Nodal-related protein (ndr2) is a member of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the zebrafish, Danio rerio, ndr2 gene lead to similar phenotypes, including loss of medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, eight of which are from chemical mutagenesis, four are radiationinduced, and the remaining alleles were obtained by random insertion, gene targeting (TALEN) or unknown. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles have small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele in detail that results in the loss of the conserved terminal cysteine coding sequences. This study is another demonstration of the utility of CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into structure-function of ndr2 gene.

Nodal-related protein (ndr2) is a member of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced, and the remaining alleles were obtained via random insertion, gene targeting (TALEN), or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domainspecific mutations and provides further insights into the structure-function of the ndr2 gene..

Private insurance is designed to provide more timely and cost-effective medical care. With the increase in healthcare deductibles, however, patients are delaying operative management of a variety of pathologies toward the end of the fiscal year with the hopes of having met their deductible. In this retrospective review, the authors assess the operative timing of common orthopedic upper extremity surgeries between publicly and privately insured patients at both university and physician-owned medical institutions.

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Medullary thyroid cancer (MTC) is a type of neuroendocrine cancer (NE) with increasing incidence worldwide. Compared with well-differentiated thyroid cancers, distant metastases are more commonly observed in MTC patients, ranging from 7-23%. The inability to treat metastatic MTC contributes to a decreased patient survival rate. Currently, at least nine mammalian voltage-gated sodium channels (VGSCs) named Nav1.1-Nav1.9 have been identified as potential safe drug targets for treating metastasis in various cancers. Herein, we investigate the effects of targeting VGSCs in MTC using small molecule inhibitors. Two MTC cell lines, TT and MZ, were treated with four compounds and the changes in mRNA and protein expression of VGSC isoform Nav1.7 were assessed through RT-PCR and western blotting. Treatment by one of the compounds exhibited a 28-fold decrease in mRNA expression of VGSC isoform 1.7. Furthermore, a cell proliferation assay, proliferation assay (MTT) a MTT, assay to study cell proliferation after treatment showed decreasing cell viability resulted from the treatment. and Additionally, morphological changes characteristic of apoptosis in fluorescently labeled MTC cell lines after 48 hours of treatment with VGSC inhibitory compounds were observed. A Boyden chamber assay was also performed on MTC cell lines to examine changes in migration and invasion after administering the potential inhibitory compounds. This study has generated preliminary evidence that voltage-gated sodium channels (VGSCs) may indeed be potential targets for treating metastatic MTC. The results may be translated to future studies focused on controlling metastasis through specified drug treatment in MTC, leading to the limitation of cancer progression by means of targeting VGSCs. Citation Format: Paras Ahuja, Rachael Guenter, Jaden Cowan, Yazen Shihab, Jason Whitt, Herbert Chen, Sadanandan Velu, Renata Jaskula-Sztul. Inhibiting voltage-gated sodium channel activity in medullary thyroid cancer using small molecule compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2001.