5'-Isoforms of miR-1246 Have Distinct Targets and Stronger Functional Impact Compared with Canonical miR-1246 in Colorectal Cancer Cells In Vitro.

Colorectal cancer (CRC) is a multifactorial disease involving genetic and epigenetic factors, such as miRNAs. Sequencing-based studies have revealed that miRNAs have many isoforms (isomiRs) with modifications at the 3'- and 5'-ends or in the middle, resulting in distinct targetomes and, consequently, functions. In the present study, we aimed to evaluate the putative targets and functional role of miR-1246 and its two 5'-isoforms (ISO-miR-1246_a and ISO-miR-1246_G) in vitro. Commercial Caco-2 cells of CRC origin were analyzed for the expression of WT-miR-1246 and its 5'-isoforms using small RNA sequencing data, and the overabundance of the two miR-1246 isoforms was determined in cells. The transcriptome analysis of Caco-2 cells transfected with WT-miR-1246, ISO-miR-1246_G, and ISO-miR-1246_a indicated the minor overlap of the targetomes between the studied miRNA isoforms. Consequently, an enrichment analysis showed the involvement of the potential targets of the miR-1246 isoforms in distinct signaling pathways. Cancer-related pathways were predominantly more enriched in dysregulated genes in ISO-miR-1246_G and ISO-miR-1246_a, whereas cell cycle pathways were more enriched in WT-miR-1246. The functional analysis of WT-miR-1246 and its two 5'-isoforms revealed that the inhibition of any of these molecules had a tumor-suppressive role (reduced cell viability and migration and promotion of early cell apoptosis) in CRC cells. However, the 5'-isoforms had a stronger effect on viability compared with WT-miR-1246. To conclude, this research shows that WT-miR-1246 and its two 5'-isoforms have different targetomes and are involved in distinct signaling pathways but collectively play an important role in CRC pathogenesis.

Determining the International Spread of B.1.1.523 SARS-CoV-2 Lineage with a Set of Mutations Highly Associated with Reduced Immune Neutralization.

Here, we report the emergence of the variant lineage B.1.1.523 that contains a set of mutations including 156_158del, E484K and S494P in the spike protein. E484K and S494P are known to significantly reduce SARS-CoV-2 neutralization by convalescent and vaccinated sera and are considered as mutations of concern. Lineage B.1.1.523 presumably originated in the Russian Federation and spread across European countries with the peak of transmission in April-May 2021. The B.1.1.523 lineage has now been reported from 31 countries. In this article, we analyze the possible origin of this mutation subset and its immune response using in silico methods.

SARS-CoV-2: Two Years in the Pandemic: What Have We Observed from Genome Sequencing Results in Lithuania?

SARS-CoV-2 has spread vastly throughout the word. In this study, we focus on the patterns of spread in Lithuania. By analysing the genetically sequenced data of different lineages and their first appearances, we were able to compare the dynamics of spreading of the lineages and recognize the main possible cause. The impact of emigration patterns and international travel on the variety of lineages was also assessed. Results showed different patterns of spread, and while a vast variety of different lineages were brought in by international travel, many of the viral outbreaks were caused by local lineages. It can be concluded that international travel had the most impact on the spread of SARS-CoV-2.

Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions.

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers.

High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts.

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1-V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

MuA-based Molecular Indexing for Rare Mutation Detection by Next-Generation Sequencing.

Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases. Here we demonstrate the remarkable tolerance of MuA transposase to the presence of multiple replacements in transposon sequence, and explore this unique feature to engineer the MuA transposome complex with randomised nucleotides in 12 transposon positions, which can be introduced as a barcode into the target molecule after transposition event. We applied the approach of Unique MuA-based Molecular Indexing (UMAMI) to assess the power of rare mutation detection by shortgun sequencing on the Illumina platform. Our results show that UMAMI allows detection of rare mutations readily and reliably, and in this paper we report error rate values for the number of thermophilic DNA polymerases measured by using UMAMI.

Exploring Viral Diversity in a Gypsum Karst Lake Ecosystem Using Targeted Single-Cell Genomics.

Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus-host interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.

Evolutionary stasis of a deep subsurface microbial lineage.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP's, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

3D Cell Culture-Based Global miRNA Expression Analysis Reveals miR-142-5p as a Theranostic Biomarker of Rectal Cancer Following Neoadjuvant Long-Course Treatment.

Altered expression of miRNAs in tumor tissue encourages the translation of this specific molecular pattern into clinical practice. However, the establishment of a selective biomarker signature for many tumor types remains an inextricable challenge. For this purpose, a preclinical experimental design, which could maintain a fast and sensitive discovery of potential biomarkers, is in demand. The present study suggests that the approach of 3D cell cultures as a preclinical cancer model that is characterized to mimic a natural tumor environment maintained in solid tumors could successfully be employed for the biomarker discovery and validation. Subsequently, in this study, we investigated an environment-dependent miRNA expression changes in colorectal adenocarcinoma DLD1 and HT29 cell lines using next-generation sequencing (NGS) technology. We detected a subset of 16 miRNAs differentially expressed in both cell lines cultivated in multicellular spheroids compared to expression levels in cells grown in 2D. Furthermore, results of in silico miRNA target analysis showed that miRNAs, which were differentially expressed in both cell lines grown in MCS, are involved in the regulation of molecular mechanisms implicated in cell adhesion, cell-ECM interaction, and gap junction pathways. In addition, integrins and platelet-derived growth factor receptors were determined to be the most significant target genes of deregulated miRNAs, which was concordant with the environment-dependent gene expression changes validated by RT-qPCR. Our results revealed that 3D microenvironment-dependent deregulation of miRNA expression in CRC cells potentially triggers essential molecular mechanisms predominantly including the regulation of cell adhesion, cell-cell, and cell-ECM interactions important in CRC initiation and development. Finally, we demonstrated increased levels of selected miR-142-5p in rectum tumor tissue samples after neoadjuvant long course treatment compared to miR-142-5p expression levels in tumor biopsy samples collected before the therapy. Remarkably, the elevation of miR-142-5p expression remained in tumor samples compared to adjacent normal rectum tissue as well. Therefore, the current study provides valuable insights into the molecular miRNA machinery of CRC and proposes a potential miRNA signature for the assessment of CRC in further clinical research.

Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions.

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria.IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique.

Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification. To be able to select for mezophilic enzymes we have employed multiple freezing-thawing cycles of emulsion as a bacterial cell wall disruption step instead of high temperature incubation. Subsequently WGA like plasmid DNA amplification could be performed by phi29 DNA polymerase applying different selection pressure conditions (temperature, buffer composition, modified dNTP, time, etc.). In our case the library of random phi29 DNA polymerase mutants was subjected to seven selection rounds of isothermal CSR (iCSR). After the selection polymerase variant containing the most frequent mutations was constructed and characterized. The mutant phi29 DNA polymerase can perform WGA at elevated temperatures (40-42°C), generate two to five times more of DNA amplification products, and has significantly increased half-life at 30 and 40°C, both in the presence or the absence of DNA substrate.

Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix.

In our previous work we showed that DNaseI-like protein from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix retained its activity at salt concentrations as high as 4 M NaCl and the key factor allowing this was the C-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs. The further investigations revealed that this domain originated from proteins related to bacterial competence ComEA/ComE proteins. It is likely that in the course of evolution the DNA-binding domain from these proteins was fused to a metallo-ß-lactamase superfamily domain. Very likely such domain organization having proteins subsequently "donated" the DNA-binding domain to bacterial DNases. In this study we have mimicked this evolutionary step by fusing bovine DNaseI and DNA-binding domains. We have created two fusions: one harboring the DNA-binding domain of DNaseI-like protein from Thioalkalivibrio sp. K90mix and the second one harboring the DNA-binding domain of bacterial competence protein ComEA from Bacillus subtilis. Both domains enhanced salt tolerance of DNaseI, albeit to different extent. Molecular modeling revealed the essential differences between their interaction with DNA shedding some light on the differences in salt tolerance. In this study we have enhanced salt tolerance of bovine DNaseI; thus, we successfully mimicked the Nature's evolutionary engineering that created the extremely halotolerant bacterial DNase. We have demonstrated that the newly engineered DNaseI variants can be successfully used in applications where activity of the wild type bovine DNaseI is impeded by buffers used.

Domain organization of DNase from Thioalkalivibrio sp. provides insights into retention of activity in high salt environments.

Our study indicates that DNA binding domains are common in many halophilic or halotolerant bacterial DNases and they are potential activators of enzymatic activity at high ionic strength. Usually, proteins adapt to high ionic strength by increasing the number of negatively charged residues on the surface. However, in DNases such adaptation would hinder the binding to negatively charged DNA, a step critical for catalysis. In our study we demonstrate how evolution has solved this dilemma by engaging the DNA binding domain. We propose a mechanism, which enables the enzyme activity at salt concentrations as high as 4 M of sodium chloride, based on collected experimental data and domain structure analysis of a secreted bacterial DNase from the extremely halotolerant bacterium Thioalkalivibrio sp. K90mix. The enzyme harbors two domains: an N-terminal domain, that exhibits DNase activity, and a C-terminal domain, comprising a duplicate DNA binding helix-hairpin-helix motif. Here we present experimental data demonstrating that the C-terminal domain is responsible for the enzyme's resistance to high ionic strength.

Draft Genome Sequence of the Cyanobacterium Aphanizomenon flos-aquae Strain 2012/KM1/D3, Isolated from the Curonian Lagoon (Baltic Sea).

We report here the de novo genome assembly of a cyanobacterium, Aphanizomenon flos-aquae strain 2012/KM1/D3, a harmful bloom-forming species in temperate aquatic ecosystems. The genome is 5.7 Mb with a G+C content of 38.2%, and it is enriched mostly with genes involved in amino acid and carbohydrate metabolism.

Analysis of DNA Methylation and Hydroxymethylation in the Genome of Crustacean Daphnia pulex.

The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants.

In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme).