Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

RATIONALE: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable. METHODS: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns. RESULTS: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families. CONCLUSIONS: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.

Genetic data from the extinct giant rat from Tenerife (Canary Islands) points to a recent divergence from mainland relatives.

Evolution of vertebrate endemics in oceanic islands follows a predictable pattern, known as the island rule, according to which gigantism arises in originally small-sized species and dwarfism in large ones. Species of extinct insular giant rodents are known from all over the world. In the Canary Islands, two examples of giant rats, †Canariomys bravoi and †Canariomys tamarani, endemic to Tenerife and Gran Canaria, respectively, disappeared soon after human settlement. The highly derived morphological features of these insular endemic rodents hamper the reconstruction of their evolutionary histories. We have retrieved partial nuclear and mitochondrial data from †C. bravoi and used this information to explore its evolutionary affinities. The resulting dated phylogeny confidently places †C. bravoi within the African grass rat clade (Arvicanthis niloticus). The estimated divergence time, 650 000 years ago (95% higher posterior densities: 373 000-944 000), points toward an island colonization during the Günz-Mindel interglacial stage. †Canariomys bravoi ancestors would have reached the island via passive rafting and then underwent a yearly increase of mean body mass calculated between 0.0015 g and 0.0023 g; this corresponds to fast evolutionary rates (in darwins (d), ranging from 7.09 d to 2.78 d) that are well above those observed for non-insular mammals.

Y chromosome sequence and epigenomic reconstruction across human populations.

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

Genomic Analysis of 18th-Century Kazakh Individuals and Their Oral Microbiome.

The Asian Central Steppe, consisting of current-day Kazakhstan and Russia, has acted as a highway for major migrations throughout history. Therefore, describing the genetic composition of past populations in Central Asia holds value to understanding human mobility in this pivotal region. In this study, we analyse paleogenomic data generated from five humans from Kuygenzhar, Kazakhstan. These individuals date to the early to mid-18th century, shortly after the Kazakh Khanate was founded, a union of nomadic tribes of Mongol Golden Horde and Turkic origins. Genomic analysis identifies that these individuals are admixed with varying proportions of East Asian ancestry, indicating a recent admixture event from East Asia. The high amounts of DNA from the anaerobic Gram-negative bacteria Tannerella forsythia, a periodontal pathogen, recovered from their teeth suggest they may have suffered from periodontitis disease. Genomic analysis of this bacterium identified recently evolved virulence and glycosylation genes including the presence of antibiotic resistance genes predating the antibiotic era. This study provides an integrated analysis of individuals with a diet mostly based on meat (mainly horse and lamb), milk, and dairy products and their oral microbiome.

Chemotherapeutic targeting of cell death pathways.

Cell death plays an important role in cancer growth and progression, as well as in the efficiency of chemotherapy. Although apoptosis is commonly regarded as the principal mechanism of programmed cell death, it has been increasingly reported that several anticancer agents do not only induce apoptosis but other forms of cell death such as necrosis, autophagy and mitotic catastrophe, as well as the state of permanent loss of proliferative capacity known as senescence. A deeper understanding of what we know about chemotherapy-induced death is rather relevant considering the emerging knowledge of non-apoptotic cell death signaling pathways, and the fact that many tumors have the apoptosis pathway seriously compromised. In this review we examine the effects that various anti-cancer agents have on pathways involved in the different cell death outcomes. Novel and specific anti-cancer agents directed toward members of the cell death signaling pathways are being developed and currently being tested in clinical trials. If we precisely activate or inhibit molecules that mediate the diversity of cell death outcomes, we might succeed in more effective and less toxic chemotherapy.

Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe.

We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50-60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity.

Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods.

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.