RESUMEN
DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
Asunto(s)
ADN Mitocondrial , Efectores Tipo Activadores de la Transcripción , Animales , Humanos , Ratones , Adenina , Citosina , ADN Mitocondrial/genética , Edición Génica , ARN , Efectores Tipo Activadores de la Transcripción/metabolismo , Ingeniería de ProteínasRESUMEN
Inducing external strains on highly oriented thin films transferred onto mechanically deformable substrates enables a drastic enhancement of their ferroelectric, magnetic, and electronic performances, which cannot be achieved in films on rigid single crystals. Herein, the growth and diffusion behaviors of BiFeO3 thin films grown at various temperatures is reported on α-MoO3 layers of different thicknesses using sputtering. When the BiFeO3 thin films are deposited at a high temperature, significant diffusion of Fe into α-MoO3 occurs, producing the Fe1.89Mo4.11O7 phase and suppressing the maintenance of the 2D structure of the α-MoO3 layers. Although lowering the deposition temperature alleviates the diffusion yielding the survival of the α-MoO3 layer, enabling exfoliation, the BiFeO3 is amorphous and the formation of the Fe1.89Mo4.11O7 phase cannot be suppressed at the crystallization temperature. High-temperature-grown BiFeO3 thin films are successfully transferred onto flexible substrates via mechanical exfoliation by introducing a blocking layer of Au and measured the ferroelectric properties of the transferred films.
RESUMEN
Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1-5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.
Asunto(s)
Acné Vulgar , Antiinfecciosos , Naftoquinonas , Infecciones Estafilocócicas , Animales , Ratones , Candida albicans/genética , Staphylococcus aureus , Biopelículas , Antiinfecciosos/farmacologíaRESUMEN
Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 cells (AT2s) act as stem cells to replenish both themselves and alveolar type 1 cells (AT1s); however, the complex orchestration of stem cell activity after injury is poorly understood. Here, we establish longitudinal imaging of AT2s in murine intact tissues ex vivo and in vivo in order to track their dynamic behavior over time. We discover that a large fraction of AT2s become motile following injury and provide direct evidence for their migration between alveolar units. High-resolution morphokinetic mapping of AT2s further uncovers the emergence of distinct motile phenotypes. Inhibition of AT2 migration via genetic depletion of ArpC3 leads to impaired regeneration of AT2s and AT1s in vivo. Together, our results establish a requirement for stem cell migration between alveolar units and identify properties of stem cell motility at high cellular resolution.