autoimmunological diseases, and probiotics. Differential abundance analysis revealed fungal species that exhibited different patterns of changes under specific conditions. The human gut mycobiota is dominated by yeast, including Saccharomyces, Malassezia, and Candida. Although intervolunteer variability was high, several fungal species persisted across most samples, which may be evidence that a core gut mycobiota exists. Moreover, we showed that host-related factors such as diet, age, and marital status influence the variability of gut mycobiota. To our knowledge, this is the first large and comprehensive study of the European cohort in terms of gut mycobiota associations with such an extensive and differentiated host-related set of factors. IMPORTANCE The human gut is inhabited by many organisms, including bacteria and fungi, that may affect human health. However, research on human gut mycobiome is still rare. Moreover, the large European-based cohort study is missing. Here, we analyzed the first large European cohort in terms of gut mycobiota associations with a differentiated host-related set of factors. Our results showed that chips, meat, sodas, sweetening, processed food, beer, alcohol consumption, age, and marital status were associated with the variability of gut mycobiota. Moreover, our analysis revealed changes in abundances at the fungal species level for many investigated factors. Our results can suggest potentially valuable paths for further, narrowly focused research on gut mycobiome and its impact on human health. In the coming era of gut microbiome-based precision medicine, further research into the relationship between different mycobial structures and host-related factors may result in new preventive approaches or therapeutic procedures.

The standardisation of the approach to metagenomic human gut analysis: from sample collection to microbiome profiling.

In recent years, the number of metagenomic studies increased significantly. Wide range of factors, including the tremendous community complexity and variability, is contributing to the challenge in reliable microbiome community profiling. Many approaches have been proposed to overcome these problems making hardly possible to compare results of different studies. The significant differences between procedures used in metagenomic research are reflected in a variation of the obtained results. This calls for the need for standardisation of the procedure, to reduce the confounding factors originating from DNA isolation, sequencing and bioinformatics analyses in order to ensure that the differences in microbiome composition are of a true biological origin. Although the best practices for metagenomics studies have been the topic of several publications and the main aim of the International Human Microbiome Standard (IHMS) project, standardisation of the procedure for generating and analysing metagenomic data is still far from being achieved. To highlight the difficulties in the standardisation of metagenomics methods, we thoroughly examined each step of the analysis of the human gut microbiome. We tested the DNA isolation procedure, preparation of NGS libraries for next-generation sequencing, and bioinformatics analysis, aimed at identifying microbial taxa. We showed that the homogenisation time is the leading factor impacting sample diversity, with the recommendation for a shorter homogenisation time (10 min). Ten minutes of homogenisation allows for better reflection of the bacteria gram-positive/gram-negative ratio, and the obtained results are the least heterogenous in terms of beta-diversity of samples microbial composition. Besides increasing the homogenisation time, we observed further potential impact of the library preparation kit on the gut microbiome profiling. Moreover, our analysis revealed that the choice of the library preparation kit influences the reproducibility of the results, which is an important factor that has to be taken into account in every experiment. In this study, a tagmentation-based kit allowed for obtaining the most reproducible results. We also considered the choice of the computational tool for determining the composition of intestinal microbiota, with Kraken2/Bracken pipeline outperforming MetaPhlAn2 in our in silico experiments. The design of an experiment and a detailed establishment of an experimental protocol may have a serious impact on determining the taxonomic profile of the intestinal microbiome community. Results of our experiment can be helpful for a wide range of studies that aim to better understand the role of the gut microbiome, as well as for clinical purposes.

Profile of Basal Cell Carcinoma Mutations and Copy Number Alterations - Focus on Gene-Associated Noncoding Variants.

Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5'UTRs, 3'UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, ~50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3'UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5'UTR, and CHCHD2 frequently showed mutations in the 5'UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified a frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general.

At-C-RNA database, a one-stop source for information on circRNAs in Arabidopsis thaliana in a unified format.

Circular RNAs (circRNAs) are a large class of noncoding RNAs with functions that, in most cases, remain unknown. Recent genome-wide analysis of circRNAs using RNA-Seq has revealed that circRNAs are abundant and some of them conserved in plants. Furthermore, it has been shown that the expression of circRNAs in plants is regulated in a tissue-specific manner. Arabidopsis thaliana circular RNA database is a new resource designed to integrate and standardize the data available for circRNAs in a model plant A. thaliana, which is currently the best-characterized plant in terms of circRNAs. The resource integrates all applicable publicly available RNA-seq datasets. These datasets were subjected to extensive reanalysis and curation, yielding results in a unified format. Moreover, all data were normalized according to our optimized approach developed for circRNA identification in plants. As a result, the database accommodates circRNAs identified across organs and seedlings of wild-type A. thaliana and its single-gene knockout mutants for genes related to splicing. The database provides free access to unified data and search functionalities, thus enabling comparative analyses of A. thaliana circRNAs between organs, variants and studies for the first time. Database URLhttps://plantcircrna.ibch.poznan.pl/.

Urban wastewater as a conduit for pathogenic Gram-positive bacteria and genes encoding resistance to ß-lactams and glycopeptides.

The emergence and spread of clinical pathogens, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment pose a direct threat to human and animal health worldwide. In this study, we analyzed qualitatively and quantitatively urban sewage resistome for the occurrence of genes encoding resistance to ß-lactams and glycopeptides in the genomes of culturable bacteria, as well as in the wastewater metagenome of the Central Wastewater Treatment Plant in Kozieglowy (Poland). Moreover, we estimated the presence of pathogenic Gram-positive bacteria in wastewater based on analysis of species-specific virulence genes in the wastewater metagenome. The results show that the final effluent contains alarm pathogens with particularly dangerous mechanisms of antibiotic resistance, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). We also noticed that during the wastewater treatment, there is an increase in the frequency of MRSA and VRE. Furthermore, the results prove the effective removal of vanA, but at the same time show that wastewater treatment increases the relative abundance of mecA and virulence genes (groES and sec), indicating the presence of clinical pathogens E. faecalis and S. aureus in the effluent released to surface waters. We also observed an increase in the relative abundance of mecA and vanA genes already in the aeration tank, which suggests accumulation of contaminants affecting enhanced selection and HGT processes in the activated sludge. Moreover, we found a relation between the taxonomic composition and the copy number of ARGs as well as the presence of pathogens at various stages of wastewater treatment. The presence of clinically relevant pathogens, ARB, including multi-resistant bacteria, and ARGs in the effluent indicates that wastewater treatment plant play a key role in the existence of pathogens and antimicrobial resistance spreading pathway in the environment and human communities, which is a direct threat to public health and environmental protection.

Expression Landscape of circRNAs in Arabidopsis thaliana Seedlings and Adult Tissues.

RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Arabidopsis thaliana cbp80, c2h2, and flk Knockout Mutants Accumulate Increased Amounts of Circular RNAs.

Circular RNAs (circRNAs) are the products of the non-canonical splicing of pre-mRNAs. In contrast to humans and animals, our knowledge of the biogenesis and function of circRNAs in plants is very scarce. To identify proteins involved in plant circRNA generation, we characterized the transcriptomes of 18 Arabidopsis thaliana knockout mutants for genes related to splicing. The vast majority (>90%) of circRNAs were formed in more than one variant; only a small fraction of circRNAs was mutant-specific. Five times more circRNA types were identified in cbp80 and three times more in c2h2 mutants than in the wild-type. We also discovered that in cbp80, c2h2 and flk mutants, the accumulation of circRNAs was significantly increased. The increased accumulation of circular transcripts was not accompanied by corresponding changes in the accumulation of linear transcripts. Our results indicate that one of the roles of CBP80, C2H2 and FLK in splicing is to ensure the proper order of the exons. In the absence of one of the above-mentioned factors, the process might be altered, leading to the production of circular transcripts. This suggests that the transition toward circRNA production can be triggered by factors sequestering these proteins. Consequently, the expression of linear transcripts might be regulated through circRNA production.

Analysis of oral microbiome from fossil human remains revealed the significant differences in virulence factors of modern and ancient Tannerella forsythia.

BACKGROUND: Recent advances in the next-generation sequencing (NGS) allowed the metagenomic analyses of DNA from many different environments and sources, including thousands of years old skeletal remains. It has been shown that most of the DNA extracted from ancient samples is microbial. There are several reports demonstrating that the considerable fraction of extracted DNA belonged to the bacteria accompanying the studied individuals before their death. RESULTS: In this study we scanned 344 microbiomes from 1000- and 2000- year-old human teeth. The datasets originated from our previous studies on human ancient DNA (aDNA) and on microbial DNA accompanying human remains. We previously noticed that in many samples infection-related species have been identified, among them Tannerella forsythia, one of the most prevalent oral human pathogens. Samples containing sufficient amount of T. forsythia aDNA for a complete genome assembly were selected for thorough analyses. We confirmed that the T. forsythia-containing samples have higher amounts of the periodontitis-associated species than the control samples. Despites, other pathogens-derived aDNA was found in the tested samples it was too fragmented and damaged to allow any reasonable reconstruction of these bacteria genomes. The anthropological examination of ancient skulls from which the T. forsythia-containing samples were obtained revealed the pathogenic alveolar bone loss in tooth areas characteristic for advanced periodontitis. Finally, we analyzed the genetic material of ancient T. forsythia strains. As a result, we assembled four ancient T. forsythia genomes - one 2000- and three 1000- year-old. Their comparison with contemporary T. forsythia genomes revealed a lower genetic diversity within the four ancient strains than within contemporary strains. We also investigated the genes of T. forsythia virulence factors and found that several of them (KLIKK protease and bspA genes) differ significantly between ancient and modern bacteria. CONCLUSIONS: In summary, we showed that NGS screening of the ancient human microbiome is a valid approach for the identification of disease-associated microbes. Following this protocol, we provided a new set of information on the emergence, evolution and virulence factors of T. forsythia, the member of the oral dysbiotic microbiome.

Metagenomic analysis of ß-lactamase and carbapenemase genes in the wastewater resistome.

The emergence and spread of resistance to antibiotics among bacteria is the most serious global threat to public health in recent and coming decades. In this study, we characterized qualitatively and quantitatively ß-lactamase and carbapenemase genes in the wastewater resistome of Central Wastewater Treatment Plant in Kozieglowy, Poland. The research concerns determination of the frequency of genes conferring resistance to ß-lactam and carbapenem antibiotics in the genomes of culturable bacteria, as well as in the wastewater metagenome at three stages of treatment: raw sewage, aeration tank, and final effluent. In the final effluent we found bacteria with genes that pose the greatest threat to public health, including genes of extended spectrum ß-lactamases - blaCTX-M, carbapenemases - blaNDM, blaVIM, blaGES, blaOXA-48, and showed that during the wastewater treatment their frequency increased. Moreover, the wastewater treatment process leads to significant increase in the relative abundance of blaTEM and blaGES genes and tend to increase the relative abundance of blaCTX-M, blaSHV and blaOXA-48 genes in the effluent metagenome. The biodiversity of bacterial populations increased during the wastewater treatment and there was a correlation between the change in the composition of bacterial populations and the variation of relative abundance of ß-lactamase and carbapenemase genes. PCR-based quantitative metagenomic analysis combined with analyses based on culture methods provided significant information on the routes of ARBs and ARGs spread through WWTP. The limited effectiveness of wastewater treatment processes in the elimination of antibiotic-resistant bacteria and resistance genes impose the need to develop an effective strategy and implement additional methods of wastewater disinfection, in order to limit the increase and the spread of antibiotic resistance in the environment.

Global Increase in Circular RNA Levels in Myotonic Dystrophy.

Splicing aberrations induced as a consequence of the sequestration of muscleblind-like splicing factors on the dystrophia myotonica protein kinase transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As muscleblind-like factors may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected 20 well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1, and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.

A mosaic genetic structure of the human population living in the South Baltic region during the Iron Age.

Despite the increase in our knowledge about the factors that shaped the genetic structure of the human population in Europe, the demographic processes that occurred during and after the Early Bronze Age (EBA) in Central-East Europe remain unclear. To fill the gap, we isolated and sequenced DNAs of 60 individuals from Kowalewko, a bi-ritual cemetery of the Iron Age (IA) Wielbark culture, located between the Oder and Vistula rivers (Kow-OVIA population). The collected data revealed high genetic diversity of Kow-OVIA, suggesting that it was not a small isolated population. Analyses of mtDNA haplogroup frequencies and genetic distances performed for Kow-OVIA and other ancient European populations showed that Kow-OVIA was most closely linked to the Jutland Iron Age (JIA) population. However, the relationship of both populations to the preceding Late Neolithic (LN) and EBA populations were different. We found that this phenomenon is most likely the consequence of the distinct genetic history observed for Kow-OVIA women and men. Females were related to the Early-Middle Neolithic farmers, whereas males were related to JIA and LN Bell Beakers. In general, our findings disclose the mechanisms that could underlie the formation of the local genetic substructures in the South Baltic region during the IA.

Small RNA fragments derived from multiple RNA classes - the missing element of multi-omics characteristics of the hepatitis C virus cell culture model.

BACKGROUND: A pool of small RNA fragments (RFs) derived from diverse cellular RNAs has recently emerged as a rich source of functionally relevant molecules. Although their formation and accumulation has been connected to various stress conditions, the knowledge on RFs produced upon viral infections is very limited. Here, we applied the next generation sequencing (NGS) to characterize RFs generated in the hepatitis C virus (HCV) cell culture model (HCV-permissive Huh-7.5 cell line). RESULTS: We found that both infected and non-infected cells contained a wide spectrum of RFs derived from virtually all RNA classes. A significant fraction of identified RFs accumulated to similar levels as miRNAs. Our analysis, focused on RFs originating from constitutively expressed non-coding RNAs, revealed three major patterns of parental RNA cleavage. We found that HCV infection induced significant changes in the accumulation of low copy number RFs, while subtly altered the levels of high copy number ones. Finally, the candidate RFs potentially relevant for host-virus interactions were identified. CONCLUSIONS: Our results indicate that RFs should be considered an important component of the Huh-7.5 transcriptome and suggest that the main factors influencing the RF biogenesis are the RNA structure and RNA protection by interacting proteins. The data presented here significantly complement the existing transcriptomic, miRnomic, proteomic and metabolomic characteristics of the HCV cell culture model.

Comprehensive analysis of microorganisms accompanying human archaeological remains.

Metagenome analysis has become a common source of information about microbial communities that occupy a wide range of niches, including archaeological specimens. It has been shown that the vast majority of DNA extracted from ancient samples come from bacteria (presumably modern contaminants). However, characterization of microbial DNA accompanying human remains has never been done systematically for a wide range of different samples. We used metagenomic approaches to perform comparative analyses of microorganism communities present in 161 archaeological human remains. DNA samples were isolated from the teeth of human skeletons dated from 100 AD to 1200 AD. The skeletons were collected from 7 archaeological sites in Central Europe and stored under different conditions. The majority of identified microbes were ubiquitous environmental bacteria that most likely contaminated the host remains not long ago. We observed that the composition of microbial communities was sample-specific and not correlated with its temporal or geographical origin. Additionally, traces of bacteria and archaea typical for human oral/gut flora, as well as potential pathogens, were identified in two-thirds of the samples. The genetic material of human-related species, in contrast to the environmental species that accounted for the majority of identified bacteria, displayed DNA damage patterns comparable with endogenous human ancient DNA, which suggested that these microbes might have accompanied the individual before death. Our study showed that the microbiome observed in an individual sample is not reliant on the method or duration of sample storage. Moreover, shallow sequencing of DNA extracted from ancient specimens and subsequent bioinformatics analysis allowed both the identification of ancient microbial species, including potential pathogens, and their differentiation from contemporary species that colonized human remains more recently.

Computational methods for prediction of RNA interactions with metal ions and small organic ligands.

In the recent years, it has become clear that a wide range of regulatory functions in bacteria are performed by riboswitches--regions of mRNA that change their structure upon external stimuli. Riboswitches are therefore attractive targets for drug design, molecular engineering, and fundamental research on regulatory circuitry of living cells. Several mechanisms are known for riboswitches controlling gene expression, but most of them perform their roles by ligand binding. As with other macromolecules, knowledge of the 3D structure of riboswitches is crucial for the understanding of their function. The development of experimental methods allowed for investigation of RNA structure and its complexes with ligands (which are either riboswitches' substrates or inhibitors) and metal cations (which stabilize the structure and are also known to be riboswitches' inhibitors). The experimental probing of different states of riboswitches is however time consuming, costly, and difficult to resolve without theoretical support. The natural consequence is the use of computational methods at least for initial research, such as the prediction of putative binding sites of ligands or metal ions. Here, we present a review on such methods, with a special focus on knowledge-based methods developed in our laboratory: LigandRNA--a scoring function for the prediction of RNA-small molecule interactions and MetalionRNA--a predictor of metal ions-binding sites in RNA structures. Both programs are available free of charge as a Web servers, LigandRNA at http://ligandrna.genesilico.pl and MetalionRNA at http://metalionrna.genesilico.pl/.

HarmonyDOCK: the structural analysis of poses in protein-ligand docking.

Molecular docking is a widely used method for lead optimization. However, docking tools often fail to predict how a ligand (the smaller molecule, such as a substrate or drug candidate) binds to a receptor (the accepting part of a protein). We present here the HarmonyDOCK, a novel method for assessing the docking software accuracy, and creating the scoring function which would determine consensus protein-ligand pose among those generated by available docking programs. Conformations for few hundred protein-ligand complexes with known three-dimensional structure were predicted on a benchmark set by set of different docking programs. On the basis of the derived ranking, the point of reference and the lower score limit were determined for subsequent investigations. The focus of the methodology is on the top-ranked poses, with the assumption being that the conformation of the docked molecules is the most accurate. We found out that some docking programs perform considerably better than the others, yet in all cases the proper selection of decoys, namely HarmonyDOCK, is needed for successful docking procedure.

LigandRNA: computational predictor of RNA-ligand interactions.

RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.