Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue’s native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids’ response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level

Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery

Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell–cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids’ crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Re-assessing the diversity of negative strand RNA viruses in insects.

The spectrum of viruses in insects is important for subjects as diverse as public health, veterinary medicine, food production, and biodiversity conservation. The traditional interest in vector-borne diseases of humans and livestock has drawn the attention of virus studies to hematophagous insect species. However, these represent only a tiny fraction of the broad diversity of Hexapoda, the most speciose group of animals. Here, we systematically probed the diversity of negative strand RNA viruses in the largest and most representative collection of insect transcriptomes from samples representing all 34 extant orders of Hexapoda and 3 orders of Entognatha, as well as outgroups, altogether representing 1243 species. Based on profile hidden Markov models we detected 488 viral RNA-directed RNA polymerase (RdRp) sequences with similarity to negative strand RNA viruses. These were identified in members of 324 arthropod species. Selection for length, quality, and uniqueness left 234 sequences for analyses, showing similarity to genomes of viruses classified in Bunyavirales (n = 86), Articulavirales (n = 54), and several orders within Haploviricotina (n = 94). Coding-complete genomes or nearly-complete subgenomic assemblies were obtained in 61 cases. Based on phylogenetic topology and the availability of coding-complete genomes we estimate that at least 20 novel viral genera in seven families need to be defined, only two of them monospecific. Seven additional viral clades emerge when adding sequences from the present study to formerly monospecific lineages, potentially requiring up to seven additional genera. One long sequence may indicate a novel family. For segmented viruses, cophylogenies between genome segments were generally improved by the inclusion of viruses from the present study, suggesting that in silico misassembly of segmented genomes is rare or absent. Contrary to previous assessments, significant virus-host codivergence was identified in major phylogenetic lineages based on two different approaches of codivergence analysis in a hypotheses testing framework. In spite of these additions to the known spectrum of viruses in insects, we caution that basing taxonomic decisions on genome information alone is challenging due to technical uncertainties, such as the inability to prove integrity of complete genome assemblies of segmented viruses

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids

Four myriapod relatives – but who are sisters? No end to debates on relationships among the four major myriapod subgroups

BackgroundPhylogenetic relationships among the myriapod subgroups Chilopoda, Diplopoda, Symphyla and Pauropoda are still not robustly resolved. The first phylogenomic study covering all subgroups resolved phylogenetic relationships congruently to morphological evidence but is in conflict with most previously published phylogenetic trees based on diverse molecular data. Outgroup choice and long-branch attraction effects were stated as possible explanations for these incongruencies. In this study, we addressed these issues by extending the myriapod and outgroup taxon sampling using transcriptome data.ResultsWe generated new transcriptome data of 42 panarthropod species, including all four myriapod subgroups and additional outgroup taxa. Our taxon sampling was complemented by published transcriptome and genome data resulting in a supermatrix covering 59 species. We compiled two data sets, the first with a full coverage of genes per species (292 single-copy protein-coding genes), the second with a less stringent coverage (988 genes). We inferred phylogenetic relationships among myriapods using different data types, tree inference, and quartet computation approaches. Our results unambiguously support monophyletic Mandibulata and Myriapoda. Our analyses clearly showed that there is strong signal for a single unrooted topology, but a sensitivity of the position of the internal root on the choice of outgroups. However, we observe strong evidence for a clade Pauropoda+Symphyla, as well as for a clade Chilopoda+Diplopoda.ConclusionsOur best quartet topology is incongruent with current morphological phylogenies which were supported in another phylogenomic study. AU tests and quartet mapping reject the quartet topology congruent to trees inferred with morphological characters. Moreover, quartet mapping shows that confounding signal present in the data set is sufficient to explain the weak signal for the quartet topology derived from morphological characters. Although outgroup choice affects results, our study could narrow possible trees to derivatives of a single quartet topology. For highly disputed relationships, we propose to apply a series of tests (AU and quartet mapping), since results of such tests allow to narrow down possible relationships and to rule out confounding signal

Neutral and Charged Polymers at Interfaces

Chain-like macromolecules (polymers) show characteristic adsorption properties due to their flexibility and internal degrees of freedom, when attracted to surfaces and interfaces. In this review we discuss concepts and features that are relevant to the adsorption of neutral and charged polymers at equilibrium, including the type of polymer/surface interaction, the solvent quality, the characteristics of the surface, and the polymer structure. We pay special attention to the case of charged polymers (polyelectrolytes) that have a special importance due to their water solubility. We present a summary of recent progress in this rapidly evolving field. Because many experimental studies are performed with rather stiff biopolymers, we discuss in detail the case of semi-flexible polymers in addition to flexible ones. We first review the behavior of neutral and charged chains in solution. Then, the adsorption of a single polymer chain is considered. Next, the adsorption and depletion processes in the many-chain case are reviewed. Profiles, changes in the surface tension and polymer surface excess are presented. Mean-field and corrections due to fluctuations and lateral correlations are discussed. The force of interaction between two adsorbed layers, which is important in understanding colloidal stability, is characterized. The behavior of grafted polymers is also reviewed, both for neutral and charged polymer brushes.Comment: a review: 130 pages, 30 ps figures; final form, added reference

Expectant parents' views of factors influencing infant feeding decisions in the antenatal period: A systematic review

Objective: To explore the factors that influence expectant parents’ infant feeding decisions in the antenatal period. Design: Mixed method systematic review focussing on participant views data. Data sources: CINAHL, Medline, Embase and PsychInfo databases were interrogated using initial keywords and then refined terms to elicit relevant studies. Reference lists were checked and hand-searching was undertaken for 2 journals (‘Midwifery’ and ‘Social Science and Medicine’) covering a 3 year time period (January 2011–March 2014). Key inclusion criteria: studies reflecting expectant parents’ views of the factors influencing their infant feeding decisions in the antenatal period; Studies in the English language published after 1990, from developed countries and of qualitative, quantitative or mixed method design. Review methods: A narrative interpretive synthesis of the views data from studies of qualitative, quantitative and mixed method design. Data were extracted on study characteristics and parents’ views, using the Social Ecological Model to support data extraction and thematic synthesis. Synthesis was influenced by the Evidence for Policy and Practice Information and Co-Ordinating Centre approach to mixed method reviews. Results: Of the 409 studies identified through search methods, 17 studies met the inclusion criteria for the review. Thematic synthesis identified 9 themes: Bonding/Attachment; Body Image; Self Esteem/Confidence; Female Role Models; Family and Support Network; Lifestyle; Formal Information Sources; Knowledge; and Feeding in front of others/Public. The review identified a significant bias in the data towards negative factors relating to the breastfeeding decision, suggesting that infant feeding was not a choice between two feeding options, but rather a process of weighing reasons for and against breastfeeding. Findings reflected the perception of the maternal role as intrinsic to the expectant mothers’ infant feeding decisions. Cultural perceptions permeated personal, familial and social influences on the decision-making process. Expectant mothers were sensitive to the way professionals attempted to support and inform them about infant feeding choices. Conclusions: By taking a Social Ecological perspective, we were able to explore and demonstrate the multiple influences impacting on expectant parents in the decision-making process. A better understanding of expectant parents’ views and experiences in making infant feeding decisions in the prenatal and antenatal periods will inform public health policy and the coordination of service provision to support infant feeding activities

Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders

Obestatin as a key regulator of metabolism and cardiovascular function with emerging therapeutic potential for diabetes

Obestatin is a 23‐amino acid C‐terminally amidated gastrointestinal peptide derived from preproghrelin and which forms an α helix. Although obestatin has a short biological half‐life and is rapidly degraded, it is proposed to exert wide‐ranging pathophysiological actions. Whilst the precise nature of many of its effects is unclear, accumulating evidence supports positive actions on both metabolism and cardiovascular function. For example, obestatin has been reported to inhibit food and water intake, body weight gain and gastrointestinal motility and also to mediate promotion of cell survival and prevention of apoptosis. Obestatin‐induced increases in beta cell mass, enhanced adipogenesis and improved lipid metabolism have been noted along with up‐regulation of genes associated with beta cell regeneration, insulin production and adipogenesis. Furthermore, human circulating obestatin levels generally demonstrate an inverse association with obesity and diabetes, whilst the peptide has been shown to confer protective metabolic effects in experimental diabetes, suggesting that it may hold therapeutic potential in this setting. Obestatin also appears to be involved in blood pressure regulation and to exert beneficial effects on endothelial function, with experimental studies indicating that it may also promote cardioprotective actions against, for example, ischaemia–reperfusion injury. This review will present a critical appraisal of the expanding obestatin research area and discuss the emerging therapeutic potential of this peptide for both metabolic and cardiovascular complications of diabetes