Faculty Bibliography

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.

Oligodendrocyte precursor cells (OPCs) differentiate during postnatal development into myelin-forming oligodendrocytes, in a process distinguished by substantial changes in morphology and the onset of myelin gene expression. A mammalian-specific CNS myelin gene, tmem10, also called Opalin, encodes a type 1 transmembrane protein that is highly upregulated during early stages of OPC differentiation; however, a function for TMEM10 has not yet been identified. Here, consistent with previous studies, we detect TMEM10 protein in mouse brain beginning at ~P10 and show that protein levels continue to increase as oligodendrocytes differentiate and myelinate axons in vivo. We show that constitutive TMEM10 overexpression in the Oli-neu oligodendroglial cell line promotes the expression of the myelin-associated genes MAG, CNP and CGT, whereas TMEM10 knock down in primary OPCs reduces CNP mRNA expression and decreases the percentage of MBP-positive oligodendrocytes that differentiate in vitro. Ectopic TMEM10 expression evokes an increase in process extension and branching, and blocking endogenous TMEM10 expression results in oligodendrocytes with abnormal cell morphology. These findings may have implications for human demyelinating disorders, as oligodendrocytes expressing TMEM10 are detected in human remyelinating multiple sclerosis lesions. Together, our findings provide evidence that TMEM10 promotes oligodendrocyte terminal differentiation and may represent a novel target to promote remyelination in demyelinating disorders.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Niche cell enwrapment of stem cells and their differentiating progeny is common and provides a specialized signaling and protective environment. Elucidating the mechanisms underlying enwrapment behavior has important basic and clinical significance in not only understanding how niches are formed and maintained but also how they can be engineered and how they are misregulated in human pathologies, such as cancer. Previous work in C. elegans found that, when germ cells, which are enwrapped by somatic gonadal niche cells, are freed into the body cavity, they embed into other tissues. We investigated this phenomenon using live-cell imaging and discovered that ectopic germ cells preferentially induce body-wall muscle to extend cellular processes that enwrap the germ cells, the extent of which was strikingly similar to the distal tip cell (DTC)-germ stem cell niche. Enwrapment was specific for escaped germ cells, and genetic analysis revealed it did not depend on pathways that control cell death and engulfment or muscle arm extension. Instead, using a large-scale RNAi screen and GFP knockin strains, we discovered that the enwrapping behavior of muscle relied upon the same suite of cell-cell adhesion molecules that functioned in the endogenous niche: the C. elegans E-cadherin HMR-1, its intracellular associates α-catenin (HMP-1) and β-catenin (HMP-2), and the L1CAM protein SAX-7. This ectopic niche-like behavior resembles the seed-and-soil model of cancer metastasis and offers a new model to understand factors regulating ectopic niche formation.

The gastrointestinal (GI) tract renews frequently to sustain nutrient digestion and absorption in the face of consistent tissue stress. In many species, proliferative intestinal stem cells (ISCs) are responsible for the repair of the damage arising from chemical and mechanical aspects of food breakdown and exposure to pathogens. As the cellular source of all mature cell types of the intestinal epithelium throughout adulthood, ISCs hold tremendous therapeutic potential for understanding and treating GI disease in humans. This review focuses on recent advances in our understanding of ISC identity, behavior, and regulation during homeostasis and injury-induced repair, as revealed by two major animal models used to study regeneration of the small intestine: Drosophila melanogaster and Mus musculus. We emphasize recent findings from Drosophila that are likely to translate to the mammalian GI system, as well as challenging topics in mouse ISC biology that may be ideally suited for investigation in flies. For context, we begin by reviewing major physiological similarities and distinctions between the Drosophila midgut and mouse small intestine.

Background: The occurrence of bifid mandibular canals is unusual but not rare. Previous reviews and case studies have described numerous types of bifid canals based on location, anatomy, and contents. Developmentally, ossification of the mandible begins at the region of the mental foramen and continues posteriorly, forming the mandibular canal around the neurovascular bundle within. This process explains the creation of multiple mandibular canals and the diversity of canal types previously recorded in the literature. However, the presence of two distinct mandibular canals, each originating from its own mandibular foramen, is even more unusual. Material(s) and Method(s): This case report describes a unilateral variant discovered during the dissection of a 92-year-old African American female. Result(s): On the left side, the cadaver presents two distinct mandibular canals, each containing a branch of the inferior alveolar artery and mandibular nerve, the third division of the trigeminal. The nerves within the two canals were distinct from each other at the level of the posterior division of the mandibular nerve, within 1 cm of foramen ovale. Conclusion(s): This is the first description of such an occurrence and emphasizes the need for identification of the contents of a bifid or accessory mandibular canal prior to invasive procedures.

Macrophages in the heart have dual roles in injury and repair after myocardial infarction, and understanding the two sides of this coin using traditional 'bulk cell' technologies has been challenging. By combining genetic fate-mapping and single-cell transcriptomics, a new study (Nat. Immunol. 2019;20:29-39) reveals how distinct macrophage populations expand and diverge across the healthy heart and after infarction.

PURPOSE/OBJECTIVE:Schools undergoing curricular reform are reconsidering the optimal timing of Step 1. This study provides a psychometric investigation of the impact on United States Medical Licensing Examination Step 1 scores of changing the timing of Step 1 from after completion of the basic science curricula to after core clerkships. METHOD/METHODS:Data from four schools that recently moved the examination were analyzed in a pre-post format using examinee scores from three years before and after the change. The sample included scores from 2008 through 2016. Several confounders were addressed, including rising national scores and potential differences in cohort abilities using deviation scores and analysis of covariance (ANCOVA) controlling for Medical College Admission Test (MCAT) scores. A resampling procedure compared study schools' score changes to similar schools' in the same time period. RESULTS:The ANCOVA indicated that post-change Step 1 scores were higher compared to pre-change (adjusted difference = 2.67, 95% confidence interval [CI]: 1.50-3.83, P < .001; effect size = 0.14) after adjusting for MCAT scores and rising national averages. The average score increase in the study schools was larger than changes seen in similar schools. Failure rates also decreased from 2.87% (n = 48) pre-change to 0.39% (n = 6) post-change (P < .001). CONCLUSIONS:Results suggest moving Step 1 after core clerkships yielded a small increase in scores and a reduction in failure rates. While these small increases are unlikely to represent meaningful knowledge gains, this demonstration of "non-inferiority" may allow schools to implement significant curricular reformsWritten work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a "work of the United States Government" for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti-PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGF-beta expression or activation increases in irradiated tissues, we tested whether TGF-beta blockade may further enhance abscopal effects in conjunction with the anti-PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ neutralizing monoclonal antibody, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the non-irradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the non-irradiated contralateral tumor which showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGF-beta hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore TGF-beta blockade in combination with radioimmunotherapy results in greater efficacy.