Occurrence and subtyping of Blastocystis in coypus (Myocastor coypus) in China

Background Blastocystis is an anaerobic unicellular protist frequently detected in the gastrointestinal tracts of humans and animals worldwide. However, the prevalence and subtype distribution of Blastocystis in the coypu (Myocastor coypus) population have not been reported so far. The aim of this study was to determine the prevalence, genetic characteristics, and zoonotic potential of Blastocystis isolates detected in coypus in China. Results A total of 308 fecal samples were collected from coypus in seven regions across China and subsequently examined. Blastocystis was detected in 44 (14.3%) specimens by nested PCR amplification of the small subunit ribosomal rRNA (SSU rRNA) gene. Further DNA sequencing and phylogenetic analyses resulted in the identification of two zoonotic known subtypes, ST4 and ST5, and an unknown subtype. ST4 was the most predominant subtype observed in the samples. ST5 infections were only observed in three coypus. Factors that were associated with prevalence of Blastocystis included age, geographical region and subtype. Interestingly, this is the first report about a potentially novel subtype infecting coypus. Conclusions This is the first comprehensive report of Blastocystis in M. coypus across a wide geographic range of China. A moderate degree of genetic divergence was observed. The presence of zoonotic subtypes in farmed M. coypus suggests that these animals have the potential to transmit blastocystosis to both humans and domestic animals. These findings provide a better understanding of the genetic diversity of Blastocystis in rodents and contribute towards the establishment of efficient blastocystosis control strategies in the investigated areas. Graphical abstract

Regulatory roles of RNA modifications in breast cancer

Collectively referred to as the epitranscriptome, RNA modifications play important roles in gene expression control regulating relevant cellular processes. In the last few decades, growing numbers of RNA modifications have been identified not only in abundant ribosomal (rRNA) and transfer RNA (tRNA) but also in messenger RNA (mRNA). In addition, many writers, erasers and readers that dynamically regulate the chemical marks have also been characterized. Correct deposition of RNA modifications is prerequisite for cellular homeostasis, and its alteration results in aberrant transcriptional programs that dictate human disease, including breast cancer, the most frequent female malignancy, and the leading cause of cancer-related death in women. In this review, we emphasize the major RNA modifications that are present in tRNA, rRNA and mRNA. We have categorized breast cancer-associated chemical marks and summarize their contribution to breast tumorigenesis. In addition, we describe less abundant tRNA modifications with related pathways implicated in breast cancer. Finally, we discuss current limitations and perspectives on epitranscriptomics for use in therapeutic strategies against breast and other cancers.

Fossil-calibrated molecular phylogeny of atlantid heteropods (Gastropoda, Pterotracheoidea)

Background The aragonite shelled, planktonic gastropod family Atlantidae (shelled heteropods) is likely to be one of the first groups to be impacted by imminent ocean changes, including ocean warming and ocean acidification. With a fossil record spanning at least 100 Ma, atlantids have experienced and survived global-scale ocean changes and extinction events in the past. However, the diversification patterns and tempo of evolution in this family are largely unknown. Results Based on a concatenated maximum likelihood phylogeny of three genes (cytochrome c oxidase subunit 1 mitochondrial DNA, 28S and 18S ribosomal rRNA) we show that the three extant genera of the family Atlantidae, Atlanta, Protatlanta and Oxygyrus, form monophyletic groups. The genus Atlanta is split into two groups, one exhibiting smaller, well ornamented shells, and the other having larger, less ornamented shells. The fossil record, in combination with a fossil-calibrated phylogeny, suggests that large scale atlantid extinction was accompanied by considerable and rapid diversification over the last 25 Ma, potentially driven by vicariance events. Conclusions Now confronted with a rapidly changing modern ocean, the ability of atlantids to survive past global change crises gives some optimism that they may be able to persist through the Anthropocene.

Fossil-calibrated molecular phylogeny of atlantid heteropods (Gastropoda, Pterotracheoidea)

Background: The aragonite shelled, planktonic gastropod family Atlantidae (shelled heteropods) is likely to be one of the first groups to be impacted by imminent ocean changes, including ocean warming and ocean acidification. With a fossil record spanning at least 100 Million years (Ma), atlantids have experienced and survived global-scale ocean changes and extinction events in the past. However, the diversification patterns and tempo of evolution in this family are largely unknown. Results: Based on a concatenated maximum likelihood phylogeny of three genes (cytochrome c oxidase subunit 1 mitochondrial DNA, 28S and 18S ribosomal rRNA) we show that the three extant genera of the family Atlantidae, Atlanta, Protatlanta and Oxygyrus, form monophyletic groups. The genus Atlanta is split into two groups, one exhibiting smaller, well ornamented shells, and the other having larger, less ornamented shells. The fossil record, in combination with a fossil-calibrated phylogeny, suggests that large scale atlantid extinction was accompanied by considerable and rapid diversification over the last 25 Ma, potentially driven by vicariance events. Conclusions: Now confronted with a rapidly changing modern ocean, the ability of atlantids to survive past global change crises gives some optimism that they may be able to persist through the Anthropocene.

Fossil-calibrated molecular phylogeny of atlantid heteropods (Gastropoda, Pterotracheoidea)

The aragonite shelled, planktonic gastropod family Atlantidae (shelled heteropods) is likely to be one of the first groups to be impacted by imminent ocean changes, including ocean warming and ocean acidification. With a fossil record spanning at least 100 Million years (Ma), atlantids have experienced and survived global-scale ocean changes and extinction events in the past. However, the diversification patterns and tempo of evolution in this family are largely unknown. Based on a concatenated maximum likelihood phylogeny of three genes (cytochrome c oxidase subunit 1 mitochondrial DNA, 28S and 18S ribosomal rRNA) we show that the three extant genera of the family Atlantidae, Atlanta, Protatlanta and Oxygyrus, form monophyletic clades. The genus Atlanta is split into two groups, one exhibiting smaller, well ornamented shells, and the other having larger, less ornamented shells. The fossil record, in combination with a fossil-calibrated phylogeny suggest that large scale atlantid extinction was accompanied by considerable and rapid diversification over the last 25 Ma, potentially driven by vicariance events. Now confronted with a rapidly changing modern ocean, the ability of atlantids to survive past global change crises gives some optimism that they may be able to persist through the Anthropocene.

Metabarcoding data allow for reliable biomass estimates in the most abundant animals on earth

Microscopic organisms are the dominant and most diverse organisms on Earth. Nematodes, as part of this microscopic diversity, are by far the most abundant animals and their diversity is equally high. Molecular metabarcoding is often applied to study the diversity of microorganisms, but has yet to become the standard to determine nematode communities. As such, the information metabarcoding provides, such as in terms of species coverage, taxonomic resolution and especially if sequence reads can be linked to the abundance or biomass of nematodes in a sample, has yet to be determined. Here, we applied metabarcoding using three primer sets located within ribosomal rRNA gene regions to target assembled mock-communities consisting of 18 different nematode species that we established in 9 different compositions. We determined abundances and biomass of all species added to examine if relative sequence abundance or biomass can be linked to relative sequence reads. We found that nematode communities are not equally represented by the three different primer sets and we found that relative read abundances almost perfectly correlated positively with relative species biomass for two of the primer sets. This strong biomass-read number correlation suggests that metabarcoding reads can reveal biomass information even amongst more complex nematode communities as present in the environment and possibly can be transferred to better study other groups of organisms. This biomass-read link is of particular importance for more reliably assessing nutrient flow through food-webs, as well as adjusting biogeochemical models through user-friendly and easily obtainable metabarcoding data.

Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.

Nucleic Acid Cleavage with a Hyperthermophilic Cas9 from an Unculturable Ignavibacterium

CRISPR-Cas9 systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrated that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA (rRNA) without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. Taken together, IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.

Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments

ABSTRACTDNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well-established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable. Here we used Pacifc Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote ribosomal SSU and LSU rRNA genes and the ITS spacer region. We developed a long-read analysis pipeline that reduced error rates to levels 15 comparable to short-read platforms. Validation using fungal isolates and a mock community indicated that our pipeline detected 98% of chimeras de novo i.e., even in the absence of reference sequences. We recovered 947 OTUs from water and sediment samples in a natural lake, 848 of which could be classifed to phylum, 486 to family, 397 to genus and 330 to species. By 20 allowing for the simultaneous use of three global databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 25 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for the study of eukaryotic diversity more broadly.