Inspiratory and sigh breathing rhythms depend on distinct cellular signalling mechanisms in the preBötzinger complex.

Breathing behaviour involves the generation of normal breaths (eupnoea) on a timescale of seconds and sigh breaths on the order of minutes. Both rhythms emerge in tandem from a single brainstem site, but whether and how a single cell population can generate two disparate rhythms remains unclear. We posit that recurrent synaptic excitation in concert with synaptic depression and cellular refractoriness gives rise to the eupnoea rhythm, whereas an intracellular calcium oscillation that is slower by orders of magnitude gives rise to the sigh rhythm. A mathematical model capturing these dynamics simultaneously generates eupnoea and sigh rhythms with disparate frequencies, which can be separately regulated by physiological parameters. We experimentally validated key model predictions regarding intracellular calcium signalling. All vertebrate brains feature a network oscillator that drives the breathing pump for regular respiration. However, in air-breathing mammals with compliant lungs susceptible to collapse, the breathing rhythmogenic network may have refashioned ubiquitous intracellular signalling systems to produce a second slower rhythm (for sighs) that prevents atelectasis without impeding eupnoea. KEY POINTS: A simplified activity-based model of the preBötC generates inspiratory and sigh rhythms from a single neuron population. Inspiration is attributable to a canonical excitatory network oscillator mechanism. Sigh emerges from intracellular calcium signalling. The model predicts that perturbations of calcium uptake and release across the endoplasmic reticulum counterintuitively accelerate and decelerate sigh rhythmicity, respectively, which was experimentally validated. Vertebrate evolution may have adapted existing intracellular signalling mechanisms to produce slow oscillations needed to optimize pulmonary function in mammals.

Assessment of guidelines for bariatric and metabolic surgery: a systematic review and evaluation using appraisal of guidelines for research and evaluation II (AGREE II).

BACKGROUND: In recent years, multiple guidelines on bariatric and metabolic surgery were published, however, their quality remains unknown, leaving providers with uncertainty when using them to make perioperative decisions. This study aims to evaluate the quality of existing guidelines for perioperative bariatric surgery care. METHODS: A comprehensive search of MEDLINE and EMBASE were conducted from January 2010 to October 2022 for bariatric clinical practice guidelines. Guideline evaluation was carried out using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) framework. RESULTS: The initial search yielded 1483 citations, of which, 26 were included in final analysis. The overall median domain scores for guidelines were: (1) scope and purpose: 87.5% (IQR: 57-94%), (2) stakeholder involvement: 49% (IQR: 40-64%), (3) rigor of development: 42.5% (IQR: 22-68%), (4) clarity of presentation: 85% (IQR: 81-90%), (5) applicability: 6% (IQR: 3-16%), (6) editorial independence: 50% (IQR: 48-67%), (7) overall impressions: 48% (IQR: 33-67%). Only six guidelines achieved an overall score >70%. CONCLUSIONS: Bariatric surgery guidelines effectively outlined their aim and presented recommendations. However, many did not adequately seek patient input, state search criteria, use evidence rating tools, and consider resource implications. Future guidelines should reference the AGREE II framework in study design.

Patient-Reported Outcomes Measures in Rhinoplasty: Need for Use and Implementation.

Patient-reported outcome metrics (PROMs) are increasingly utilized to capture data about patients' quality of life. PROMs play an important role in the value-based health care movement by providing a patient-centered metric of quality. There are many barriers to the implementation of PROMs, and widespread adoption requires buy-in from numerous stakeholders including patients, clinicians, institutions, and payers. Several validated PROMs have been utilized by facial plastic surgeons to measure both functional and aesthetic outcomes among rhinoplasty patients. These PROMs can help clinicians and rhinoplasty patients participate in shared decision making (SDM), a process via which clinicians and patients arrive at treatment decisions together through a patient-centered approach. However, widespread adoption of PROMs and SDM has not yet been achieved. Further work should focus on overcoming barriers to implementation and engaging key stakeholders to increase the utilization of PROMs in rhinoplasty.

Development of consensus-derived quality indicators for laparoscopic sleeve gastrectomy operative reports.

BACKGROUND: Synoptic operative reporting has been used as a solution to the poor quality of narrative reports. The aim of this study was to develop operative report quality indicators for the laparoscopic sleeve gastrectomy and to generate parameters by which these reports can be evaluated and improved. METHODS: A Delphi protocol was used to determine quality indicators for LSG. Bariatric surgeons across Canada were recruited along with key physician stakeholders to participate via a secure web-based platform. Transferrable consensus items for LSG from previously developed Roux-en-Y gastric bypass operative indictors were put forward for consideration. Participants also initially submitted potential QIs. These were grouped by theme. Items were rated on 5-point Likert scales in subsequent rounds. Scores of 70% or higher were used for inclusion and 30% or less denoted exclusion. Elements scoring 30% to 70% agreement were recirculated by runoff in subsequent rounds to generate the final list of quality indicators. RESULTS: Seven bariatric surgeons, representing all regions preforming LSG in Canada, were invited to participate in the Delphi group. Multidisciplinary invitees included one academic minimally invasive/acute care surgeon, one tertiary abdominal radiologist, and one academic gastroenterologist with bariatric expertise. Two rounds were required to achieve consensus. Both rounds achieved a 100% response (10/10). In round 1, forty items reached consensus. In Round 2, an additional 28 items reached consensus, with three items excluded, bringing the total number of quality indicators to 65. CONCLUSION: This study establishes consensus-derived multidisciplinary quality indicators for LSG operative reports. Application of these findings aims to advance the quality and completeness of operative reporting in LSG in order to improve communication of important surgical details and quality measures to the multidisciplinary team involved in bariatric surgery care.

Extracellular vesicle biomarkers of Alzheimer's disease associated with sub-clinical cognitive decline in late middle age.

INTRODUCTION: Neuronal extracellular vesicle (nEV) tau and insulin signaling biomarkers may detect preclinical Alzheimer's disease and age-associated cognitive decline. METHODS: This case-control study used repeated serum samples from 73 cognitively declining and 73 stable Wisconsin Registry for Alzheimer's Prevention participants (62.4 ± 6.3 years old). We immunocaptured nEVs; measured tau and insulin signaling biomarkers; and examined biomarker differences by group, their performance in group classification in training and test datasets (97, 49 individuals, respectively), and whether they predict cognitive performance change. RESULTS: Declining compared to stable individuals showed higher baseline total, p231-, and p181-tau with older age and higher annualized change for p-IR and p-IGF-1R. Combining biomarkers classified decliners with 94% area under the curve (AUC), 86.0% sensitivity and 86.7% specificity, in training data, and 75% AUC, 71.4% sensitivity, and 77.3% specificity, in test data. Insulin biomarkers predicted cognitive performance change prospectively. DISCUSSION: Combining nEV biomarkers can identify individuals with age-associated cognitive decline.

Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD.

Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson's disease (PD), while normal physiological levels of SNCA are needed to maintain neuronal function. We ought to develop new therapeutic strategies targeting the regulation of SNCA expression. DNA methylation at SNCA intron 1 regulates SNCA transcription, and PD brains showed differential methylation levels compared to controls. Thus, DNA methylation at SNCA intron 1 is an attractive target for fine-tuned downregulation of SNCA levels. Here we developed a system, comprising an all-in-one lentiviral vector, for targeted DNA methylation editing within intron 1. The system is based on CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A). Applying the system to human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons from a PD patient with the SNCA triplication resulted in fine downregulation of SNCA mRNA and protein mediated by targeted DNA methylation at intron 1. Furthermore, the reduction in SNCA levels by the guide RNA (gRNA)-dCas9-DMNT3A system rescued disease-related cellular phenotype characteristics of the SNCA triplication hiPSC-derived dopaminergic neurons, e.g., mitochondrial ROS production and cellular viability. We established that DNA hypermethylation at SNCA intron 1 allows an effective and sufficient tight downregulation of SNCA expression levels, suggesting the potential of this target sequence combined with the CRISPR-dCas9 technology as a novel epigenetic-based therapeutic approach for PD.

Creation of a new clinical framework - why women choose mastectomy versus breast conserving therapy.

BACKGROUND: Clinical medicine has lagged behind other fields in understanding and utilizing frameworks to guide research. In this article, we introduce a new framework to examine why women choose mastectomy versus breast conserving therapy in early stage breast cancer, and highlight the importance of utilizing a conceptual framework to guide clinical research. METHODS: The framework we present was developed through integrating previous literature, frameworks, theories, models, and the author's past research. RESULTS: We present a conceptual framework that illustrates the central domains that influence women's choice between mastectomy versus breast conserving therapy. These have been organized into three broad constructs: clinicopathological factors, physician factors, and individual factors with subgroups of sociodemographic, geographic, and individual belief factors. The aim of this framework is to provide a comprehensive basis to describe, examine, and explain the factors that influence women's choice of mastectomy versus breast conserving therapy at the individual level. CONCLUSION: We have developed a framework with the purpose of helping health care workers and policy makers better understand the multitude of factors that influence a patient's choice of therapy at an individual level. We hope this framework is useful for future scholars to utilize, challenge, and build upon in their own work on decision-making in the setting of breast cancer. For clinician-researchers who have limited experience with frameworks, this paper will highlight the importance of utilizing a conceptual framework to guide future research and provide an example.

The Effects of Perioperative Tamoxifen Therapy on Microvascular Flap Complications in Transverse Rectus Abdominis Myocutaneous/Deep Inferior Epigastric Perforator Flap Breast Reconstruction.

BACKGROUND: Tamoxifen is an important adjunct therapy in breast cancer treatment; however, it has been implicated in increasing microvascular flap complications. Current recommendations on stopping tamoxifen are conflicting and do not address tamoxifen therapy that is continued perioperatively. The purpose of this study is to determine whether tamoxifen taken at the time of free transverse rectus abdominis myocutaneous (TRAM) and deep inferior epigastric perforator (DIEP) flap breast reconstruction affects thrombotic complication rates. METHODS: A retrospective review of microvascular breast reconstruction with TRAM/DIEP flaps over the last 20 years was carried out at a single institution. Patients were divided into 2 cohorts: those receiving tamoxifen at the time of reconstruction (tamoxifen cohort) and those not taking tamoxifen (control). Demographic information, procedural characteristics, and rates of microvascular flap complications were compared. RESULTS: Forty-three patients (56 flaps) received tamoxifen at the time of microvascular breast reconstruction, and 185 patients (267 flaps) did not. Patients in the tamoxifen cohort had a lower mean age of 48.9 years (P = 0.013). A greater percentage of patients in the tamoxifen cohort had preoperative radiation (P < 0.0001) and chemotherapy (P = 0.018) and underwent delayed reconstruction (P < 0.0001). There were no significant differences between the 2 cohorts with regard to flap complications including both arterial and venous thrombosis, flap failure, and other local flap complications. CONCLUSIONS: Patients receiving tamoxifen during TRAM/DIEP flap breast reconstruction did not have increased rates of flap thrombosis or failure; therefore, stopping tamoxifen prior to these procedures may not be necessary.

Direct Neck Lift for Men.

Direct neck lift offers an excellent surgical technique for men seeking to rejuvenate the neck and avoid a full rhytidectomy. In this chapter, we provide an overview of direct submentoplasty techniques, as well as clinical pearls to consider in the preoperative, intraoperative, and postoperative periods. Different surgical incisions and resultant scars in the anterior neck are discussed and illustrated with figures. Given the degree of variation of submental fullness with which patients present, it is beneficial to be familiar with several different techniques to address the submental and submandibular areas.

Decision-making in the management of obesity: a scoping review protocol.

OBJECTIVE: This scoping review will evaluate the current published literature on decision-making in obesity management. INTRODUCTION: Obesity is increasing in incidence worldwide. Although indications have been established for a variety of available treatment modalities, treatment selection must also factor in patient preferences, clinician expertise, and resource availability. Such considerations are crucial given the exponential expansion of new surgical techniques and pharmacologic options in the last decade. Although literature exists for decision-making on various obesity management topics, there are no scoping reviews systematically mapping the literature. This scoping review is timely given that the treatment of obesity has evolved into a multidisciplinary endeavor with myriad management decisions that both patients and clinicians must navigate. INCLUSION CRITERIA: The review will consider for inclusion full-text primary studies, published in English from the year 2000 onwards, pertaining to decision-making in obesity management for health care providers involved in obesity management for patients aged ≥18 years. METHODS: This scoping review will be conducted in accordance with the JBI methodology for scoping reviews. Embase (Elsevier), MEDLINE (PubMed), Scopus (Elsevier), Web of Science (Clarivate), CINAHL Complete (EBSCO), PsycINFO (EBSCO), and Cochrane Central (Wiley) will be systematically searched using a predefined strategy. Two independent reviewers will conduct a 3-tiered screen of identified articles, with a third reviewer resolving disputes. Data extraction will be performed using a predefined, yet flexible form. Descriptive summaries and mapping will be provided for included studies. Available evidence and knowledge gaps will be identified and summarized as they relate to specific concepts, populations, and contexts in obesity management decision-making.

Characterization of the tumor immune microenvironment of sinonasal squamous-cell carcinoma.

BACKGROUND: Treatment and prognosis of sinonasal squamous-cell carcinoma (SNSCC) have not significantly improved despite improvements in radical therapy. Characterization of the tumor immune microenvironment (TiME) may identify patient subgroups associated with disease recurrence, and provide new biomarkers for improved patient stratification and treatment. METHODS: The TiME was quantitatively evaluated by multiplex immunohistochemistry (mIHC) in archived tissue sections from 38 patients with SNSCC, and were assessed for differences between recurrent (n = 20) and nonrecurrent (n = 18) groups. Hierarchical clustering analyses were performed to identify phenotypic TiME subgroups within the cohort and were used to compare survival outcomes. RESULTS: Our mIHC analysis revealed increased T-cell populations and decreased myeloid-cell populations in SNSCC patients without recurrent disease, as compared with patients with recurrent disease. Within T-cell subsets, there was a significantly higher percentage of granzyme B+ , T-bet+ , Eomes+ T cells, as well as higher proliferation of CD8+ T cells within the nonrecurrent group relative to the recurrent group. Furthermore, immune-cell complexity profiles of SNSCC revealed hyper- and hypo-T-cell-inflamed, myeloid-inflamed, B-cell-inflamed, and broadly hypoinflamed subtypes not previously identified by gene expression analyses. Our study revealed that presence of either hyper- or hypo-T-cell-inflamed TiME subtypes were associated with increased survival outcomes as compared with broadly hypoinflamed TiME subtypes (p = 0.035 and 0.0376, respectively). CONCLUSIONS: The TiME of SNSCC reveals distinct subtypes, which may correlate with recurrence and survival outcomes.

Cell-Type Specific Changes in DNA Methylation of SNCA Intron 1 in Synucleinopathy Brains.

Parkinson's disease (PD) and dementia with Lewy body (DLB) are the most common synucleinopathies. SNCA gene is a major genetic risk factor for these diseases group, and dysregulation of its expression has been implicated in the genetic etiologies of several synucleinopathies. DNA methylation at CpG island (CGI) within SNCA intron 1 has been suggested as a regulatory mechanism of SNCA expression, and changes in methylation levels at this region were associated with PD and DLB. However, the role of DNA methylation in the regulation of SNCA expression in a cell-type specific manner and its contribution to the pathogenesis of PD and DLB remain poorly understood, and the data are conflicting. Here, we employed a bisulfite pyrosequencing technique to profile the DNA methylation across SNCA intron 1 CGI in PD and DLB compared to age- and sex-matched normal control subjects. We analyzed homogenates of bulk post-mortem frozen frontal cortex samples and a subset of neuronal and glia nuclei sorted by the fluorescence-activated nuclei sorting (FANS) method. Bulk brain tissues showed no significant difference in the overall DNA methylation across SNCA intron 1 CGI region between the neuropathological groups. Sorted neuronal nuclei from PD frontal cortex showed significant lower levels of DNA methylation at this region compared to normal controls, but no differences between DLB and control, while sorted glia nuclei exhibited trends of decreased overall DNA methylation in DLB only. In conclusion, our data suggested disease-dependent cell-type specific differential DNA methylation within SNCA intron 1 CGI. These changes may affect SNCA dysregulation that presumably mediates disease-specific risk. Our results can be translated into the development of the SNCA intron 1 CGI region as an attractive therapeutics target for gene therapy in patients who suffer from synucleinopathies due to SNCA dysregulation.

Diabetes improvement and bariatric surgery-review of laparoscopic Roux-en-Y gastric bypass vs. laparoscopic vertical sleeve gastrectomy.

Obesity and type 2 diabetes mellitus (T2DM) are globally escalating major health care issues. For both obesity and T2DM management, it has been well established that bariatric surgery is superior to lifestyle and medical management alone. Over the past two decades, the introduction of laparoscopic vertical sleeve gastrectomy (LVSG) has seen a marked rise in usage, and combined with laparoscopic Roux-en-Y gastric bypass (LRYGB), these two procedures represent more than 80% of all bariatric surgeries globally. However, the differences in effectiveness between these two procedures have been less clearly defined. This article will serve as a focused review of the literature comparing LRYGB and LVSG for T2DM management. Based on our review, we believe that both procedures are very effective at improving T2DM care, especially compared with conventional medical management. However, there may be a modest benefit to be had by using LRYGB over LVSG.

Objective measures and the standardized letter of recommendation in the otolaryngology residency match.

OBJECTIVES/HYPOTHESIS: The objective of this study were to determine if any of the 10 attributes of the standardized letter of recommendation (SLOR) correlate significantly with objective measures of each applicant and to determine whether professor characteristics were associated with reported SLOR attributes. STUDY DESIGN: Retrospective cohort study. METHODS: We reviewed 339 SLORs from 187 otolaryngology residency applicants. The 10 SLOR attributes were converted into a percentile score and statistically analyzed. RESULTS: All 10 SLOR attributes had a mean ± standard deviation above the 80th percentile (0.82-0.93 ± 0.08-0.52) and negative skewness. United States Medical Licensing Examination step 1 and 2 scores correlated most strongly with match potential (B = 93.1,P < .001 and B = 96.3, P = .002, respectively). Students from top 40 National Institutes of Health-funded institutions had a significant difference in length of relationship with their letter writer (B = -0.51, P = .02) and interpersonal communication skills (B = -12.9, P = .011). Alpha Omega Alpha (AOA) and non-AOA members differed in medical knowledge (B = 21.0, P = .007), research (B = -8.80, P = .036), and commitment to otolaryngology (B = -25.89, P = .020). Total number of research experiences correlated most strongly with medical knowledge (B = -16.55, P = .011). There was a significant difference in means between length of relationship and professorial rank (F[4, 339] = 3.61, P = .023), administrative position (F[5, 339] = 5.43, P = .002), and institution of the letter writer (P < .001). CONCLUSIONS: Overall, the lack of correlation between SLOR attributes and objective measures suggests that the SLOR adds little to the residency application. Professor characteristics were similarly not associated with any of the 10 SLOR attributes; however, there were significant differences in the length of relationship between the professor and students with respect to professor rank, administrative rank, and institution of the letter writer. LEVEL OF EVIDENCE: NA Laryngoscope, 130:603-608, 2020.

Mitochondrial Protrusions in Neuronal Cells.

Mitochondrial function relies on multiple quality control mechanisms, including the release of mitochondrial vesicles. To investigate the ultrastructure and prevalence of mitochondrial membranous protrusions (and, by extension, vesicles) in neurons, we surveyed mitochondria in rat and planarian brains using transmission electron microscopy (EM). We observed that mitochondrial protrusions mostly extend from the outer membrane. Leveraging available 3D EM datasets of the brain, we further analyzed mitochondrial protrusions in neurons of mouse and Drosophila brains, identifying high-resolution spatial views of these protrusions. To assess whether the abundance of mitochondrial protrusions and mitochondria-derived vesicles respond to cellular stress, we examined neurons expressing fluorescently tagged mitochondrial markers using confocal microscopy with Airyscan and found increased numbers of mitochondrial protrusions and vesicles with mild stress. Future studies using improved spatial resolution with added temporal information may further define the functional implications of mitochondrial protrusions and vesicles in neurons.

Validation of a septoplasty deformity grading system for the evaluation of nasal obstruction.

OBJECTIVES/HYPOTHESIS: We developed and validated a septal deformity grading (SDG) system that accounts for anatomic location and grading of deformity severity. STUDY DESIGN: Retrospective cohort study. METHODS: Subjects were patients with nasal obstruction presenting to University of California, Irvine Medical Center. Subjects were given pre- and postoperative Nasal Obstruction Symptom Evaluation (NOSE) questionnaires and were evaluated by a facial plastic surgeon using our septal deformity grading (SDG) system. Validity and reliability analyses were conducted on the SDG results. Statistical analyses were conducted on SDG and NOSE data to assess and compare instruments, and to validate the SDG instrument using the NOSE instrument. RESULTS: One hundred thirty-five patients met inclusion criteria. Cronbach's α was ≥ 0.7 for SDG and pre- and postoperative NOSE scores. There was a significant difference in pre- and postoperative NOSE scores (Z score = -7.21, P < .001). Correlations between postoperative NOSE and SDG scores were significant (P = .014), and convergent construct validity was achieved. There was a significant difference in SDG scores between primary versus revision operations (P < .001), history versus no history of nasal trauma, and nasal/septal surgery (P = .025, P = .003, respectively). The odds of having a revision operation were 2.3 times higher for high SDG scores (P < .001), of having a history of nasal trauma were 1.33 times higher for high SDG scores (P = .014), and of having a history of nasal/septal surgery were 2.9 times higher for low SDG scores. CONCLUSIONS: Our SDG system addresses the challenge of providing objective anatomic information on the severity of nasal septal deformities, and may be valuable when used in conjunction with subjective data gathered from the NOSE questionnaire. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:586-593, 2019.

Functional Outcomes, Quantitative Morphometry, and Aesthetic Analysis of Articulated Alar Rim Grafts in Septorhinoplasty.

IMPORTANCE: The design, use, and indications for the articulated alar rim graft (AARG) and the functional and aesthetic improvements that can be achieved have not been fully characterized. OBJECTIVE: To analyze the functional and aesthetic outcomes of AARG placement on nasal airway function, nasal base shape change, and appearance. DESIGN, SETTING, AND PARTICIPANTS: A case series study of patients who underwent septorhinoplasty with placement of AARG at University of California, Irvine Medical Center, from 2015 to 2018 was carried out. Surgical data recorded included stage of rhinoplasty (primary vs revision), use of spreader grafts, rim grafts (and dimensions), caudal septal extension graft (CSEG), lateral crural tensioning (LCT), and turbinate reductions. MAIN OUTCOMES AND MEASURES: Preoperative and postoperative Nasal Obstruction Symptom Evaluation Survey (NOSE) surveys were analyzed and correlated with AARG geometry, use of CSEG, and the LCT maneuver. Preoperative and postoperative alar base views were evaluated by fitting base shape to a parametric numerical model to categorize each to 1 of 6 shape categories. Blinded reviewers rated alar furrow severity and the alar ridge presence using a Likert scale for both preoperative and postoperative images to subjectively gauge aesthetic outcomes. RESULTS: Overall, 90 patients with both preoperative and postoperative NOSE scores who underwent septorhinoplasty and placement of an AARG were included. Of the 90 patients, 60 were women (mean age, 38.2 years). Patient NOSE scores (70.4 preoperatively to 25.1 postoperatively) significantly improved from preoperation to postoperation (P < .001), regardless of AARG size, CSEG, or LCT. Alar base shape parametric analysis showed preoperative to postoperative improvements were significant for anterior-to-posterior ratio mass distribution (95% CI, -0.16 to 0.02; P = .05) and vertical projection-to-horizontal base width ratio (95% CI, 0.01-0.32; P = .02) in flat noses and cloverleafing for narrow noses (95% CI, -0.05 to -0.01; P = .001); enhancement approached significance for reduction in lateral scalloping in cloverleaf noses (P = .06). Aesthetic analysis showed that there was a statistically significant improvement for the alar furrow (95% CI, -0.68 to -0.29 for rater 1; -0.54 to -0.27 for rater 2; and -0.59 to -0.27 for rater 3; P < .001) for all raters and for the alar ridge (95% CI, 0.16-0.48; P < .001) for 1 rater. CONCLUSIONS AND RELEVANCE: To our knowledge, this is the first study to demonstrate that AARG use is associated with statistically significant improvement in NOSE scores. Placement of AARGs may improve posterior mass ratios in flat noses and lateral cloverleafing in narrow noses as suggested by quantitative shape change parameter analysis. The placement of AARGs was associated with aesthetic and functional enhancement in the cloverleaf deformity, which is associated with a prominent alar furrow, and often external nasal valve collapse. Patient selection is key when placing AARGs. LEVEL OF EVIDENCE: NA.

Development and evaluation of rhinoplasty spreader graft suture simulator for novice surgeons.

OBJECTIVE: Surgical simulators aimed at mimicking elements of rhinoplasty surgery, specifically those aimed at improving cartilage suturing, are not available. Here, we present a surgical simulator for spreader graft placement that uses cartilage rather than synthetic materials and gauge improvement using objective measures for suture placement accuracy, speed, and efficiency of hand motion. METHODS: Twenty-two otolaryngologists in two groups (residents [10] and experts [12]) were instructed to secure the two spreader graft specimen into position with three mattress sutures on a nose model that used porcine septal cartilage as a proxy for the human counterpart. Hand motion was tracked using an electromagnetic position sensing device. The time required to complete the suture task, total hand displacement, cumulative number of hand motion direction changes, and accuracy of suture insertion were measured. These measurements were compared between the two cohort groups for construct validity. The subjects completed a survey to evaluate realism and value of the model. RESULTS: The expert group had a lower mean time required to complete the task (P < 0.05), total hand displacement (P < 0.01), and number of hand motion direction changes (P < 0.001). No significant difference was observed between the two groups in suture precision measurement. The subjects agreed on the face validity and usefulness of the trainer. CONCLUSIONS: Our study suggests that the simulator may be a useful tool to objectively gauge suturing efficiency. Devices such as this may be useful for developing skill with suturing cartilage tissue and potentially be used to assess resident acquisition of surgical skill. LEVEL OF EVIDENCE: NA Laryngoscope, 129:344-350, 2019.

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson's disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.