Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367.

Artificial transcription factors are powerful tools for regulating gene expression. Here we report results with engineered zinc-finger transcription factors (ZF-TFs) targeting four protein-coding genes, OCT4, SOX2, KLF4 and c-MYC, and one noncoding ribonucleic acid (RNA) gene, the microRNA (miRNA) miR302/367 cluster. We designed over 300 ZF-TFs whose targets lie within 1 kb of the transcriptional start sites (TSSs), screened them for increased messenger RNA or miRNA levels in transfected cells, and identified potent ZF-TF activators for each gene. Furthermore, we demonstrate that selected ZF-TFs function with alternative activation domains and in multiple cell lines. For OCT4, we expanded the target range to -2.5 kb and +500 bp relative to the TSS and identified additional active ZF-TFs, including three highly active ZF-TFs targeting distal enhancer, proximal enhancer and downstream from the proximal promoter. Chromatin immunoprecipitation (FLAG-ChIP) results indicate that several inactive ZF-TFs targeting within the same regulatory region bind as well as the most active ZF-TFs, suggesting that efficient binding within one of these regulatory regions may be necessary but not sufficient for activation. These results further our understanding of ZF-TF design principles and corroborate the use of ZF-TFs targeting enhancers and downstream from the TSS for transcriptional activation.

Surgeon Perceptions of Performing Transforaminal Lumbar Interbody Fusion in an Ambulatory Surgical Center vs Hospital Setting in the Elderly Population: Results of a Surgeon Survey.

BACKGROUND: There is an increasing acceptance of conducting minimally invasive transforaminal lumbar interbody fusion (TLIF) in ambulatory surgical centers (ASCs). The Centers for Medicare and Medicaid Services (CMS) introduced the Hospitals Without Walls (HWW) program in March 2020. This program granted hospitals regulatory flexibility to offer services and procedures in nontraditional locations, including ASCs. However, implementation hurdles persist. METHODS: A survey was sent to 235 surgeons regarding the use of ASCs for performing TLIF surgeries on elderly patients. Multiple-choice questions covering various aspects of TLIF practice preferences, including surgical indications, decision factors for choosing ASCs over hospitals, implementation hurdles, reimbursement concerns, staffing issues, and the impact of CMS rules and regulations on TLIF in ASCs, particularly concerning physician ownership and self-referral conflicts governed by the Stark law, were asked. RESULTS: The survey completion rate was 25.8% (Figure 1). The most common surgical indications for TLIF in ASCs were spondylolisthesis (80%), spinal stenosis (62.5%), and low back pain (47.5%). Most surgeons (78%) believed TLIF could be safely performed in ASCs. Streamlined workflow, lower infection rates, and cost-effectiveness were advantages listed by 58.5% of surgeons. Patient's medical history (75.8%), followed by ASC resources and capabilities (61%) and surgeon preference (61%), were relevant factors. Higher efficiencies at ASCs (14.6%), contractual issues (9.8%), and ownership issues (7.3%) were less relevant to surgeons. About 65.9% of surgeons reported lower reimbursement in ASCs, and 43.9% said it was an implementation hurdle. Lower direct costs were reported by 53.7% of surgeons. Other hurdles included a lack of trained staff (24.4%), inadequate staffing (22.0%), cost overruns (26.8%), high Joint Commission or the Accreditation Association for Ambulatory Health Care credentialing costs, and surgeons feeling uncomfortable performing TLIF in ASCs (22.0%). Only 17.1% listed medical problems as a reason their patient was considered unsuitable for the ASC environment. A majority (53.7%) stated that their ASCs complied with strict Stark requirements by disclosing physician ownership interests. However, 22% of surgeons reported self-referrals under the "In-Office Ancillary Services Exception" allowed by the Stark law. CONCLUSION: Our survey data show that surgeons' perceptions of current CMS rules and regulations may hinder the transition into the ASC setting because they think the reimbursement is too low and the regulatory burden is too high. ASCs have disproportionally higher initial acquisition and ongoing costs related to staff training and maintenance of the TLIF technology that CMS should consider when determining the appropriate financial remuneration for these complex procedures. CLINICAL RELEVANCE: ASC offers a viable and attractive option for their TLIF procedure with the advantage of same-day discharge and at-home recovery.

Endoscopic posterior cricoid split with costal cartilage graft placement in patients under one year of age.

Endoscopic posterior cricoid split and costal rib graft placement (EPCSCG) is an important tool in enlarging the glottic and subglottic airway, both of which can be disproportionally affected in the small airways of neonates and early infants. We present a series of 8 patients under the age of one who successfully underwent EPCSCG, with 7/8 patients avoiding tracheostomy entirely. Of these patients, the indication for EPCSCG was isolated bilateral vocal fold immobility (6/8), bilateral vocal fold immobility with subglottic stenosis (1/8), and isolated subglottic stenosis (1/8). EPCSCG can be safely applied to select patients less than one year of age.

International pediatric otolaryngology group (IPOG) consensus on approach to aspiration.

OBJECTIVE: To provide recommendations for a comprehensive management approach for infants and children presenting with symptoms or signs of aspiration. METHODS: Three rounds of surveys were sent to authors from 23 institutions worldwide. The threshold for the critical level of agreement among respondents was set at 80 %. To develop the definition of "intractable aspiration," each author was first asked to define the condition. Second, each author was asked to complete a 5-point Likert scale to specify the level of agreement with the definition derived in the first step. RESULTS: Recommendations by the authors regarding the clinical presentation, diagnostic considerations, and medical and surgical management options for aspiration in children. CONCLUSION: Approach to pediatric aspiration is best achieved by implementing a multidisciplinary approach with a comprehensive investigation strategy and different treatment options.

Comprehensive management and classification of first branchial cleft anomalies: An International Pediatric Otolaryngology Group (IPOG) consensus statement.

OBJECTIVE: First branchial cleft anomalies are rare congenital head and neck lesions. Literature pertaining to classification, work up and surgical treatment of these lesions is limited and, in some instances, contradictory. The goal of this work is to provide refinement of the classification system of these lesions and to provide guidance for clinicians to aid in the comprehensive management of children with first branchial cleft anomalies. MATERIALS AND METHODS: Delphi method survey of expert opinion under the direction of the International Pediatric Otolaryngology Group (IPOG) was conducted to generate recommendations for the definition and management of first branchial cleft anomalies. The recommendations are the result of expert consensus and critical review of the literature. RESULTS: Consensus recommendations include evaluation and diagnostic considerations for children with first branchial cleft anomalies as well as recommendations for surgical management. The current Work classification system was reviewed, and modifications were made to it to provide a more cogent categorization of these lesions. CONCLUSION: The mission of the International Pediatric Otolaryngology Group (IPOG) is to develop expertise-based recommendations based on review of the literature for the management of pediatric otolaryngologic disorders. These consensus recommendations are aimed at improving care of children presenting with first branchial cleft anomalies. Here we present a revised classification system based on parotid gland involvement, with a focus on avoiding stratification based on germ layer, in addition to guidelines for management.

Comparison of Transforaminal Lumbar Interbody Fusion in the Ambulatory Surgery Center and Traditional Hospital Settings, Part 1: Multi-Center Assessment of Surgical Safety.

(1) Background: The technological advances achieved with minimally-invasive surgery have enabled procedures to be undertaken in outpatient settings, and there has been growing acceptance of performing minimally-invasive transforaminal interbody fusion (TLIF) in the ambulatory surgery center (ASC). The purposeof this study was to determine the comparative 30 day safety profile for patients treated with TLIF in the ASC versus the hospital setting. (2) Methods: This multi-center study retrospectively collected baseline characteristics, perioperative variables, and 30 day postoperative safety outcomes for patients having a TLIF using the VariLift®-LX expandable lumbar interbody fusion device. Outcomes were compared between patients undergoing TLIF in the ASC (n = 53) versus in the hospital (n = 114). (3) Results: Patients treated in-hospital were significantly older, frailer and more likely to have had previous spinal surgery than ASC patients. Preoperative back and leg pain scores were similar between study groups (median, 7). ASC patients had almost exclusively one-level procedures (98%) vs. 20% of hospital procedures involving two-levels (p = 0.004). Most procedures (>90%) employed a stand-alone device. The median length of stay for hospital patients was five times greater than for ASC patients (1.4 days vs. 0.3 days, p = 0.001). Emergency department visits, re-admissions and reoperations were rare whether the patients were managed in the traditional hospital setting or the ASC. (4) Conclusions: There were equivalent 30 day postoperative safety profiles for patients undergoing a minimally-invasive TLIF irrespective of surgical setting. For appropriately selected surgical candidates, the ASC offers a viable and attractive option for their TLIF procedure with the advantage of same-day discharge and at-home recovery.

Slide Tracheoplasty: Complete Tracheal Rings and Beyond.

Congenital tracheal stenosis is a rare but potentially life-threatening condition that is most commonly caused by complete tracheal rings. Slide tracheoplasty was initially introduced as a surgical treatment for congenital tracheal stenosis in 1989 and has significantly improved outcomes and overall survival rates for these patients. It has subsequently been adapted to treat other conditions such as laryngotracheal stenosis, tracheoesophageal fistula, and bronchial stenosis. This article reviews the history, the variety of applications, perioperative management, surgical techniques, potential complications, and new frontiers in slide tracheoplasty surgery.

Pediatric tonsillectomy: A short-term and long-term comparison of intracapsular versus extracapsular techniques.

OBJECTIVE: To review a cohort of over 2500 patients and investigate the short and long-term outcomes of intracapsular as compared to extracapsular tonsillectomy, and show if the complication rates are comparable between methods. STUDY DESIGN: A multicenter, retrospective chart review was conducted, evaluating pediatric tonsillectomies performed from 2004 to 2014. The electronic medical record was reviewed through December 2018, providing up to 14 years of follow-up data. SETTING: Two tertiary care, academic medical centers. SUBJECTS AND METHODS: A retrospective chart review was conducted to identify children undergoing tonsillectomy and adenotonsillectomy. A chart review was first performed of patients by a single surgeon (MEG) and then the analysis was repeated using enterprise data warehouse (EDW) to search for complications and interventions using International Classification of Diseases, ninth revision, (ICD-9) and Current Procedural Terminology (CPT) codes. The second surgeon's patients (JLC) patients were added to increase the cohort. Patients were excluded from the review of long-term outcomes if there was less than two-year follow-up. Short-term outcomes examined included rate of post-tonsillectomy hemorrhage and re-presentation for dehydration, while long-term outcomes included rates of peritonsillar abscess and tonsillar regrowth requiring revision tonsillectomy. RESULTS: A total of 2508 pediatric patients were identified who had undergone tonsillectomy or adenotonsillectomy. In 1456 (58.1%) of these patients, the intracapsular technique was used and in 1052 (41.9%) patients, the extracapsular technique was used. The mean documented follow-up time was 8.2 years. Thirty-five patients (1.4%) were identified with post-tonsillectomy hemorrhage, 2 of these patients (5.7%) with primary hemorrhage and 33 patients (94.3%) with secondary hemorrhage. 11 underwent intracapsular tonsillectomy and 24 underwent extracapsular tonsillectomy (p = 0.0042). The rate of post-tonsillectomy hemorrhage with intracapsular tonsillectomy was 0.76%, compared to 2.3% in the extracapsular group. Three patients (0.12%) undergoing intracapsular tonsillectomy required revision tonsillectomy; no patients in the extracapsular group required revision surgery. Three patients (0.12%) developed peritonsillar abscess post-operatively, two following intracapsular tonsillectomy and one following extracapsular tonsillectomy. CONCLUSION: This retrospective review comparing the intracapsular and extracapsular techniques for tonsillectomy provides further evidence of the benefits of this technique. It is worthwhile to continue offering intracapsular tonsillectomy to patients and their families during pre-operative discussions.

Simulation of Pediatric Endoscopic Cricoid Reduction and Expansion.

Endoscopic cricoid expansion and reduction are newer approaches to the management of pediatric bilateral vocal fold immobility and postlaryngotracheal reconstruction glottic insufficiency, respectively. These procedures offer a less invasive, endoscopic alternative to procedures that typically required open management with a more prolonged recovery. These technically demanding procedures are currently performed only in select centers, and there is no currently described training model for practicing them. We present a modification to a laryngeal dissection station that allows for simulation of endoscopic cricoid reduction and expansion with excised larynges. The model allows trainees to practice endoscopic posterior cricoid exposure, incision of the cricoid cartilage, placement of a simulated costal cartilage graft for expansion, and endoscopic suturing for reduction. Development of simulators for procedures that are infrequently performed have the potential to help trainees reach surgical competency faster and more safely.

Mutational analysis of an RNA polymerase II elongation factor in Drosophila melanogaster.

The ELL family of proteins function in vitro as elongation factors for RNA polymerase II. Deletion studies have defined domains in mammalian ELL required for transcription elongation activity and RNA polymerase binding in vitro, for transformation of cultured cells when overexpressed, and for leukemogenesis and cell proliferation as part of a leukemic fusion protein. The goal of this study was to identify domains required for chromosome targeting and viability in the unique Drosophila ELL (dELL) protein. Here, we show that an N-terminal domain of dELL is necessary and sufficient for targeting to transcriptionally active puff sites in chromatin, supporting a role for this domain in recruiting dELL to elongating RNA polymerase II. We demonstrate that a central domain of dELL is required for rapid mobilization of ELL during the heat shock response, suggesting a regulatory function for this domain. Unexpectedly, transgenic dELL in which the N-terminal chromosome binding domain is deleted can complement the recessive lethality of mutations in ELL, suggesting that Drosophila ELL has an essential activity in development distinct from its role as an RNA polymerase II elongation factor.

Endoscopic posterior costal cartilage graft placement for acute management of pediatric bilateral vocal fold paralysis without tracheostomy.

Endoscopic posterior cricoid split with costal cartilage graft stabilization has previously been shont to allow for glottic/infraglottic expansion in children with long standing vocal fold paralysis. We report on an extension of this technique to use in the acute setting in the management of acute BVP in children with acute upper airway obstructive symptoms.

Protein 4.1B/differentially expressed in adenocarcinoma of the lung-1 functions as a growth suppressor in meningioma cells by activating Rac1-dependent c-Jun-NH(2)-kinase signaling.

Meningiomas are the second most common brain tumor in adults, yet comparatively little is presently known about the dysregulated growth control pathways involved in their formation and progression. One of the most frequently observed genetic changes in benign meningioma involves loss of protein 4.1B expression. Previous studies from our laboratory have shown that protein 4.1B growth suppression in meningioma is associated with the activation of the c-Jun-NH(2)-kinase (JNK) pathway and requires localization of a small unique region (U2 domain) of protein 4.1B to the plasma membrane. To define the relationship between protein 4.1B expression and JNK activation, as well as to determine the mechanism of JNK activation by protein 4.1B, we used a combination of genetic and pharmacologic approaches. In this report, we show that protein 4.1B/differentially expressed in adenocarcinoma of the lung-1 (DAL-1) expression in meningioma cells in vitro results in JNK activation, which requires the sequential activation of Src, Rac1, and JNK. In addition, inhibition of Rac1 or JNK activation abrogates protein 4.1B/DAL-1 growth suppression and cyclin A regulation. Last, protein 4.1B/DAL-1 regulation of this critical growth control pathway in meningioma cells requires the presence of the U2 domain. Collectively, these observations provide the first mechanistic insights into the function of protein 4.1B as a growth regulator in meningioma cells.

Adenoidectomy With Balloon Catheter Sinuplasty: A Randomized Trial for Pediatric Rhinosinusitis.

OBJECTIVES/HYPOTHESIS: To compare treatment outcomes in pediatric patients with chronic rhinosinusitis (CRS) using adenoidectomy and maxillary sinus irrigation with or without balloon catheter sinuplasty (BCS). METHODS: Prospective, randomized, and blinded. Patients between 2 and 12 years who met clinical and computed tomography criteria for CRS unresponsive to maximal medical therapy and no prior sinus- or adenoid-related procedures were eligible. Twenty-five patients were randomized, had surgery, and completed the follow-up. The control group underwent adenoidectomy with maxillary sinus irrigation alone; the treatment group underwent the same, with the addition of maxillary BCS for the maxillary sinus irrigation. Patients and parents were blinded to group assignment throughout the follow-up time frame. A validated questionnaire, the Sinus and Nasal Quality of Life Survey (SN-5), was used to measure health status changes and quality of life (QOL) at baseline, 6 months, and 12 months postintervention. Data was analyzed using t tests and multivariate linear regression. RESULTS: There were 12 patients in the BCS group and 13 in the control group. Other than a higher atopic history in the control group (P = 0.047), there were no significant differences (P = 0.07) in the two groups (demographics and baseline SN-5 scores). Following surgery, both groups demonstrated similar improvement in QOL scores and median SN-5 scores in all domains (number of sinus infections, nasal obstruction, allergy symptoms, emotional distress, and activity limitations). CONCLUSION: The addition of BCS to adenoidectomy/maxillary sinus irrigation did not provide additional QOL and sinonasal symptom improvement in the surgical treatment of pediatric CRS. LEVEL OF EVIDENCE: 1b Laryngoscope, 128:2893-2897, 2018.

Ideal Characteristics of a Laser-Protected Endotracheal Tube: ABEA and AHNS Member Survey and Biomechanical Testing.

OBJECTIVES: To determine the characteristics of laser-protected endotracheal tubes (LPETs) valued by otolaryngologists performing transoral laser surgery in the head and neck and to measure LPET stiffness. METHODS: An online questionnaire was completed by American Broncho-Esophagological Association (ABEA) and American Head and Neck Society (AHNS) members. LPET distal end compliance was measured in a biomechanics laboratory. RESULTS: A total of 228 out of 2109 combined ABEA and AHNS members completed the survey. The following LPET characteristics, which were properties of the Medtronic Laser-Shield II tube (MLST), were highly valued: softness and flexibility, surface smoothness, and a tight-to-shaft balloon (all P < .01). Prior to industry-driven discontinuation of the MLST, 52% of surgeons (78% of fellowship-trained laryngologists [FTLs]) reported using it; afterward, 58% reported using the stainless steel, Mallinckrodt Laser-Flex tube (MLFT). Forty-six percent of all respondents (69% of FTLs) did not consider cost being a factor in LPET choice. Biomechanical testing revealed the distal end of the MLST to be 3.45 times more compliant than the MLFT ( P < .01). CONCLUSION: Members of the ABEA and AHNS, particularly FTLs, highly value distinguishing properties of the now discontinued MLST. Manufacturers should consider this in the design of new LPETs.

In vivo requirement of the RNA polymerase II elongation factor elongin A for proper gene expression and development.

A number of transcription factors that increase the catalytic rate of mRNA synthesis by RNA polymerase II (Pol II) have been purified from higher eukaryotes. Among these are the ELL family, DSIF, and the heterotrimeric elongin complex. Elongin A, the largest subunit of the elongin complex, is the transcriptionally active subunit, while the smaller elongin B and C subunits appear to act as regulatory subunits. While much is known about the in vitro properties of elongin A and other members of this class of elongation factors, the physiological role(s) of these proteins remain largely unclear. To elucidate in vivo functions of elongin A, we have characterized its Drosophila homologue (dEloA). dEloA associates with transcriptionally active puff sites within Drosophila polytene chromosomes and exhibits many of the expected biochemical and cytological properties consistent with a Pol II-associated elongation factor. RNA interference-mediated depletion of dEloA demonstrated that elongin A is an essential factor that is required for proper metamorphosis. Consistent with this observation, dEloA expression peaks during the larval stages of development, suggesting that this factor may be important for proper regulation of developmental events during these stages. The discovery of the role of elongin A in an in vivo model system defines the novel contribution played by RNA polymerase II elongation machinery in regulation of gene expression that is required for proper development.

Membrane localization of the U2 domain of Protein 4.1B is necessary and sufficient for meningioma growth suppression.

Meningiomas are common central nervous system tumors; however, the molecular mechanisms underlying their pathogenesis are largely undefined. Previous work has implicated Protein 4.1B as an important tumor suppressor involved in the development of these neoplasms. In this report, we demonstrate that the U2 domain is necessary and sufficient for the ability of Protein 4.1B to function as a meningioma growth suppressor. Using a series of truncation and deletion constructs of DAL-1 (a fragment of Protein 4.1B that retains all the growth suppressive properties), we narrowed the domain required for 4.1B growth suppression to a fragment containing a portion of the FERM domain and the U2 domain using clonogenic assays on meningioma cells. Deletion of the U2 domain in the context of the full-length DAL-1 molecule eliminated growth suppressor function, as measured by thymidine incorporation and caspase-3 activation. Moreover, targeting the U2 domain to the plasma membrane using a membrane localization signal (MLS) reduced cell proliferation, similar to wild-type DAL-1. Collectively, the data suggest that the U2 domain, when properly targeted to the plasma membrane, contains all the residues necessary for mediating Protein 4.1B growth suppression.

An adolescent girl with Crohn's disease, fever, and sore throat.

An adolescent female with a past medical history significant for Crohn's disease presented with fevers, tonsillitis without exudate, and tender posterior cervical lymphadenopathy. Laboratory results showed transaminitis, leukocytosis with a left shift, and atypical lymphocytes on a blood smear. The patient did not respond to supportive care or dexamethasone, necessitating a tonsillectomy and adenoidectomy. Although her presentation was consistent with infectious mononucleosis, diagnosis was not confirmed until Epstein-Barr virus (EBV) polymerase chain reaction (PCR) from tonsillar tissue was positive. False-negative results on the heterophile antibody test are common in pediatric populations and the detection of EBV antibodies is further complicated in immunocompromised patients. Studies indicate PCR is a more sensitive test, although there is no consensus regarding ideal material to use or quantitative levels necessitating intervention.