A journey in the world of DNA rings and beyond.

I was born in China and would have remained there but for the tumultuous events that led many of my generation to the United States for graduate studies. Norman Davidson introduced me to DNA when I became a postdoctoral fellow in his group at the California Institute of Technology in 1964, and a fortuitous conversation there ignited my interest in DNA ring formation, which later led me to study different topological forms of DNA rings-catenanes, knots, and supercoils. In 1968, a chance observation led me to identify a new enzyme capable of converting one DNA ring form to another, an enzyme now known as a DNA topoisomerase. My interest in DNA rings and DNA topoisomerases continued throughout my years at the University of California, Berkeley, and Harvard. The fascinating ability of the topoisomerases in passing DNA strands or double helices through one another and their importance in cellular processes have kept me and many others excited in their studies.

Effects of Essential Oils on Scars and Wound Healing: A Systematic Review.

Essential oils (EOs) have been considered as a potential alternative therapy for wound healing and scar reduction. The aim of this article was to provide a comprehensive review examining the effects of EOs on wound healing and scars. PubMed, Cochrane, Ovid, and Embase computerized searches were performed through June 2020. Two independent reviewers conducted data extraction, with search results reviewed by the senior author following the PRISMA protocol. Three manuscripts examining three different EO-containing topical agents were analyzed. Outcomes include healing rate, erythema, pain, pruritus, patient discomfort, physician satisfaction, percent wound reduction, wound/scar surface perimeter area, and qualitative dermatological evaluation. All articles concluded that the EO-containing topical agents resulted in either superior or noninferior outcomes in comparison with controls. Hypericum-Calendula oil obtained lower wound surface perimeter area. Erythema (p = 0.001) was significantly decreased by the peppermint EO-containing topical agent. Physicians also reported greater satisfaction (p < 0.001) in wound appearance with use of the peppermint EO-containing topical agent. A paucity of studies have examined EO use for wound healing and scar reduction. Treatment with EO-containing topical agents resulted in decreased erythema with increased physician satisfaction of wound appearance. Future studies should assess what level of purity is needed for improved results and which EO, or combination of EOs, is most beneficial.

A Report on the Use of Telehealth in Otolaryngology in the Pre COVID-19 Era.

Objective: To investigate the integration of and barriers to the utilization of telehealth technology and its components (telemedicine, e-Health, m-health) in daily otolaryngologic practice before the SARS CoV-2 (COVID-19) pandemic. Methods: This cross-sectional study was conducted at a tertiary academic center. A national survey of members of the American Academy of Otolaryngology-Head and Neck Surgery was administered. Descriptive analyses were performed to determine how telehealth was employed in otolaryngologists' practices. Results: A total of 184 surveys were completed. Telehealth technology was used by 50% of otolaryngologists surveyed. Regions with the largest percentage of physicians using telehealth were the Mid-Atlantic region (84%) and West Coast (67%). Most otolaryngologists indicated that they were familiar with telehealth or any of its components and how it is used in practice (52-83%), they had heard of telehealth or any of its components but were unsure what the terms specifically entailed (17-42%); 53% were satisfied with their current use of telehealth and electronic medical record (EMR); and 72% were comfortable utilizing smart devices for patient care. Most otolaryngologists (65%) indicated reimbursement as the biggest limitation to implementing telehealth, and 67% believed that typing was a hindrance to EMR utility. Conclusion: Half of the surveyed otolaryngologists used some form of telehealth at the time of the survey. The most commonly cited obstacle to physician adoption of telehealth was reimbursement. Although the adoption of telehealth technology was still limited in the field of otolaryngology based on this study, we are now seeing significant change due to the COVID-19 pandemic.

Geographic Distribution of Facial Plastic and Reconstructive Surgeons.

Background: Distribution of the general otolaryngology workforce has been described, but not specifically for the facial plastic and reconstructive surgeon (FPRS) workforce. Objective: To describe the distribution of FPRS within the United States. Methods: The 2022 American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS) registry was used to identify active FPRSs. Member addresses were converted into coordinates and overlayed onto a geographic representation of 2020 census data within ArcGIS software. A centroid model of U.S. counties was constructed to determine the average distances residents were from the nearest FPRS. Results: In total, 1312 AAFPRS active members practiced in 373 counties. Thirty-three percent of all residents (115 million) resided in counties without an FPRS and 15.3% of FPRSs practiced in New York City or the Greater Los Angeles Area, which accounted for 8% of the total U.S. population. The mean and median distances a resident in a county without an FPRS was from the nearest FPRS are 63 and 49 miles (101 and 79 kilometers), respectively. Conclusions: Metropolitan areas have greater concentrations of FPRSs than the national average and the distances U.S. residents are from FPRS services are quantifiable.

Localized interleukin-12 delivery for immunotherapy of solid tumours.

Interleukin (IL)-12 is the key cytokine in the initiation of a Th1 response and has shown promise as an anti-cancer agent; however, clinical trials involving IL-12 have been unsuccessful due to toxic side-effects. To address this issue, lentiviral vectors were used to transduce tumour cell lines that were injected as an autologous tumour cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumour model. SCCVII cells that were transduced to produce IL-12 at different concentrations were then isolated. Subcutaneous injection of parental SCCVII cells results in tumour development, while a mixture of IL-12-producing and non-producing cells results in tumour clearance. Interestingly, when comparing mice injected a mixture of SCCVII and either high IL-12-producing tumour cells or low IL-12-producing tumour cells, we observed that mixtures containing small amounts of high producing cells lead to tumour clearance, whereas mixtures containing large amounts of low producing cells fail to elicit protection, despite the production of equal amounts of total IL-12 in both mixtures. Furthermore, immunizing mice with IL-12-producing cells leads to the establishment of both local and systemic immunity against challenge with SCCVII. Using depletion antibodies, it was shown that both CD4(+) and CD8(+) cells are crucial for therapy. Lastly, we have established cell clones of other solid tumour cell lines (RM-1, LLC1 and moto1.1) that produce IL-12. Our results show that the delivery of IL-12 by cancer cells is an effective route for immune activation.

Biopolymers and the fellowship of DNA rings.

This article presents a brief account of the historical backdrop of the study of interlocked DNA rings (DNA catenanes), their formation in cells, and the importance of resolving the component rings of an intracellular DNA catenane if they are to be properly partitioned into a pair of progeny cells. In humans, for example, aberrant segregation of intertwined chromosomes is a major cause of birth defects, as well as termination of pregnancy in utero. Some yet unresolved issues of DNA catenation, including plausible structural and/or functional roles of DNA interlacing in chromosomes, are briefly mentioned.

Engineering lentiviral vectors for modulation of dendritic cell apoptotic pathways.

BACKGROUND: Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. Unfortunately, cancer cells can often disarm differentiated DCs by rendering them incapable of maturation or by promoting their apoptosis. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. In addition to undergoing cancer-induced apoptosis, it is well established that DCs are intrinsically short-lived cell types. It is likely that a significant portion of infused DCs undergo apoptosis prior to locating and activating naïve TAA-reactive T cells. METHODS: In our current study, we constructed and investigated novel bicistronic lentivectors (LVs) encoding the cDNA for the xeno-TAA, rat HER-2/neu (rHER-2), along with five candidate mouse DC survival factors (c-FLIPS, c-FLIPL, Bcl-XL, M11L, and AKT-1) that operate in both the extrinsic and intrinsic cycles of apoptosis. The murine DC cell line, DC2.4 was transduced separately with each novel LV construct. Infected cells were enriched via flow cytometric methods based on rHER-2 expression. Transduced DC2.4 cell lines were then exposed to Fetal Calf Serum (FCS) withdrawal and to specific pharmacological apoptosis-inducing agents. DC2.4 cell death was assayed based on Annexin V and PI double-positive staining via flow cytometry. The phenotype and function of transduced DC2.4 cells and primary bone marrow-derived DCs were then assessed via expression and secretion of DC markers and cytokines, respectively. RESULTS: DC2.4 cells transduced with LVs encoding cDNAs for c-FLIPS, c-FLIPL, Bcl-XL, and M11L were protected from apoptosis when exposed to low FCS-containing culture media. When treated with an anti-CD95 antibody, only DC2.4 cells transduced with LVs encoding c-FLIPS and c-FLIPL were protected from apoptosis. In contrast, only DC2.4 cells transduced with LVs encoding Bcl-XL and M11L were protected from effects of staurosporine (STS) treatment. Also, LV-modified DCs maintained their original phenotype and function. CONCLUSIONS: We present evidence that by employing novel recombinant bicistronic LVs we can simultaneously load DCs with a relevant TAA and block apoptosis; thereby confirming the usage of such LVs in the modulation of DC lifespan and function.

Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase.

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Lentivector transduction improves outcomes over transplantation of human HSCs alone in NOD/SCID/Fabry mice.

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb(3)) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34(+) cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb(3) quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb(3) reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.

Estrogen receptor-positive adenocarcinoma of the cervix presenting during pregnancy: Two case reports and review of the literature.

The incidence of adenocarcinoma of the cervix in pregnancy is exceptionally rare, and thus there is no consensus on its management. Here, we report two cases of adenocarcinoma of the cervix diagnosed in the context of pregnancy. In our first case, a patient referred to colposcopy for atypical glandular cells of undetermined significance was subsequently diagnosed with well differentiated endocervical adenocarcinoma on cone biopsy. Just prior to the cone biopsy, she was incidentally found to have a first trimester pregnancy loss. The patient subsequently underwent a radical hysterectomy and bilateral sentinel lymph node dissection. Final pathology revealed a stage 1B1 (FIGO 2009) well differentiated adenocarcinoma of the cervix. Interestingly, the tumour was positive for estrogen receptor, which is unusual for cervical adenocarcinoma. In our second case, a patient presented with a pedunculated, exophytic cervical neoplasm at 31 weeks GA with self-limiting antepartum hemorrhage. The primary lesion measured 52 mm in diameter on MRI and was amputated at the base during the patient's elective repeat cesarean section. Final pathology revealed a stage IB2 (FIGO 2009) mucinous adenocarcinoma of the cervix. The patient subsequently underwent a radical hysterectomy and bilateral pelvic lymph node dissection 17 weeks after initial presentation. The depth of invasion was 2.2 mm, restricted to the inner third of the cervical wall, and there was no lymphovascular space invasion in the surgical specimen. Surgical margins, parametria, and lymph nodes were all negative for adenocarcinoma. This tumour was also found to be estrogen receptor/progesterone receptor (ER/PR) positive, again unusual for cervical adenocarcinoma. P16 was strongly positive and HPV DNA studies were also positive for human papilloma virus 18. The patient received adjuvant external beam radiotherapy to the pelvis and currently remains in remission.

A multi-institutional study evaluating and describing atypical parathyroid tumors discovered after parathyroidectomy.

Objective: To describe common intraoperative and pathologic findings of atypical parathyroid tumors (APTs) and evaluate clinical outcomes in patients undergoing parathyroidectomy. Methods: In this multi-institutional retrospective case series, data were collected from patients who underwent parathyroidectomy from 2000 to 2018 from three tertiary care institutions. APTs were defined according to the AJCC eighth edition guidelines and retrospective chart review was performed to evaluate the incidence of recurrent laryngeal nerve injury, recurrence of disease, and disease-specific mortality. Results: Twenty-eight patients were identified with a histopathologic diagnosis of atypical tumor. Mean age was 56 years (range, 23-83) and 68% (19/28) were female. All patients had an initial diagnosis of primary hyperparathyroidism with 21% (6/28) exhibiting clinical loss of bone density and 32% (9/28) presenting with nephrolithiasis or renal dysfunction. Intraoperatively, 29% (8/28) required thyroid lobectomy, 29% (8/28) had gross adherence to adjacent structures and 46% (13/28) had RLN adherence. The most common pathologic finding was fibrosis 46% (13/28). Postoperative complications include RLN paresis/paralysis in 14% (4/28) and hungry bone syndrome in 7% (2/28). No patients with a diagnosis of atypical tumor developed recurrent disease, however there was one patient that had persistent disease and hypercalcemia that is being observed. There were 96% (27/28) patients alive at last follow-up, with one death unrelated to disease. Conclusion: Despite the new AJCC categorization of atypical tumors staged as Tis, we observed no recurrence of disease after resection and no disease-specific mortality. However, patients with atypical tumors may be at increased risk for recurrent laryngeal nerve injury and incomplete resection.

Location Propensity for Keloids in the Head and Neck.

Importance: The difficulty associated with concealing keloids located in the face and neck regions often results in disfigurement and psychological stress for patients. Objective: The purpose of this review is to determine if specific regions on the head and neck have a greater propensity to develop keloids to alert the facial plastic surgeon of regional keloid risk. Evidence Review: PubMed, Ovid, Cochrane, and Embase computerized searches were performed through January 2020. Two independent reviewers conducted data extraction following a predetermined protocol identifying 1445 keloid studies. Findings: The prevalence of keloids in different regions of the head and neck was evaluated among 1598 head and neck keloids in the final analysis. Three of nine studies evaluated keloids specific to the ear and described a combined 1194 ear keloids locations (53.0% lobule, 22.9% cartilaginous region, 0.3% both, and 23.7% unspecified). One study reported exclusively on 82 neck keloids 72% of which were located in the submental or submandibular region. The remaining five studies evaluated 322 head and neck keloids more generally and showed 70.2% ear, 9.6% unspecified, 6.5% peri-/postauricular, 5.6% beard area, 2.2% scalp/forehead, 2.2% chin, 1.6% lateral face, 1.6% neck, and 0.3% central face keloids. Conclusions and Relevance: The ear, periauricular regions, bearded facial regions, and submandibular and submental sites show the highest propensity for keloid development in the head and neck region. The lowest keloid risk occurred in the central face region. Risk factors in relation to facial and neck regions associated with the formation of keloids should be taken into consideration before performing elective procedures in keloid susceptible individuals.

Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies.

Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the beta rather than the alpha isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2beta(+) than in skin-specific top2beta-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2beta-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2beta-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2alpha-dependent. These results point to the importance of developing Top2alpha-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies.

Association of the sinonasal bacterial microbiome with clinical outcomes in chronic rhinosinusitis: a systematic review.

BACKGROUND: The association between sinonasal microbiome and clinical outcomes of patients with chronic rhinosinusitis (CRS) is unclear. We performed a systematic review of prior studies evaluating the CRS microbiome in relation to clinical outcomes. METHODS: Computerized searches of PubMed/Medline, Cochrane, and EMBASE were updated through October 2019 revealing a total of 9 studies including 244 CRS patients. A systematic review of the literature was performed, including data extraction focusing on sample region, sequencing platforms, predominant organisms, and outcomes measures. RESULTS: Nine criterion-meeting studies included 244 CRS patients, with varied results. Eight studies used 16s-ribosomal RNA (16s-rRNA) gene sequencing to assess the sinonasal microbiome and 1 used 16s-rRNA PhyloChip analysis. Seven studies used Sino-Nasal Outcome Test scores, 1 applied another CRS symptom metric, and 1 used need for additional procedures/antibiotics as the primary clinical outcome. Three studies suggest that baseline abundance of phylum Actinobacteria (specifically genus Corynebacterium) was predictive of better surgical outcome. One study found C. tuberculostearicum was positively correlated with symptom severity. Another study revealed genus Escherichia was overrepresented in CRS and had positive correlation with increased symptom scores. In addition, 1 study identified Acinetobacter johnsonii to be associated with improvement in symptom scores while supporting Pseudomonas aeruginosa as having a negative impact on quality of life. CONCLUSION: Microbiome data are varied in their association with clinical outcomes of CRS patients. Further research is required to identify if predominance of certain microbes within the microbiome is predictive of CRS patients' outcomes.

Sinonasal pathophysiology of SARS-CoV-2 and COVID-19: A systematic review of the current evidence.

OBJECTIVE: The ongoing pandemic of coronavirus disease (2019 coronavirus disease [COVID-19]), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus, is highly contagious with high morbidity and mortality. The role of the nasal and paranasal sinus cavities is increasingly recognized for COVID-19 symptomatology and transmission. We therefore conducted a systematic review, synthesizing existing scientific evidence about sinonasal pathophysiology in COVID-19. STUDY DESIGN: Systematic review. METHODS: Systematic searches were performed of all indexed studies in PubMed/Medline and Cochrane databases through 28 March 2020 and studies searchable on preprints.com (including ArXiv and Scilit repositories) through 30 March 2020. Data extraction focused on sinonasal pathophysiology in COVID-19. RESULTS: A total of 19 studies were identified. The sinonasal cavity may be a major site of infection by SARS-CoV-2, where susceptibility genes required for infection are expressed at high levels and may be modulated by environmental and host factors. Viral shedding appears to be highest from the nose, therefore reflecting a major source for transmission. This has been highlighted by multiple reports of health care-associated infection (HAI) during rhinologic procedures, which are now consequently considered to be high risk for SARS-CoV-2 transmission to health care workers. While sinonasal symptomatology, such as rhinorrhea or congestion, appears to be a rarer symptom of COVID-19, anosmia without nasal obstruction is reported as highly specific predictor of COVID-19+ patients. CONCLUSION: Sinonasal pathophysiology is increasingly important in our understanding of COVID-19. The sinonasal tract may be an important site of infection while sinonasal viral shedding may be an important transmission mechanism-including HAI. Anosmia without nasal obstruction may be a highly specific indicator of COVID-19. LEVEL OF EVIDENCE: 2a.

Competitiveness of Otolaryngology Residency Applicants without a Home Program.

PURPOSE: Investigate if otolaryngology residency home programs (HP) are associated with advantages in National Resident Matching Program match compared to applicants without HPs. METHODS: Surveys were distributed to fourth-year medical students applying to otolaryngology residency (2015-2016 cycle) via OHNS (2015-2016) Applicants Closed Facebook Page and Otomatch. Applicant data analyzed included HP, United States Medical Licensing Examination (USMLE) scores, number of away rotations, and matching at top choice. RESULTS: Applicants were grouped: (1) HP, (2) no HP but have ENT staff (staff), and (3) no HP or staff (none). Ninety-five percent of survey participants matched into otolaryngology (n = 62). A sub-analysis of match preference among matching applicants revealed 63% of participants with HP matched to their first choice compared to 56% (staff) and 14% (none) (P = .058). Match rate between those with any staff (HP or staff) versus those without was statistically significant (P = .037). Applicants without HPs went on more away rotations than students with HPs (mean: 2.5 ± 0.5 vs 1.7 ± 0.07, P = .0002). No statistical significance was seen between applicants with/without HP in regards to USMLE scores, publications, or number of interviews. CONCLUSION: Applicants applying to otolaryngology residency without HPs are as competitive as those who have HPs. However, without HPs, applicants tend to participate in more away rotations and are less likely to match at their top choice.

Role of topoisomerase IIbeta in the expression of developmentally regulated genes.

Mice lacking topoisomerase IIbeta (TopIIbeta) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2beta knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2beta knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIbeta is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIbeta and histone deacetylase 2, a known TopIIbeta-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIbeta binding to the 5' region of a number of TopIIbeta-sensitive genes. Further studies of a TopIIbeta-sensitive gene, Kcnd2, revealed the presence of TopIIbeta in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIbeta in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.

Feasibility of shotgun metagenomics to assess microbial ecology of pediatric tracheostomy tubes.

OBJECTIVE: Biofilm formation on medical devices such as tracheostomy tubes (TTs) is a serious problem. The clinical impact of biofilms on the airway is still unclear. Biofilms may play a role in granulation tissue development, recurrent airway infections, and failure of laryngotracheal reconstructions. The microbial ecology on TTs has yet to be elucidated. The purpose of this study was to determine the feasibility of shotgun metagenomics to assess the biodistribution of microorganisms on TTs. METHODS: Four TTs were collected from pediatric patients (1.4-10.2 years) with (n = 2) and without (n = 2) granulation tissue formation. Duration of TT placement prior to retrieval from patients ranged from 5 to 365 days. DNA extraction was performed using the MO BIO UltraClean Microbial Isolation (Mo Bio Laboratories, Carlsbad, CA). Library generation using Nextera XT adapters (Illumina Inc., San Diego, CA) and metagenomic shotgun sequencing was performed using the Illumina NextSeq500 (Illumina Inc, San Diego, CA). Salinibacter ruber, a species not found in mammalian microbiome communities, was used as a DNA standard and represented 0.7% to 5.7% of the microbiome, ensuring good quality and abundance of sample DNA. RESULTS: Metagenomic shotgun sequencing was successful for all patients. In TTs associated with granuloma, Fusobacterium nucleatum, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae were predominant, most of which are considered pathogens. From TTs without granulomas, Neisseria mucosa, Neisseria sicca, Acinetobacter baumannii, and Haemophilus parainfluenzae were identified, primarily consistent with respiratory microbiome. CONCLUSION: This study reveals that metagenomic shotgun sequencing of biofilms formed on pediatric TTs is feasible with an apparent difference in microbiome for patients with granulation tissue. Further studies are necessary to elucidate the pathogenesis of microbial ecology and its role in airway disease in patients with TTs. LEVEL OF EVIDENCE: 2c Laryngoscope, 129:317-323, 2019.

Are Standardized Letters of Recommendation in Residency Applications Correlated With Objective Data?

OBJECTIVE: To evaluate whether the standardized letter of recommendation (SLOR), commonly used for medical students applying to otolaryngology residency, correlates with objective data in the application. BACKGROUND: Standardized letters of recommendation using Likert-type scales for different attributes are commonly used by evaluators because of their high interrater reliability and efficiency in preparation and interpretation. Given that these are subjectively scored, it is unknown how well these correlate with objective data. METHODS: Applications to the University of Cincinnati otolaryngology residency were evaluated in the academic cycle of 2017-2018. Standardized letters of recommendation were scored to determine whether certain attributes were correlated with objective data (United States Medical Licensing Examination [USMLE] scores and number of presentations/publications) provided in their application. Spearman correlations were used to evaluate the strength of the relationship between the subjective score in certain attributes with objective data. RESULTS: There were 217 applications to the University of Cincinnati that contained SLORs. Of these applications, 474 standardized letters were scored in categories of medical knowledge, research, and commitment to academic medicine. Total publications and presentations were weakly correlated with commitment to academic medicine (0.35, P < .0001, n = 369) and with research (0.44, P < .0001, n = 355). Medical knowledge was weakly correlated with Step 1 scores (0.20, P < .0001) and Step 2 scores (0.18, P = .0002). CONCLUSIONS: Subjective research and commitment to academic medicine rating scores were weakly correlated with greater academic productivity. Similarly, medical knowledge scores were weakly correlated with Step 1/2 scores. Further research may be needed to assess how to interpret SLOR scores in addition to the information available in an otolaryngology application. LEVEL OF EVIDENCE: 4.

Prospective observational case series evaluating middle ear fluid and tympanostomy tubes through pyrosequencing.

OBJECTIVE: One of the most common sequelae of tympanostomy tube (TT) placement is post-tympanostomy tube otorrhea (PTTO). Granulation tissue formation has been reported in 5-13.8% of patients with TT placement. The purpose of this study is to determine the biodistribution of microorganisms on TTs and middle ear fluid obtained from patients with PTTO. METHODS: This was a prospective observational study of subjects (6-months-16-years) who underwent standard of care treatment for chronic PTTO. Data was collected on diagnosis, existence of antibiotic resistance, duration of tube placement, and evidence of recurrent infection and/or PTTO. TTs and middle ear fluid were subjected to pyrosequencing; additionally, ear fluid samples were sent for culture-based laboratory diagnostics. RESULTS: DNA-pyrosequencing analysis of bacteria from fluid and TTs of pediatric subjects with PTTO revealed a mixture of both aerobic and anaerobic populations. Retained tubes with minimal otorrhea revealed a predominance of Staphylococcus species, normal external auditory canal (EAC) microbiome, within middle ear fluid as well as on TTs. However, TTs with active mucopurulent otorrhea and granulation tissue unveiled prominence of Gram-negative facultative anaerobes such as Pseudomonas and Eikenella. Discrepancies in prominent bacteria were seen between standard culture-based techniques versus pyrosequencing. CONCLUSION: Retained tympanostomy tubes are colonized primarily with normal flora of the EAC. However, mucopurulent otorrhea associated with granulation tissue formation revealed a prominence of Gram-negative facultative anaerobes. Standard culture-based diagnostics may identify bacteria, which are not predominant species of infection. Future studies are necessary to assess the association of Gram-negative facultative anaerobes with granulation tissue formation.