Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3–V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles.

Numerous studies have demonstrated the efficacy of extremely low frequency‐pulsed electromagnetic fields (ELF‐PEMF) in accelerating the wound healing process in vitro and in vivo. Our study focuses specifically on ELF‐PEMF applied with the Magnomega® device and aims to assess their effect during the main stages of the proliferative phase of dermal wound closure, in vitro. Thus, after the characterization of the EMFs delivered by the Magnomega® unit, primary culture of human dermal fibroblasts (HDFs) were exposed, or not for the control culture, to 10–12 and 100 Hz ELF‐PEMF. These parameters are used in clinical practice by physiotherapists in order to enhance healing of dermal lesions in patients. HDFs proliferation was first assessed and revealed an increase in the expression of one of the two genetic markers of cell proliferation tested (PCNA and MKI67), after initial exposure of the cells to 10–12 Hz PEMF. Next, migration of HDFs was investigated by performing scratch assays on HDF layers. The observed wound closure kinetics corroborate the early organization of actin stress fibers that was revealed in the cytoplasm of HDFs exposed to 100 Hz ELF‐PEMF. Also, maturation of HDFs into myofibroblasts was significantly increased in cells exposed to 10–12 or to 100 Hz PEMF. The present study is the first to demonstrate, in vitro, an early stimulation of HDFs, after their exposure to ELF‐PEMF delivered by the Magnomega® device, which could contribute to an acceleration of the wound healing process.

Transient overexpression of genes involved in lung regulation might prevent alveolar developmental disorders (ADDs) in premature neonates. However, adenovirus 5 (Ad5) vectors per se, and not isolated capsid proteins, induce ADDs after tracheal administration to newborn rats. To test the hypothesis that Ad5 capsid components are mainly responsible for ADDs, we evaluated newborn rats' lung development by morphometry after tracheal administration of a panel of Ad5 vectors with mutations in the fiber or penton base. Three distinct patterns of lung response were observed on postnatal day (PD) 21: (i) emphysematous-like lesions, common to Ad5 overexposing RGD motifs; (ii) altered septation, representative of the wild-type capsid Ad5 lesion; (iii) absence of lung toxicity, shown by Ad5 vectors with fibers shortened to seven repeats. None of these patterns correlated with the degree of lung inflammation or gene transduction. In contrast, a more impaired elastogenesis associated with emphysema was preceded by a significantly increased level of activated caspase 3 on PD11. Moreover, the altered septation was associated with a persistent and significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive alveolar septal cells on PD21. Our results underline the deleterious effects of Ad-induced apoptosis, which is not only responsible for limited transgene expression but also involved in lung development disorders.

Electroporation (EPN) is the process by which cell membranes become transiently or permanently permeable upon exposure to pulsed electric fields of suitable intensity and duration. Depending on the pulse parameters, permeabilization can be reversible or irreversible, enabling a wide range of biomedical applications. To improve our understanding of EPN effects on tissues and select efficient treatments and parameters, relevant in vitro tumour models are required. Three-dimensional (3D) cell spheroids have emerged as valuable systems, as they more accurately replicate tumour microenvironment and cell-to-cell interactions than conventional 2D cultures. In this work, we present a new microdevice designed for the culture and gradual electroporation of a population of several hundred uniformly sized spheroids, allowing the systematic study of electroporation over a wide range of electric field intensities within a single experiment. Gradual permeabilization of HT-29 colorectal cancer cell spheroids was performed using a standard electrochemotherapy protocol, and electroporation efficiency was assessed by analysing propidium iodide (PI) uptake. Spheroids were treated with electric fields ranging from 800 V cm⁻¹ to 3400 V cm⁻¹. In toto analysis of PI distribution within spheroids by confocal microscopy revealed highly heterogeneous permeabilization patterns throughout the spheroid volume, for all intensities investigated, even at the highest one of 3400 V cm⁻¹. This study introduces a robust 3D in vitro assay for the systematic evaluation of electroporation-based treatments, providing new insights into the influence of electric field heterogeneity, electrical protocol, and estimation of molecular uptake in the spheroid volume.