Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.

Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.

Epidermoid Cyst in the Submandibular Region Mimicking Plunging Ranula: A Case Report.

Epidermoid cysts rarely present in the submandibular area, constituting approximately less than 7% of all cystic lesions in the head and neck region and less than 0.01% of all oral cavity cysts. Therefore, epidermoid cysts can be easily misdiagnosed, as the differential diagnosis for a submandibular area mass is very broad. Imaging can help define characteristics of the mass; however, a pathologic specimen is usually required for the final diagnosis. Surgical excision is often required and tolerated well by most patients. However, there is a risk of recurrence of the cyst after excision, as well as a rare chance for malignant transformation if not excised, which must be discussed with the patient at the time of diagnosis of epidermoid cyst. We present a 33-year-old Caucasian female with a left submandibular cystic mass measuring 4.7 cm x 2.9 cm, that was originally thought to be a plunging ranula and subsequently diagnosed as an epidermoid cyst. This report is meant to raise awareness of the possibility of a submandibular mass being an epidermoid cyst as well as appropriate workup, treatment, and prognosis of epidermoid cysts in the submandibular region. This report also describes a unique approach to a submandibular epidermoid cyst of which the submandibular gland is divided for access to the cyst for safe and effective excision. To the author's knowledge, this surgical approach has not been described in the literature for a submandibular epidermoid cyst.

Induction Chemotherapy Prior to Endoscopic Resection of Alveolar Rhabdomyosarcoma.

Head and neck rhabdomyosarcoma (HNRMS) is a rare type of soft tissue tumor that affects both adults and children with an overall incidence of 0.041 per 100,000 people. Adults make up approximately 31.2% of all HNRMS diagnoses and have an overall survival rate between 20% and 40%. We present a case of a 46-year-old male who initially presented with nasal congestion and vision changes. Maxillofacial computed tomography and magnetic resonance imaging of the brain showed involvement of the orbital apex, abutment of the planum sphenoidale, and extension to the foramen rotundum (FR). Nasal endoscopy with biopsy confirmed the diagnosis of T2aN0M0 parameningeal HNRMS. The patient underwent induction chemotherapy, followed by endoscopic resection, which resulted in negative intraoperative margins. Subsequently, he underwent adjuvant concurrent chemotherapy and proton beam radiation after positive microscopic positive margins were found on the optic nerve. The patient did not experience any significant complications, and he is currently without radiographic or clinical recurrence 18 months after the treatment. He was able to maintain his vision throughout the treatment. In adults, HNRMS is usually treated with chemoradiotherapy based on pediatric protocols, since there are limited data available for adult treatment protocols and outcomes. Although surgery has been associated with positive outcomes in adult patients, there are no previous reports of its use with either neoadjuvant or adjuvant treatment. This type of treatment protocol has never been described for adult HNRMS. We hope that our report can add more data to the growing body of literature on HNRMS treatment protocols.

A Rare Case of Vocal Cord Paralysis in the Setting of Ramsay Hunt Syndrome.

Ramsay Hunt syndrome (RHS) with concomitant vocal cord paralysis (VCP) is a rare finding. This case is particularly rare because the patient lacked the symptoms of otalgia or hearing loss when in fact, a majority of cases typically demonstrate both hearing loss and otalgia. Unique to this case is also the fact that it was complicated by a concomitant infarction of the splenium corpus callosum and a right temporal meningioma. The purpose of this study was to bring awareness to the fact that RHS can cause multiple cranial nerve neuropathies including VCP and should be included in the differential diagnosis for VCP.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Bovine herpesvirus 1 modified live virus vaccines for cattle reproduction: Balancing protection with undesired effects.

Bovine herpesvirus 1 (BoHV-1) has long been associated with reproductive failure in cattle following infection of the ovary and/or fetus. Vaccination prior to breeding has been an effective approach to lessen the impact of BoHV-1 on reproduction. Prior studies in the 1980s and 1990s established the susceptibility of the ovary and particularly the corpus luteum (CL) to BoHV-1 infection. A series of studies at breeding time established that: (1) in naïve animals, the CL was the major target of BoHV-1 pathology; (2) CL lesions occurred within 4-9 days after estrus; (3) similar lesions was seen with BoHV-1 MLV vaccines; (4) ovarian lesions varied by the vaccine strain used; (5) progesterone decreased with or without CL lesions; and (6) following reactivation of BoHV-1 latent infection, ovaries could become reinfected in the face of BoHV-1 immunity. Large scale field studies demonstrated that conception was highest in animals previously vaccinated and boostered with inactivated vaccine compared to animals revaccinated with MLV. In the early 2000s, to get a label claim to vaccinate calves nursing pregnant cows, safety study outlines were approved by USDA-APHIS CVB. These studies were designed to determine the effect of revaccination with MLV during pregnancy on previously vaccinated cows and were not rigorous enough to confirm complete fetal safety. As designed these studies showed no difference in reproductive loss between the previously vaccinated animals and the animals revaccinated ∼4, 7 and 9 months later, leading to the label approval for MLV vaccination in pregnant cows. Subsequent investigations by diagnostic laboratories found an increase in BoHV-1 reproductive loss after the approval for use in pregnant animals. A method was developed to differentiate IBR vaccine strains from field strains. Analysis of viruses from 31 cases from 2009-2016 indicated that all 31 isolates matched with vaccine strains. Going forward, it will be necessary to develop vaccine approaches that use non-abortifacient, nonlatent BoHV-1 vaccines that develop lifelong immunity, protecting the animal while doing no harm to the fetus.