Genetic history of East-Central Europe in the first millennium CE.

BACKGROUND: The appearance of Slavs in East-Central Europe has been the subject of an over 200-year debate driven by two conflicting hypotheses. The first assumes that Slavs came to the territory of contemporary Poland no earlier than the sixth century CE; the second postulates that they already inhabited this region in the Iron Age (IA). Testing either hypothesis is not trivial given that cremation of the dead was the prevailing custom in Central Europe from the late Bronze Age until the Middle Ages (MA). RESULTS: To address this problem, we determined the genetic makeup of representatives of the IA Wielbark- and MA Slav-associated cultures from the territory of present-day Poland. The study involved 474 individuals buried in 27 cemeteries. For 197 of them, genome-wide data were obtained. We found close genetic affinities between the IA Wielbark culture-associated individuals and contemporary to them and older northern European populations. Further, we observed that the IA individuals had genetic components which were indispensable to model the MA population. CONCLUSIONS: The collected data suggest that the Wielbark culture-associated IA population was formed by immigrants from the north who entered the region of contemporary Poland most likely at the beginning of the first millennium CE and mixed with autochthons. The presented results are in line with the hypothesis that assumes the genetic continuation between IA and MA periods in East-Central Europe.

Human AGEs: an interactive spatio-temporal visualization and database of human archeogenomics.

Archeogenomics is a rapidly growing interdisciplinary research field driven by the development of techniques that enable the acquisition and analysis of ancient DNA (aDNA). Recent advances in aDNA studies have contributed significantly to increasing our understanding of the natural history of humans. One of the most significant challenges facing archeogenomics is the integration of highly heterogeneous genomic, archeological, and anthropological data and their comprehensive analysis, considering changes that occur in time and space. Only this complex approach can explain the relationship between past populations in the context of migration or cultural development. To address these challenges, we developed a Human AGEs web server. It focuses on creating comprehensive spatiotemporal visualizations of genomic, archeogenomic, and archeological information, which can be provided by the user or loaded from a graph database. The interactive map application at the center of Human AGEs can display multiple layers of data in various forms, such as bubble charts, pie charts, heatmaps, or tag clouds. These visualizations can be modified using various clustering, filtering, and styling options, and the map state can be exported to a high-resolution image or saved as a session file for later use. Human AGEs, along with their tutorial, are accessible at https://archeogenomics.eu/.

Dimensionality reduction by UMAP for visualizing and aiding in classification of imaging flow cytometry data.

Recent advances in imaging flow cytometry (IFC) have revolutionized high-throughput multiparameter analyses at single-cell resolution. Although enabling the discovery of population heterogeneities and the detection of rare events, IFC generates hyperdimensional datasets that demand innovative analytical approaches. Current methods work in a supervised manner, utilize only limited information content, or require large annotated reference datasets. Dimensionality reduction algorithms, including uniform manifold approximation and projection (UMAP), have been successfully applied to analyze the large number of parameters generated in various high-throughput techniques. Here, we apply a workflow incorporating UMAP to analyze different IFC datasets. We demonstrate that it out-competes other popular dimensionality reduction methods in speed and accuracy. Moreover, it enables fast visualization, clustering, and tagging of unannotated objects in large-scale experiments. We anticipate that our workflow will be a robust method to address complex IFC datasets, either alone or as an upstream addition to the deep learning approaches.

From Alpha to Delta-Genetic Epidemiology of SARS-CoV-2 (hCoV-19) in Southern Poland.

In Poland, the first case of SARS-CoV-2 infection was confirmed in March 2020. Since then, many circulating virus lineages fueled rapid pandemic waves which inflicted a severe burden on the Polish healthcare system. Some of these lineages were associated with increased transmissibility and immune escape. Mutations in the viral spike protein, which is responsible for host cell recognition and serves as the primary target for neutralizing antibodies, are of particular importance. We investigated the molecular epidemiology of the SARS-CoV-2 clades circulating in Southern Poland from February 2021 to August 2021. The 921 whole-genome sequences were used for variant identification, spike mutation, and phylogenetic analyses. The Pango B.1.1.7 was the dominant variant (n = 730, 89.68%) from March 2021 to July 2021. In July 2021, the B.1.1.7 was displaced by the B.1.617.2 lineage with 66.66% in July 2021 and 92.3% in August 2021 frequencies, respectively. Moreover, our results were compared with the sequencing available on the GISAID platform for other regions of Poland, the Czech Republic, and Slovakia. The analysis showed that the dominant variant in the analyzed period was B.1.1.7 in all countries and Southern Poland (Silesia). Interestingly, B.1.1.7 was replaced by B.1.617.2 earlier in Southern Poland than in the rest of the country. Moreover, in the Czech Republic and Slovakia, AY lineages were predominant at that time, contrary to the Silesia region.

Polymorphism rs368234815 of interferon lambda 4 gene and spontaneous clearance of hepatitis C virus in haemodialysis patients: a case-control study.

BACKGROUND: In non-uremic subjects, IFNL4 rs368234815 predicts HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in dialysis subjects. We also evaluated an association of rs368234815 with patients` survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms. METHODS: Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. We used the Kaplan-Meier method with the log-rank test for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software. RESULTS: The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ΔG allele carriers (2.63, 1.38-5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45-5.43, 0.002 for rs12980275 and 2.44, 1.27-4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69-24.2, 0.003). Survival analysis suggested an association of the ΔG allele with mortality due to neoplasms (log-rank P = 0.005). The rs368234815 ∆G allele caused TFBS removal for PLAGL1. CONCLUSIONS: In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, identifying the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that we observe in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harbouring the rs368234815 ∆G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.

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.

Is the T-G-CT-G SNRNP70 haplotype another proof that mixed connective tissue disease is distinct from systemic lupus erythematosus and systemic sclerosis? A novel gene variant in SNRNP70 gene.

OBJECTIVES: U1-70K, encoded by the SNRNP70 gene, is a key early immunogen in connective tissue disease. The aim of the study was the genetic analysis of the SNRNP70 gene in mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) patients. METHODS: SNRNP70 genetic variants were detected using 3730 DNA Analyzer. SNRNP70 rs560811128 G/A (c.476-252 G/A), rs78616533delCT (c.475+130_475+131delCT) and rs117167710 T/C (c.393+326 T/C) variants were genotyped using the technique of sequence-specific hybridisation probe binding assays. SNRNP70 393_47 G/A mutation was detected using TaqMan SNP genotyping assay. RESULTS: We found one novel c.393+47G>A and three, c.476-252 G/A, c.475+130_475+131delCT and c.393+326 T/C, previously recorded variants. The present study revealed that T-G-CT-G haplotype demonstrated significantly higher frequencies in MCTD patients than in SLE and SSc patients. In MCTD patients c.475+130_475+131delCT distribution of genotype was gender-dependent and showed association with thrombo-/leukocytopenia. Mutation at position c.476-252G>A was predicted to possibly have an impact on splicing of the SNRNP70 transcript and it was present only in one MCTD patient. CONCLUSIONS: Our results demonstrated that the T-G-CT-G SNRNP70 haplotype is another proof that MCTD may be distinct from SLE and SSc. The novel c.476-252G>A mutation in SNRNP70 gene created a new acceptor splice site and may potentially alert of splicing of the SNRNP70 transcript.

Functional characterization of RNA fragments using high-throughput interactome screening.

Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules such as miRNAs or siRNAs, also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interactions of seven RFs originating from tRNA, snoRNA and snRNA. By integrating our results with publicly available data on physical protein-protein interactions, we constructed an RF interactome network. We determined that the RF interactome comprises proteins generally different from those that interact with their parental full length RNAs. Proteins captured by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carboxylic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in recently revealed RNA-protein regulatory networks. SIGNIFICANCE: In the recent decade it has become evident that RNAs with well-known functions (for example tRNA, snoRNA or rRNA) can be cleaved to yield short fragments, whose role in cells remains only partially characterized. At the same time, unconventional interactions between mRNA and proteins without RNA-binding domains have been demonstrated, revealing novel layers of possible RNA-mediated regulation. Considering the above, we hypothesized that RNA fragments (RFs) can be endogenous aptamer-like molecules that unconventionally interact with proteins. In this study we identified protein partners of seven selected RFs. We found that RFs bind different set of proteins than their parental full length RNAs and identified proteins differentially bound by the particular RFs. These observations suggest biological relevance of the discovered interactions. Our data provide a novel perspective on the significance of RFs and point to this pool of molecules as to a rich collection of potential components of the recently discovered RNA-protein regulatory networks.

ENHO, RXRA, and LXRA polymorphisms and dyslipidaemia, related comorbidities and survival in haemodialysis patients.

BACKGROUND: The energy homeostasis-associated gene (ENHO), retinoid X receptor alpha gene (RXRA), and liver X receptor alpha gene (LXRA) are involved in adipogenic/lipogenic regulation. We investigated whether single-nucleotide polymorphisms in these genes (ENHO rs2281997, rs72735260; RXRA rs749759, rs10776909, rs10881578; LXRA rs2279238, rs7120118, rs11039155) are associated with dyslipidaemia, related comorbidities and survival of haemodialysis (HD) patients also tested for T-helper (Th) cell interleukin genes (IL). METHODS: The study was carried out in 873 HD patients. Dyslipidaemia was diagnosed by the recommendations of the Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines (2003); atherogenic dyslipidaemia was referred to if the TG/HDL cholesterol ratio was equal to or higher than 3.8. Genotyping of ENHO SNPs, LXRA SNPs, and IL12A rs568408 was carried out using HRM analysis. RXRA SNPs, IL12B rs3212227, and IL18 rs360719 were genotyped using PCR-RFLP analysis. The circulating adropin concentration was determined in 126 patients by enzyme-linked immunosorbent assay. Survival probability was analysed using the Kaplan-Meier method in 440 patients followed through 7.5 years. RESULTS: Dyslipidaemia by K/DOQI was diagnosed in 459 patients (91% revealed hyper-LDL- cholesterolaemia), atherogenic dyslipidaemia was diagnosed in 454 patients, and 231 patients were free of dyslipidaemia by both criteria. The variant allele (T) of ENHO rs2281997 was associated with the hyper-LDL cholesterolaemic pattern of dyslipidaemia by K/DOQI. The frequency of atherogenic dyslipidaemia was lower in T-allele bearers than in CC-genotype patients. The rs2281997 T allele was associated with lower cardiovascular mortality in HD patients showing atherogenic dyslipidaemia. ENHO, RXRA, and LXRA showed epistatic interactions in dyslipidaemia. Circulating adropin was lower in atherogenic dyslipidaemia than in non-atherogenic conditions. RXRA rs10776909 was associated with myocardial infarction. Bearers of LXRA rs2279238, rs7120118 or rs11039155 minor alleles showed higher mortality. ENHO SNP positions fell within the same DNase 1 hypersensitivity site expressed in the Th1 cell line. Epistatic interactions occurred between rs2281997 and Th1 IL SNPs (rs360719, rs568408). CONCLUSIONS: Atherogenic dyslipidaemia occurs in HD patients in whom ENHO encodes less adropin. ENHO, RXRA, and LXRA SNPs, separately or jointly, are associated with dyslipidaemia, myocardial infarction, and survival in HD patients. Differences in the availability of transcription binding sites may contribute to these associations.

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.

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.

Associations of the calcium-sensing receptor gene CASR rs7652589 SNP with nephrolithiasis and secondary hyperparathyroidism in haemodialysis patients.

Nephrolithiasis, secondary hyperparathyroidism (sHPT), and cardiovascular complications are associated with disturbances in Ca handling and contribute to morbidity/mortality during haemodialysis (HD). Calcimimetics, activators of the calcium-sensing receptor (CaSR), provide an effective means of reducing parathyroid hormone (PTH) secretion in sHPT. Polymorphism in CaSR gene (CASR) influences Ca-related parameters, however it was not shown in HD patients for CASR rs7652589. The minor allele at this polymorphism modifies the binding sites of transcription factors and CaSR expression. We hypothesized that CASR rs7652589 variants may also influence CaSR in end stage renal disease (ESRD). We aimed to determine the associations of rs7652589 with nephrolithiasis-related ESRD, Ca, P, ALP, PTH, response to treatment with cinacalcet, prevalence of coronary artery disease, and all-cause/cardiovascular mortality in HD patients (n = 1162). Healthy individuals (n = 918) were controls. This study shows that the A allele of rs7652589 is a risk allele for nephrolithiasis-related ESRD. The AA genotype is associated with more severe sHPT (higher Ca and PTH concentrations). The A allele is associated with reduced CaSR transcript level in peripheral blood mononuclear cells. According to computational analysis, potential binding sites for GLI3, AHR and TP53 are removed by the A allele, whereas binding sites for SOX18 and TP63 are created.