Cactus: A user-friendly and reproducible ATAC-Seq and mRNA-Seq analysis pipeline for data preprocessing, differential analysis, and enrichment analysis.

The ever decreasing cost of Next-Generation Sequencing coupled with the emergence of efficient and reproducible analysis pipelines has rendered genomic methods more accessible. However, downstream analyses are basic or missing in most workflows, creating a significant barrier for non-bioinformaticians. To help close this gap, we developed Cactus, an end-to-end pipeline for analyzing ATAC-Seq and mRNA-Seq data, either separately or jointly. Its Nextflow-, container-, and virtual environment-based architecture ensures efficient and reproducible analyses. Cactus preprocesses raw reads, conducts differential analyses between conditions, and performs enrichment analyses in various databases, including DNA-binding motifs, ChIP-Seq binding sites, chromatin states, and ontologies. We demonstrate the utility of Cactus in a multi-modal and multi-species case study as well as by showcasing its unique capabilities as compared to other ATAC-Seq pipelines. In conclusion, Cactus can assist researchers in gaining comprehensive insights from chromatin accessibility and gene expression data in a quick, user-friendly, and reproducible manner.

Scalable and efficient DNA sequencing analysis on different compute infrastructures aiding variant discovery.

DNA variation analysis has become indispensable in many aspects of modern biomedicine, most prominently in the comparison of normal and tumor samples. Thousands of samples are collected in local sequencing efforts and public databases requiring highly scalable, portable, and automated workflows for streamlined processing. Here, we present nf-core/sarek 3, a well-established, comprehensive variant calling and annotation pipeline for germline and somatic samples. It is suitable for any genome with a known reference. We present a full rewrite of the original pipeline showing a significant reduction of storage requirements by using the CRAM format and runtime by increasing intra-sample parallelization. Both are leading to a 70% cost reduction in commercial clouds enabling users to do large-scale and cross-platform data analysis while keeping costs and CO2 emissions low. The code is available at https://nf-co.re/sarek.

Domestic cat larynges can produce purring frequencies without neural input.

Most mammals produce vocal sounds according to the myoelastic-aerodynamic (MEAD) principle, through self-sustaining oscillation of laryngeal tissues.1,2 In contrast, cats have long been believed to produce their low-frequency purr vocalizations through a radically different mechanism involving active muscle contractions (AMC), where neurally driven electromyographic burst patterns (typically at 20-30 Hz) cause the intrinsic laryngeal muscles to actively modulate the respiratory airflow. Direct empirical evidence for this AMC mechanism is sparse.3 Here, the fundamental frequency (fo) ranges of eight domestic cats (Felis silvestris catus) were investigated in an excised larynx setup, to test the prediction of the AMC hypothesis that vibration should be impossible without neuromuscular activity, and thus unattainable in excised larynx setups, which are based on MEAD principles. Surprisingly, all eight excised larynges produced self-sustained oscillations at typical cat purring rates. Histological analysis of cat larynges revealed the presence of connective tissue masses, up to 4 mm in diameter, embedded in the vocal fold.4 This vocal fold specialization appears to allow the unusually low fo values observed in purring. While our data do not fully reject the AMC hypothesis for purring, they show that cat larynges can easily produce sounds in the purr regime with fundamental frequencies of 25 to 30 Hz without neural input or muscular contraction. This strongly suggests that the physical and physiological basis of cat purring involves the same MEAD-based mechanisms as other cat vocalizations (e.g., meows) and most other vertebrate vocalizations but is potentially augmented by AMC.

The Swedish childhood tumor biobank: systematic collection and molecular characterization of all pediatric CNS and other solid tumors in Sweden.

The Swedish Childhood Tumor Biobank (BTB) is a nonprofit national infrastructure for collecting tissue samples and genomic data from pediatric patients diagnosed with central nervous system (CNS) and other solid tumors. The BTB is built on a multidisciplinary network established to provide the scientific community with standardized biospecimens and genomic data, thereby improving knowledge of the biology, treatment and outcome of childhood tumors. As of 2022, over 1100 fresh-frozen tumor samples are available for researchers. We present the workflow of the BTB from sample collection and processing to the generation of genomic data and services offered. To determine the research and clinical utility of the data, we performed bioinformatics analyses on next-generation sequencing (NGS) data obtained from a subset of 82 brain tumors and patient blood-derived DNA combined with methylation profiling to enhance the diagnostic accuracy and identified germline and somatic alterations with potential biological or clinical significance. The BTB procedures for collection, processing, sequencing, and bioinformatics deliver high-quality data. We observed that the findings could impact patient management by confirming or clarifying the diagnosis in 79 of the 82 tumors and detecting known or likely driver mutations in 68 of 79 patients. In addition to revealing known mutations in a broad spectrum of genes implicated in pediatric cancer, we discovered numerous alterations that may represent novel driver events and specific tumor entities. In summary, these examples reveal the power of NGS to identify a wide number of actionable gene alterations. Making the power of NGS available in healthcare is a challenging task requiring the integration of the work of clinical specialists and cancer biologists; this approach requires a dedicated infrastructure, as exemplified here by the BTB.

Immune Checkpoint Inhibitor-Induced Myositis/Myocarditis with Myasthenia Gravis-like Misleading Presentation: A Case Series in Intensive Care Unit.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) are a major breakthrough in cancer treatment. Their increasingly frequent use leads to an uprising incidence of immune-related adverse events (irAEs). Among those, myocarditis is the most reported fatal cardiovascular irAE, frequently associated with ICI-related myositis. CASE SERIES: Here, we report three cases of ICI-induced myocarditis/myositis with an extremely severe myasthenia gravis-like (MG-like) presentation, highlighting the main challenges in irAEs management. These patients were over 60 years old and presented an ongoing melanoma, either locally advanced or metastatic, treated with ICI combinations. Shortly after the first or second ICI infusion, they were admitted in an intensive care unit (ICU) for grade 3 ICI-induced MG-like symptoms leading to acute respiratory failure (ARF) requiring invasive mechanical ventilation (IMV). The initial misdiagnosis was later corrected to severe ICI-induced seronegative myocarditis/myositis upon biological results and histopathology from muscular/endomyocardial biopsies. All of them received urgent high-dose corticosteroids pulses. The oldest patient died prematurely, but the two others received targeted therapies leading to complete recovery for one of them. DISCUSSION: These cases highlight the four main challenges of irAEs, encompassing the lack of knowledge among physicians, the risk of misdiagnosis due to numerous and non-specific symptoms, the frequent overlapping forms of irAEs, and the extremely rare MG-like misleading presentation of myocarditis/myositis. The exact pathophysiology of irAEs remains unclear, although a major involvement of the lymphoid compartment (specifically T lymphocytes) was evidenced. Therapeutic management is based on urgent high-dose corticosteroids. For the severest forms of irAEs, case-by-case targeted immunosuppressive therapies should be urgently administered upon multidisciplinary meetings. CONCLUSION: These cases highlight the lack of knowledge of irAEs among physicians, aggravated by misleading overlapping forms, requiring specific management in trained units and multidisciplinary care. Severe MG-like presentation of irAEs constitutes an absolute therapeutic emergency with high-dose corticosteroids and targeted immunosuppressive therapy.

Vocal tract allometry in a mammalian vocal learner.

Acoustic allometry occurs when features of animal vocalisations can be predicted from body size measurements. Despite this being considered the norm, allometry sometimes breaks, resulting in species sounding smaller or larger than expected for their size. A recent hypothesis suggests that allometry-breaking mammals cluster into two groups: those with anatomical adaptations to their vocal tracts and those capable of learning new sounds (vocal learners). Here, we tested which mechanism is used to escape from acoustic allometry by probing vocal tract allometry in a proven mammalian vocal learner, the harbour seal (Phoca vitulina). We tested whether vocal tract structures and body size scale allometrically in 68 young individuals. We found that both body length and body mass accurately predict vocal tract length and one tracheal dimension. Independently, body length predicts vocal fold length while body mass predicts a second tracheal dimension. All vocal tract measures are larger in weaners than in pups and some structures are sexually dimorphic within age classes. We conclude that harbour seals do comply with anatomical allometric constraints. However, allometry between body size and vocal fold length seems to emerge after puppyhood, suggesting that ontogeny may modulate the anatomy-learning distinction previously hypothesised as clear cut. We suggest that seals, and perhaps other species producing signals that deviate from those expected from their vocal tract dimensions, may break allometry without morphological adaptations. In seals, and potentially other vocal learning mammals, advanced neural control over vocal organs may be the main mechanism for breaking acoustic allometry.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

A cross-species framework to identify vocal learning abilities in mammals.

Vocal production learning (VPL) is the experience-driven ability to produce novel vocal signals through imitation or modification of existing vocalizations. A parallel strand of research investigates acoustic allometry, namely how information about body size is conveyed by acoustic signals. Recently, we proposed that deviation from acoustic allometry principles as a result of sexual selection may have been an intermediate step towards the evolution of vocal learning abilities in mammals. Adopting a more hypothesis-neutral stance, here we perform phylogenetic regressions and other analyses further testing a potential link between VPL and being an allometric outlier. We find that multiple species belonging to VPL clades deviate from allometric scaling but in the opposite direction to that expected from size exaggeration mechanisms. In other words, our correlational approach finds an association between VPL and being an allometric outlier. However, the direction of this association, contra our original hypothesis, may indicate that VPL did not necessarily emerge via sexual selection for size exaggeration: VPL clades show higher vocalization frequencies than expected. In addition, our approach allows us to identify species with potential for VPL abilities: we hypothesize that those outliers from acoustic allometry lying above the regression line may be VPL species. Our results may help better understand the cross-species diversity, variability and aetiology of VPL, which among other things is a key underpinning of speech in our species. This article is part of the theme issue 'Voice modulation: from origin and mechanism to social impact (Part II)'.

Developing an Artificial Intelligence (A.I)-based descriptor of facial appearance that fits with the assessments of makeup experts.

OBJECTIVE: To develop an A.I-based automatic descriptor that detects and grades, from selfie pictures, 23 facial signs, hairs included, as a help to making-up procedures. MATERIAL AND METHODS: The selfie images taken in very different conditions by 3326 women and men were used to create (90% of dataset) and validate (10% of dataset) a new algorithm architecture to appraise and grade 23 different facial signs such as lips, nose, eye color, eyebrows, eyelashes, and hair color as defined by makeup artists. Each selfie image was annotated by 12 experts and defined references to train Artificial Intelligence (A.I)-based algorithm. RESULTS: As some the 23 signs present a continuous or discontinuous feature, these were analyzed by two different statistical approaches. The results provided by the automatic descriptor system were not only in good agreement with the expert's assessments but were even found of a better precision and reproducibility. This automatic descriptor system has proven a good and robust accuracy despite the very variable conditions in the acquisition of selfie pictures. CONCLUSION: Such automatic descriptor system seems providing a valuable help in making-up procedures and may extend to other activities such as Skincare or Haircare. As such it should allow large investigations to better evaluate the consumers' needs of esthetical improvements.

Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager.

The broadening utilisation of ancient DNA to address archaeological, palaeontological, and biological questions is resulting in a rising diversity in the size of laboratories and scale of analyses being performed. In the context of this heterogeneous landscape, we present an advanced, and entirely redesigned and extended version of the EAGER pipeline for the analysis of ancient genomic data. This Nextflow pipeline aims to address three main themes: accessibility and adaptability to different computing configurations, reproducibility to ensure robust analytical standards, and updating the pipeline to the latest routine ancient genomic practices. The new version of EAGER has been developed within the nf-core initiative to ensure high-quality software development and maintenance support; contributing to a long-term life-cycle for the pipeline. nf-core/eager will assist in ensuring that a wider range of ancient DNA analyses can be applied by a diverse range of research groups and fields.

Study of intracellular anabolism of 5-fluorouracil and incorporation in nucleic acids based on an LC-HRMS method.

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.

Effect of Ventricular Folds on Vocalization Fundamental Frequency in Domestic Pigs (Sus scrofa domesticus).

This study investigates the effect of the ventricular folds on fundamental frequency (fo) in the voice production of domestic pigs (Sus scrofa domesticus). The excised larynges of six subadult pigs were phonated in two preparation stages, with the ventricular folds present (PS1) and removed (PS2). Vocal fold resonances were tested with a laser vibrometer, and a four-mass computational model was created. Highly significant fo differences were found between PS1 and PS2 (means at 93.7 and 409.3 Hz, respectively). Two tissue resonances were found at 115 Hz and 250-290 Hz. The computational model had unique solutions for abducted and adducted ventricular folds at about 150 and 400 Hz, roughly matching the fo measured ex vivo for PS1 and PS2. The differing fo encountered across preparation stages PS1 and PS2 is explained by distinct activation of either a high or a low eigenfrequency mode, depending on the engagement of the ventricular folds. The inability of the investigated larynges to vibrate at frequencies below 250 Hz in PS2 suggests that in vivo low-frequency calls of domestic pigs (pre-eminently grunts) are likely produced with engaged ventricular folds. Allometric comparison suggests that the special, mechanically coupled "double oscillator" has evolved to prevent signaling disadvantages. Given these traits, the porcine larynx might - apart from special applications relating to the involvement of ventricular folds - not be an ideal candidate for emulating human voice production in excised larynx experimentation.

Evolution of communication signals and information during species radiation.

Communicating species identity is a key component of many animal signals. However, whether selection for species recognition systematically increases signal diversity during clade radiation remains debated. Here we show that in woodpecker drumming, a rhythmic signal used during mating and territorial defense, the amount of species identity information encoded remained stable during woodpeckers' radiation. Acoustic analyses and evolutionary reconstructions show interchange among six main drumming types despite strong phylogenetic contingencies, suggesting evolutionary tinkering of drumming structure within a constrained acoustic space. Playback experiments and quantification of species discriminability demonstrate sufficient signal differentiation to support species recognition in local communities. Finally, we only find character displacement in the rare cases where sympatric species are also closely related. Overall, our results illustrate how historical contingencies and ecological interactions can promote conservatism in signals during a clade radiation without impairing the effectiveness of information transfer relevant to inter-specific discrimination.

Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants.

Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.

Rapid evolution of the primate larynx?

Tissue vibrations in the larynx produce most sounds that comprise vocal communication in mammals. Larynx morphology is thus predicted to be a key target for selection, particularly in species with highly developed vocal communication systems. Here, we present a novel database of digitally modeled scanned larynges from 55 different mammalian species, representing a wide range of body sizes in the primate and carnivoran orders. Using phylogenetic comparative methods, we demonstrate that the primate larynx has evolved more rapidly than the carnivoran larynx, resulting in a pattern of larger size and increased deviation from expected allometry with body size. These results imply fundamental differences between primates and carnivorans in the balance of selective forces that constrain larynx size and highlight an evolutionary flexibility in primates that may help explain why we have developed complex and diverse uses of the vocal organ for communication.

Acoustic allometry and vocal learning in mammals.

Acoustic allometry is the study of how animal vocalizations reflect their body size. A key aim of this research is to identify outliers to acoustic allometry principles and pinpoint the evolutionary origins of such outliers. A parallel strand of research investigates species capable of vocal learning, the experience-driven ability to produce novel vocal signals through imitation or modification of existing vocalizations. Modification of vocalizations is a common feature found when studying both acoustic allometry and vocal learning. Yet, these two fields have only been investigated separately to date. Here, we review and connect acoustic allometry and vocal learning across mammalian clades, combining perspectives from bioacoustics, anatomy and evolutionary biology. Based on this, we hypothesize that, as a precursor to vocal learning, some species might have evolved the capacity for volitional vocal modulation via sexual selection for 'dishonest' signalling. We provide preliminary support for our hypothesis by showing significant associations between allometric deviation and vocal learning in a dataset of 164 mammals. Our work offers a testable framework for future empirical research linking allometric principles with the evolution of vocal learning.

Bound for Specific Sounds: Vocal Predisposition in Animal Communication.

Mechanical constraints imposed by anatomical adaptations are a ubiquitous feature of animal sound production. They can give rise to 'vocal predispositions' (i.e., acoustic structures strictly determined by vocal anatomy). Such predispositions are crucial to the investigation of the cognitive and evolutionary processes underlying acoustic communication in vertebrates, including human speech.