Leveraging the virtual landscape to promote diversity, equity, and inclusion in Otolaryngology-Head & Neck Surgery.

The pandemic era has wrought disruptive changes across all aspects of academic medicine, transforming clinical care systems, research enterprises, and educational practices. Among these sweeping changes, some of the most significant for Otolaryngology-Head & Neck Surgery (OHNS) relate to innovative use of virtual communication. The virtual landscape has not only redefined the delivery of patient care but also expanded educational interactions across the career continuum. As the architecture of relationships has been reimagined, the traditional Halstedian teaching paradigm has evolved, now integrating in-person, virtual, and hybrid learning. Once formidable barriers to distance learning have diminished, giving way to social networks that support mentorship, coaching, and sponsorship. Creative use of technology supports collaboration, feedback, spaced learning, scaffolding, and interleaving. These advances have immediate significance for OHNS, a highly collaborative specialty that leads in technological innovation and aspires to improve diversity. Whereas traditional efforts to grow the ranks of underrepresented in medicine (URiM) individuals in OHNS yielded only incremental progress, the virtual landscape is unveiling new strategies for reengineering narrow or leaky pipelines. Strategies that can help attract URiM applicants include inclusive departmental web presence, interactive online clinical experiences, virtual interviews, and remote research opportunities. As students, surgeons, and scientists collectively embrace technology, accelerated progress toward diversity, equity, and inclusion (DEI) becomes possible. Maximizing these opportunities requires aligning national and institutional imperatives for diversity with departmental priorities and mission. Finally, intentional outreach and holistic assessment support growth of structured virtual communities that foster equitable access for those who need it most.

Bilateral sensorineural hearing loss after opioid overdose in an adolescent male.

Opioid-induced hearing loss has been described as a form of sudden sensorineural hearing loss that can occur with chronic or acute opioid use. Here, we report a case of a 16-year-old patient with sudden onset hearing loss after opioid overdose requiring prolonged intubation with fentanyl sedation.

Higher Intercellular Variation in Genome-Wide Recombination Rate in Female Mice.

Meiotic recombination affects fertility, shuffles genomes, and modulates the effectiveness of natural selection. Despite conservation of the recombination pathway, the rate of recombination varies among individuals and along chromosomes. Recombination rate also differs among cells from the same organism, but this form of variation has received less attention. To identify patterns that characterize intercellular variation in the genome-wide recombination rate, we counted foci of the MLH1 recombination-associated protein in oocytes and spermatocytes from a panel of wild-derived inbred strains of house mice. Females show higher intercellular variation in MLH1 focus count than males from the same inbred strains. This pattern is consistent across strains from multiple subspecies, including 2 strains in which the average MLH1 focus count is higher in males. The sex difference in genome-wide recombination rate we report suggests that selection targeting recombination rate will be more efficient in males than in females.

An investigation into country of origin labeling, species authentication and short weighting of commercially sold frozen fish fillets.

Proper labeling of seafood is important to prevent economic deception and protect public health. The goal of this research was to investigate prepackaged frozen fish for Country of Origin Labeling (COOL) compliance, species labeling, net weights/short weighting, and percent glaze. A total of 111 frozen prepackaged fish fillets were purchased from grocery stores in Southern California (USA). Samples were designated as COOL compliant if they displayed both procurement method and country of origin in accordance with COOL requirements. Species labeling was examined by comparing the species identified with DNA barcoding to the acceptable market names provided in the FDA Seafood List. Net weights and percent glaze were determined by recording the weight of each product before and after deglazing. Of the 111 samples, only 1 was noncompliant with COOL and 10 samples (9%) were short-weighted. The average percent glaze was 5%, with seven samples having >10% glaze. Most fish (95.5%) were correctly labeled with regards to species. Species substitution was discovered in two samples and three samples had unacceptable market names. The results of this study indicate high COOL compliance and minimal species mislabeling in prepackaged frozen fish fillets. However, there is a need for increased focus on short weighting and/or overglazing of frozen fish products.

Sex-specific variation in the genome-wide recombination rate.

In most species that reproduce sexually, successful gametogenesis requires recombination during meiosis. The number and placement of crossovers (COs) vary among individuals, with females and males often presenting the most striking contrasts. Despite the recognition that the sexes recombine at different rates (heterochiasmy), existing data fail to answer the question of whether patterns of genetic variation in recombination rate are similar in the two sexes. To fill this gap, we measured the genome-wide recombination rate in both sexes from a panel of wild-derived inbred strains from multiple subspecies of house mice (Mus musculus) and from a few additional species of Mus. To directly compare recombination rates in females and males from the same genetic backgrounds, we applied established methods based on immunolocalization of recombination proteins to inbred strains. Our results reveal discordant patterns of genetic variation in the two sexes. Whereas male genome-wide recombination rates vary substantially among strains, female recombination rates measured in the same strains are more static. The direction of heterochiasmy varies within two subspecies, Mus musculus molossinus and Mus musculus musculus. The direction of sex differences in the length of the synaptonemal complex and CO positions is consistent across strains and does not track sex differences in genome-wide recombination rate. In males, contrasts between strains with high recombination rate and strains with low recombination rate suggest more recombination is associated with stronger CO interference and more double-strand breaks. The sex-specific patterns of genetic variation we report underscore the importance of incorporating sex differences into recombination research.

Conservation of the genome-wide recombination rate in white-footed mice.

Despite being linked to the fundamental processes of chromosome segregation and offspring diversification, meiotic recombination rates vary within and between species. Recent years have seen progress in quantifying recombination rate evolution across multiple temporal and genomic scales. Nevertheless, the level of variation in recombination rate within wild populations-a key determinant of evolution in this trait-remains poorly documented on the genomic scale. To address this notable gap, we used immunofluorescent cytology to quantify genome-wide recombination rates in males from a wild population of the white-footed mouse, Peromyscus leucopus. For comparison, we measured recombination rates in a second population of male P. leucopus raised in the laboratory and in male deer mice from the subspecies Peromyscus maniculatus bairdii. Although we found differences between individuals in the genome-wide recombination rate, levels of variation were low-within populations, between populations, and between species. Quantification of synaptonemal complex length and crossover positions along chromosome 1 using a novel automated approach also revealed conservation in broad-scale crossover patterning, including strong crossover interference. We propose stabilizing selection targeting recombination or correlated processes as the explanation for these patterns.

Safety profile of ultrasound enhancing agents in echocardiography.

INTRODUCTION: Ultrasound enhancing agents (UEAs) are often utilized to enhance ultrasound image quality; however, concerns about adverse reactions have limited their use. Moreover, these agents had been either contraindicated or are labeled with a warning in patients with intra-cardiac shunts because of a theoretic risk of systemic microvascular obstruction. This labeling was recently removed in the United States, but data in these patients are lacking. METHODS: Over a 15-month period, patients receiving three different FDA-approved UEAs at our center were prospectively evaluated for clinically significant adverse events (AEs). RESULTS: A total of 5521 UEA administrations were performed (Definity® : 3306, Lumason® : 2137, Optison® : 78). There were 14 AEs (0.25%) reported (Lumason® : 0.05% [n = 1] vs Definity® : 0.39% [n = 13], P = 0.02). Back pain was the most common complaint (n = 9), followed by headache (n = 2), rash (n = 2), dyspnea (n = 2), and palpitations (n = 1). Among the 33 patients known to have intra-cardiac shunts, there were no AEs. Known right-to-left shunts with positive saline bubble study were present in 20 patients (Lumason® : n = 9, Definity® : n = 11). Left-to-right atrial shunts based on color Doppler were present in 10 patients (Lumason® : n = 5, Definity® n = 5). Three patients were known to have ventricular septal defect with left-to-right flow (Definity® : n = 2, Optison® : n = 1). CONCLUSION: Adverse events were significantly higher with Definity® ; however, overall incidences were low, and AEs were minor. Furthermore, no AEs were reported in patients with known intra-cardiac shunts. UEAs showed a good safety profile in our study and should be afforded to all appropriate patients, including those with known intra-cardiac shunts.

Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+) subtype and fourteen triple negative (TN) subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing.

BACKGROUND: Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. RESULTS: We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells' DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant "normal" cells or "aberrant cells of unknown origin" that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. CONCLUSIONS: Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

A novel mechanism of inherited TBG deficiency: mutation in a liver-specific enhancer.

CONTEXT: T4-binding globulin (TBG), a protein secreted by the liver, is the main thyroid hormone (TH) transporter in human serum. TBG deficiency is characterized by reduced serum TH levels, but normal free TH and TSH and absent clinical manifestations. The inherited form of TBG deficiency is usually due to a mutation in the TBG gene located on the X-chromosome. OBJECTIVE: Among the 75 families with X-chromosome-linked TBG deficiency identified in our laboratory, no mutations in the TBG gene were found in four families. The aim of the study was to identify the mechanism of TBG deficiency in these four families using biochemical and genetic studies. DESIGN: Observational cohort, prospective. SETTING: University research center. PATIENTS: Four families with inherited TBG deficiency and no mutations in the TBG gene. INTERVENTION: Clinical evaluation, thyroid function tests, and targeted resequencing of 1 Mb of the X-chromosome. RESULTS: Next-generation sequencing identified a novel G to A variant 20 kb downstream of the TBG gene in all four families. In silico analysis predicted that the variant resides within a liver-specific enhancer. In vitro studies confirmed the enhancer activity of a 2.2-kb fragment of genomic DNA containing the novel variant and showed that the mutation reduces the activity of this enhancer. The affected subjects share a haplotype of 8 Mb surrounding the mutation, and the most recent common ancestor among the four families was estimated to be 19.5 generations ago (95% confidence intervals, 10.4-37). CONCLUSIONS: To our knowledge, the present study is the first report of an inherited endocrine disorder caused by a mutation in an enhancer region.

The spectrum of somatic mutations in high-risk acute myeloid leukaemia with -7/del(7q).

-7/del(7q) occurs in half of myeloid malignancies with adverse-risk cytogenetic features and is associated with poor survival. We identified the spectrum of mutations that co-occur with -7/del(7q) in 40 patients with de novo or therapy-related myeloid neoplasms. -7/del(7q) leukaemias have a distinct mutational profile characterized by low frequencies of alterations in genes encoding transcription factors, cohesin and DNA-methylation-related proteins. In contrast, RAS pathway activating mutations occurred in 50% of cases, a significantly higher frequency than other acute myeloid leukaemias and higher than previously reported. Our data provide guidance for which pathways may be most relevant in the treatment of adverse-risk myeloid leukaemia.

Next-generation sequencing of disseminated tumor cells.

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.

A conserved eEF2 coding variant in SCA26 leads to loss of translational fidelity and increased susceptibility to proteostatic insult.

The autosomal dominant spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders exhibiting cerebellar atrophy and Purkinje cell degeneration whose subtypes arise from 31 distinct genetic loci. Our group previously published the locus for SCA26 on chromosome 19p13.3. In this study, we performed targeted deep sequencing of the critical interval in order to identify candidate causative variants in individuals from the SCA26 family. We identified a single variant that co-segregates with the disease phenotype that produces a single amino acid substitution in eukaryotic elongation factor 2. This substitution, P596H, sits in a domain critical for maintaining reading frame during translation. The yeast equivalent, P580H EF2, demonstrated impaired translocation, detected as an increased rate of -1 programmed ribosomal frameshift read-through in a dual-luciferase assay for observing translational recoding. This substitution also results in a greater susceptibility to proteostatic disruption, as evidenced by a more robust activation of a reporter gene driven by unfolded protein response activation upon challenge with dithiothreitol or heat shock in our yeast model system. Our results present a compelling candidate mutation and mechanism for the pathogenesis of SCA26 and further support the role of proteostatic disruption in neurodegenerative diseases.