Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Mitochondria, the energy supply factories for cell-life activities, play important roles in controlling epigenetics, differentiation and initiation, and the execution of apoptosis. These functions of the mitochondria contribute to cell adaptation to challenging microenvironment conditions. In past decades, mitochondrial malfunction has been revealed to be closely related to the occurrence and development of a variety of human disorders, including cancer and multiple neurodegenerative diseases. The disturbance of the mitochondrial genome (mtDNA) or mitochondrial vital functions, e.g., the production of adenosine triphosphate (ATP) and the generation of reactive oxygen species (ROS), can potentially be involved in disease pathogenesis. Recent research has shown that the precise monitoring of mitochondrial environments can provide potential directions for cancer diagnosis. Furthermore, mitochondrial-targeted cancer treatment exhibits unparalleled superiority for enhanced tumor therapy. Therefore, in this review, we focus on mitochondrial-based cancer diagnosis via monitoring mitochondrial respiration or mitophagy. Current approaches using mitochondrial-based cancer treatments, including targeting mitochondrial ATP, mitochondrial membrane permeability, and mitochondrial ROS levels and mtDNA, are also summarized. This review will provide insights into mitochondrial-mediated tumor monitoring and mitochondrial-based therapy.

The aquaporins (AQPs) are a family of water channel proteins with at least 13 mammalian members (AQPs 0-12) expressed in diverse fluid transporting tissues. AQP1, AQP4, and AQP9 have been identified in the central nervous system and demonstrated or proposed to play important roles in brain water homeostasis. Aquaporin expression in the peripheral nervous system is poorly studied. Here we report that the AQP1 water channel is specifically localized to glial cells of the peripheral nervous system by immunohistochemistry, RT-PCR, and immunoblotting. Paraffin-embedded biopsies of human pancreas, esophagus, and sciatic nerves were accessed by immunoperoxidase staining using affinity-purified AQP1, AQP4, and AQP9 antibodies. Strong AQP1 expression was identified in pancreatic nerve plexuses and in the submucosal and myenteric nerve plexuses in the esophagus. AQP1 was localized to the same cell population expressing glial fibrillary acidic protein (GFAP), but not to the neurons in the plexuses, indicating glial cell-specific expression. RT-PCR and immunoblot analysis of microdissected pancreatic ganglia confirmed the expression of AQP1 transcript and protein. Pancreatic and sciatic nerve bundles, which contain nonmyelinating and myelinating Schwann cells, respectively, were also selectively labeled by AQP1 antibody. AQP4 and AQP9, which are broadly expressed in astroglial cells in brain and spinal cord, were not localized in glial cells in the peripheral nerve plexuses. These results suggest that AQPs are differentially expressed in the peripheral versus central nervous system and that channel-mediated water transport mechanisms may be involved in peripheral neuronal activity by regulating water homeostasis in nerve plexuses and bundles.

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The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100 nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR–porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease. Biological significance In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR–porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.

The purpose of this study was to elucidate the mechanisms by which histone acetylation participates in transcriptional regulation of hsp70 (heat-shock protein 70) genes SSA3 and SSA4 in yeast. Our results indicated that histone acetylation was required for the transcriptional activation of SSA3 and SSA4. The HATs (histone acetyltransferases) Gcn5 (general control non-derepressible 5) and Elp3 (elongation protein 3) modulated hsp70 gene transcription by affecting the acetylation status of histone H3. Although the two HATs possessed overlapping function regarding the acetylation of histone H3, they affected hsp70 gene transcription in different ways. The recruitment of Gcn5 was Swi/Snf-dependent and was required for HSF (heat-shock factor) binding and affected RNAPII (RNA polymerase II) recruitment, whereas Elp3 exerted its roles mainly through affecting RNAPII elongation. These results provide insights into the effects of Gcn5 and Elp3 in hsp70 gene transcription and underscore the importance of histone acetylation for transcriptional initiation and elongation in hsp genes.

The function of tumor suppressor p53 has been under intense investigation. Acute stresses such as DNA damage are able to trigger a high level of p53 activity, leading to cell cycle arrest or apoptosis. In contrast, the cellular response of mild p53 activity induced by low-level stress in vivo remains largely unexplored. Murine double minute (MDM)2 and MDM4 are two major negative regulators of p53. Here, we used the strategy of haploinsufficiency of Mdm2 and Mdm4 to induce mild p53 activation in vivo and found that Mdm2+/−Mdm4+/− double-heterozygous mice exhibited normal embryogenesis. However, closer examination demonstrated that the Mdm2+/−Mdm4+/− cells exhibited a growth disadvantage and were outcompeted during development in genetic mosaic embryos that contained wild-type cells. Further study indicated the out-competition phenotype was dependent on the levels of p53. These observations revealed that cells with mild p53 activation were less fit and exhibited altered fates in a heterotypic environment, resembling the cell competition phenomenon first uncovered in Drosophila. By marking unfit cells for elimination, p53 may exert its physiological role to ensure organ and animal fitness.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short life spans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of ~ 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to ~ 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1 year-old CF rabbits as compared to WT littermates, which were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.