A cell type-specific approach to elucidate the role of miR-96 in inner ear hair cells.

Introduction: Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Methods: Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes. Results: We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs. Conclusion: Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

Corrigendum: Transcriptomic Profiling of Zebrafish Hair Cells Using RiboTag.

[This corrects the article DOI: 10.3389/fcell.2018.00047.].

Transcriptomic Profiling of Zebrafish Hair Cells Using RiboTag.

The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure.

The astrocytic transporter SLC7A10 (Asc-1) mediates glycinergic inhibition of spinal cord motor neurons.

SLC7A10 (Asc-1) is a sodium-independent amino acid transporter known to facilitate transport of a number of amino acids including glycine, L-serine, L-alanine, and L-cysteine, as well as their D-enantiomers. It has been described as a neuronal transporter with a primary role related to modulation of excitatory glutamatergic neurotransmission. We find that SLC7A10 is substantially enriched in a subset of astrocytes of the caudal brain and spinal cord in a distribution corresponding with high densities of glycinergic inhibitory synapses. Accordingly, we find that spinal cord glycine levels are significantly reduced in Slc7a10-null mice and spontaneous glycinergic postsynaptic currents in motor neurons show substantially diminished amplitudes, demonstrating an essential role for SLC7A10 in glycinergic inhibitory function in the central nervous system. These observations establish the etiology of sustained myoclonus (sudden involuntary muscle movements) and early postnatal lethality characteristic of Slc7a10-null mice, and implicate SLC7A10 as a candidate gene and auto-antibody target in human hyperekplexia and stiff person syndrome, respectively.

In-hospital outcomes of percutaneous coronary interventions in extremely obese and normal-weight patients: findings from the NCDR (National Cardiovascular Data Registry).

OBJECTIVES: The purpose of this study was to compare in-hospital outcomes of percutaneous coronary intervention (PCI) in extreme obesity (EO) (body mass index [BMI] ≥ 40 kg/m²) with those of normal-weight (NW) patients and to examine the influence of access site on outcomes. BACKGROUND: Little is known about the outcomes of PCI in EO patients. METHODS: We analyzed CathPCI Registry data from patients who underwent radial or femoral PCI and were discharged between July 2009 and June 2011 and compared in-hospital outcomes of EO (N = 83,861) with those of NW patients (BMI 20 to 25 kg/m²; N = 217,616). Outcomes included in-hospital mortality and procedural and bleeding complications. Multivariable logistic regression models were used to assess the independent association of EO with outcomes, using previously validated risk models derived from the CathPCI Registry. The role of access site was specifically examined. RESULTS: Compared with NW patients, EO patients were younger (median age 60 vs. 69 years), more likely female (47% vs. 37%), and more likely African American (12% vs. 7%). EO patients had lower unadjusted mortality (1.2% vs. 2.0%); however, after multivariable adjustment, EO was independently associated with increased risk of in-hospital mortality (odds ratio: 1.22; 95% CI: 1.08 to 1.39) in those presenting with ST-segment elevation myocardial infarction (STEMI). Access site had no effect on bleeding or outcome. CONCLUSIONS: EO patients who underwent PCI were younger and had less bleeding compared with NW patients. After multivariable adjustment for risk, EO was independently associated with higher in-hospital mortality overall and particularly in the patients undergoing STEMI.