Abstract Chronic suppurative otitis media (CSOM) is one of the most common infectious diseases of the middle ear especially affecting children, leading to delay in language development and communication. Although Staphylococcus aureus is the most common pathogen associated with CSOM, its interaction with middle ear epithelial cells is not well known. In the present study, we observed that otopathogenic S. aureus has the ability to invade human middle ear epithelial cells (HMEECs) in a dose and time dependent manner. Scanning electron microscopy demonstrated time dependent increase in the number of S. aureus on the surface of HMEECs. We observed that otopathogenic S. aureus primarily employs a cholesterol dependent pathway to colonize HMEECs. In agreement with these findings, confocal microscopy showed that S. aureus colocalized with lipid rafts in HMEECs. The results of the present study provide new insights into the pathogenesis of S. aureus induced CSOM. The availability of in vitro cell culture model will pave the way to develop novel effective treatment modalities for CSOM beyond antibiotic therapy.
The inner ear is derived from the otic placode, one of numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE). However, the molecular dynamics underlying how the PPE is induced and regionalized are poorly resolved. We used stem cell-derived inner ear organoids to investigate the effects of Wnt signaling on otic placode development and found that modulating Wnt signaling to give intermediate activation of its downstream pathway significantly increased inner ear organoid induction efficiency. Single cell RNA-sequencing revealed that the Wnt modulation induces 1) gene signatures of the posterior PPE (pPPE), 2) expansion of the caudal neural plate border (NPB), and 3) a suitable caudal head microenvironment. Our data also suggest that precursors of the pPPE committed to caudal fate during the NPB stage. Taken together, this study improves our understanding of the role of Wnt signaling in the early development of the inner ear.
Abstract Neurofibromatosis Type 2 (NF2) is a tumor predisposition syndrome caused by germline inactivating mutations in the NF2 gene encoding the merlin tumor suppressor. Patients develop multiple benign tumor types in the nervous system including bilateral vestibular schwannomas (VS). Standard treatments include surgery and radiation therapy, which may lead to loss of hearing, impaired facial nerve function, and other complications. Kinase inhibitor monotherapies have been evaluated clinically for NF2 patients with limited success, and more effective nonsurgical therapies are urgently needed. Schwannoma model cells treated with PI3K inhibitors upregulate activity of the focal adhesion kinase (FAK) family as a compensatory survival pathway. We screened combinations of 13 clinically relevant PI3K and FAK inhibitors using human isogenic normal and merlin-deficient Schwann cell lines. The most efficacious combination was PI3K/mTOR inhibitor omipalisib with SRC/FAK inhibitor dasatinib. Sub-GI50 doses of the single drugs blocked phosphorylation of their major target proteins. The combination was superior to either single agent in promoting a G1 cell-cycle arrest and produced a 44% decrease in tumor growth over a 2-week period in a pilot orthotopic allograft model. Evaluation of single and combination drugs in six human primary VS cell models revealed the combination was superior to the monotherapies in 3 of 6 VS samples, highlighting inter-tumor variability between patients consistent with observations from clinical trials with other molecular targeted agents. Dasatinib alone performed as well as the combination in the remaining three samples. Preclinically validated combination therapies hold promise for NF2 patients and warrants further study in clinical trials.
In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu).
Methods
Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics.
Results
All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production.
Conclusion
Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.
