Thomas Hankeln

The high commercial value and the increasing demand for caviar make it susceptible to illegal trade and fraudulent activities, while mislabelling can occur both deliberately and unintentionally. Therefore, reliable methods are urgently needed to distinguish even closely related sturgeon species for the authentication of declared species in caviar products. In this study, we explore the effectiveness of ONT sequencing combined with nanopore adaptive sampling (NAS) to recover mitochondrial genomes from genomic DNA extracted from two caviar samples. NAS yielded similar percentages of mitochondrial reads as sequencing experiments without using the NAS option. However, NAS substantially increased the percentage of bases that could be mapped against a mitochondrial reference genome, demonstrating its effectiveness in enriching ONT sequencing data for mitochondrial sequences. The percentage of mapped mitochondrial reads varied between two samples in our sequencing experiments, ranging between 2.37% and 10.46%, which is exceptionally high compared to previous studies focusing on the recovery of mitochondrial genomes from genomic DNA. This increase may be attributed to the extraction of genomic DNA from individual fish eggs. To determine the taxonomic identities of the two caviar samples, a phylogenetic analysis was performed that included all available acipenseriform mitochondrial genomes from GenBank, as well as the newly recovered mitochondrial genomes. During this process, four problematic mitochondrial genomes obtained from GenBank were identified, which were characterized by either suggestively low sequence quality, chimeric sequence information, or potential misidentification. This underscores the need for reviewing sequencing data before database submission to avoid negative impacts on research and DNA-based species authentication.

Anthracyclines such as doxorubicin (DOX) remain central components of breast cancer (BC) chemotherapy, although their efficacy is frequently limited by drug resistance. Myoglobin (MB), an oxygen-binding heme protein expressed in breast tumors, has been implicated in the detoxification of DOX in cardiomyocytes, but its role in BC remains unclear. Using MB-expressing and MB-knockout (MBKO) MDA-MB-468 BC cells, we demonstrate that MB confers hypoxia-dependent resistance to DOX. Under hypoxia, MB-expressing cells exhibited reduced intracellular DOX-associated fluorescence, enhanced superoxide generation, and decreased sensitivity to DOX, findings consistent with altered redox cycling and oxidative processing of the drug. Re-expression of MB in MBKO cells restored resistance, whereas pharmacological modulation of MB function using carbon monoxide-releasing molecule-3 and tert-butoxycarbonyl-alanine reversed MB-dependent reductions in intracellular DOX accumulation. In contrast, aclarubicin, an anthracycline lacking the hydroquinone moiety required for efficient redox cycling, failed to reproduce MB-dependent effects. Analyses of four independent neoadjuvant BC cohorts further demonstrated that elevated MB expression was consistently associated with reduced probability of achieving pathological complete response following anthracycline-containing chemotherapy. Collectively, these findings identify MB as a previously unrecognized modulator of BC response to redox-active anthracyclines and support its potential utility as both a predictive biomarker and therapeutic target.

Androglobin (Adgb) is the most distinctive member of the globin superfamily. Its characteristic globin domain is permuted, interrupted by a calmodulin-binding motif, and embedded within a large multidomain protein of ∼1500 amino acids that also contains a calpain protease domain. Initially described as testis-specific in mammals, Adgb is also expressed in ciliated epithelia of the female reproductive tract, lung, and brain, and knockout studies reveal its pivotal role during spermatogenesis. To trace its evolutionary origin, we performed comprehensive phylogenetic analysis across diverse eukaryotic taxa. Adgb is present in all major flagellated eukaryotic lineages but absent from nonflagellated clades. Orthology analysis indicates Adgb has been maintained as a predominantly single-copy gene across >1 billion years of evolution—a pattern contrasting sharply with other globins that underwent repeated duplication and functional diversification. Analysis of publicly available transcriptomes from early-branching metazoans confirmed robust Adgb expression in ciliated cell types and provides evidence for its regulation by the ancient ciliogenic transcription factor cRFXa in the choanoflagellate Salpingoeca rosetta. RNA in situ hybridization validated these findings, and comparative analyses suggest that ancestral Adgb homologs lacked the permuted globin domain found in metazoans. Collectively, our results demonstrate that Adgb exemplifies a rare evolutionary trajectory where structural innovation (domain permutation and fusion) enabled functional specialization while being an integral part of the nonredundant ciliary machinery. Adgb thus illustrates how constraint and innovation combine to shape the long-term fate of a protein.

Background: All-Food-Sequencing (AFS) is a method for untargeted metagenomic analysis that allows for the detection and quantification of food ingredients. While this approach avoids some of the shortcomings of targeted PCR-based methods, its performance depends on sequencing technologies, taxonomic classification tools, and genomic reference databases. Results: AFS-MetaCache2 implements an improved reference database construction mechanism compared to prior approaches. To demonstrate the effectiveness to AFS, we sequenced sausages composed of mammalian and avian species using both short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms. While both approaches reliably detect the main components, our comparison shows that long-read sequencing is superior in terms of both quantification accuracy and false positive rates. The evaluation of representative metagenomic tools (Kraken2+Bracken, KrakenUniq, AFS-MetaCache1) demonstrates that AFS-MetaCache2 yields the best accuracy and fastest database build times, while reducing peak main memory consumption. It thus allows for efficient scaling to large reference genome sets. Conclusion: Our study suggests that deep sequencing of total genomic DNA from samples with heterogeneous taxon composition, using 3rd generation sequencing technology followed by metagenomic analysis with AFS-MetaCache2, is a valuable approach for bio-surveillance of food ingredients. Our software is available at https://github.com/muellan/metacache.

Within the last 800 million years, animals evolved a vast range of diversity of species exhibiting an enormous disparity of forms and lifestyles. The process involved an increase in complexity from life forms with few cell types to organisms with many hundreds of cell types. However, neither genome size nor number of protein-coding genes can explain these differences, and their biological basis remains elusive. Yet, recent studies suggest that the evolution of complexity is closely linked to the acquisition of a class of noncoding gene regulators called microRNAs. To test this hypothesis, we investigated the association between loss of organismal complexity and microRNAs in Syndermata, an invertebrate group including free-living wheel animals (Monogononta, Bdelloidea), epibiotic Seisonidea, and endoparasitic thorny-headed worms (Acanthocephala). Analyses of genomic, transcriptomic, and morphological data of altogether 25 syndermatan species revealed strong correlations of microRNA losses with reductions of protein-coding genes and morphological traits. The hierarchical pattern sums up to ∼85% loss of microRNAs and a ∼50% loss of conserved metazoan core genes (Benchmarking Universal Single-Copy Orthologs) on the lineage to thorny-headed worms. Extraordinarily reduced microRNA complements were confirmed by small RNA sequencing data. Endoparasitic Acanthocephala was additionally distinguished by the most morphological reductions of ancestral features, such as the digestive tract. Together, we observed that reductions of ∼400 protein-coding genes and 10 metazoan core genes tended to accompany the loss of single microRNA families. Furthermore, 4 microRNA families and 34 metazoan core genes appeared to be associated, on average, with the reduction of a single morphological trait.

In the subterranean rodent (Nanno)spalax galili, evolutionary adaptation to hypoxia is correlated with longevity and tumor resistance. Adapted gene-regulatory networks of Spalax might pinpoint strategies to maintain health in humans. Comparing liver, kidney and spleen transcriptome data from Spalax and rat at hypoxia and normoxia, we identified differentially expressed gene pathways common to multiple organs in both species. Body-wide interspecies differences affected processes like cell death, antioxidant defense, DNA repair, energy metabolism, immune response and angiogenesis, which may play a crucial role in Spalax’s adaptation to environmental hypoxia. In all organs, transcription of genes for genome stability maintenance and DNA repair was elevated in Spalax versus rat, accompanied by lower expression of aerobic energy metabolism and proinflammatory genes. These transcriptomic changes might account for the extraordinary lifespan of Spalax and its cancer resistance. The identified gene networks present candidates for further investigating the molecular basis underlying the complex Spalax phenotype.

Figure: Graphical abstract During the last 800 million years of evolution animals radiated into a vast range of diversity of species and disparity of forms and lifestyles. The process involved a near hierarchical increase in complexity from life forms with few cell types to organisms with many hundreds of cell-types. However, neither genome size nor number of protein-coding genes can explain these differences and their biological basis remains elusive. Yet, recent studies have suggested that the evolution of complexity is closely linked to the acquisition of a class of protein coding gene-regulators called microRNAs. In a regressive approach, to investigate the association between loss of organismal complexity and microRNAs, we here studied Syndermata, an invertebrate group including free-living rotifers (Monogononta, Bdelloidea), the epibiotic Seisonidea and the endoparasitic Acanthocephala. Genomic, transcriptomic and morphological characterization and comparisons across 25 syndermatan species revealed a strong correlation between loss of microRNAs, loss of protein-coding genes and decreasing morphological complexity. The near hierarchical loss extends to ∼85% loss of microRNAs and a ∼50% loss of BUSCO genes in the endoparasitic Acanthocephala, the most reduced group we studied. Together, the loss of ∼400 protein-coding genes and ∼10 metazoan core gene losses went along with one microRNA family loss. Furthermore, the loss of ∼4 microRNA families or ∼34 metazoan core genes associated with one lost morphological feature. These are the first quantitative insights into the regulatory impact of microRNAs on organismic complexity as a predictable consequence in regressive evolution of parasites.