Faculty Bibliography

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.

Atlantic tomcod in the Hudson River Estuary bioaccumulate high hepatic burdens of environmental toxicants. Previously, we demonstrated that Hudson River tomcod developed resistance to TCDD and PCB toxicity probably through strong natural selection during their early life-stages for a variant of the Aryl Hydrocarbon Receptor2 (AHR2). Here, we evaluated the genomic consequences of the resistant genotype by comparing global gene expression in larval tomcod from the Hudson River with expression in larvae from a nearby sensitive population (Shinnecock Bay). We developed an annotated draft tomcod genome to explore the effects of multigenerational exposure to toxicants and a functionally impaired AHR2 on the transcriptome. We used the tomcod genome as a reference in RNA-Seq to compare global gene expression in tomcod larvae from the Hudson River and Shinnecock Bay after experimental exposure of larvae to graded doses of TCDD. We found dramatic differences between offspring from the two populations in the number of genes that were differentially expressed at all doses (0.01, 0.1, and 1 ppb) and even in the vehicle controls. At the two lowest TCDD doses, 250 and 1,141 genes were differentially expressed in Shinnecock Bay larvae compared with 14 and 12, respectively, in Hudson River larvae. At the highest dose (1.0 ppb), 934 genes were differentially expressed in Shinnecock Bay larvae and 173 in Hudson River larvae, but only 28 (16%) of affected genes were shared among both populations. Given the large difference between the two populations in the number and identity of differentially expressed genes, it is likely that the AHR2 pathway interacts directly or indirectly with many genes beyond those known in the AHR2 battery and that other regulatory systems may also respond to TCDD exposure. The effects of chronic multi-generational exposure to environmental toxicants on the genome of Hudson River tomcod are much greater than previously expected.

INTRODUCTION: The operative management of tibial plateau fractures is challenging and post-operative complications do occur. The purpose of this study was three-fold. 1). To report complications and unplanned outcomes in patients who had sustained tibial plateau fractures and were operatively managed 2). To report predictors of these post-operative events 3). To report if differences in clinical outcomes exist in patients who sustained a post-operative event. METHODS: Over 11 years, all tibial plateau fractures were prospectively followed. Clinical outcomes were assessed using the validated Short Musculoskeletal Functional Assessment (SMFA) score. Demographics, initial injury characteristics, surgical details and post-operative events were prospectively recorded. Student's t-tests were used for continuous variables and chi-squared analysis was used for categorical variables. Binary logistic regression and multivariate linear regression were conducted for independent predictors of post-operative events and complications and functional outcomes, respectively. RESULTS: 275 patients with 279 tibial plateau fractures were included in our analysis. Ten patients (3.6%) sustained a deep infection. Six patients (2.2%) developed a superficial infection. One patient (0.4%) presented with early implant failure. Two patients (0.7%) developed a fracture nonunion. Eight patients (2.9%) developed a venous thromboembolism. Seventeen patients (6.2%) went on to re-operation for symptomatic implant removal. Nine patients (3.3%) underwent a lysis of adhesions procedure. Univariate analysis demonstrated bicondylar tibial plateau fractures (P<0.001), Moore fracture-dislocations (P=0.005), open fractures (P=0.022), and compartment syndrome (P=0.001) to be associated with post-operative complications and unplanned outcomes. Long-term functional outcomes were worse among patients who developed a post-operative complication or unplanned outcome (P=0.031). CONCLUSION: Orthopaedic trauma surgeons should be aware of complications and unplanned outcomes following operatively managed tibial plateau fractures, along with having the knowledge of factors that are associated with development of post-operative events.

We simulate deformable red blood cells in the microcirculation using the immersed boundary method with a cytoskeletal model that incorporates structural details revealed by tomographic images. The elasticity of red blood cells is known to be supplied by both their lipid bilayer membranes, which resist bending and local changes in area, and their cytoskeletons, which resist in-plane shear. The cytoskeleton consists of spectrin tetramers that are tethered to the lipid bilayer by ankyrin and by actin-based junctional complexes. We model the cytoskeleton as a random geometric graph, with nodes corresponding to junctional complexes and with edges corresponding to spectrin tetramers such that the edge lengths are given by the end-to-end distances between nodes. The statistical properties of this graph are based on distributions gathered from three-dimensional tomographic images of the cytoskeleton by a segmentation algorithm. We show that the elastic response of our model cytoskeleton, in which the spectrin polymers are treated as entropic springs, is in good agreement with the experimentally measured shear modulus. By simulating red blood cells in flow with the immersed boundary method, we compare this discrete cytoskeletal model to an existing continuum model and predict the extent to which dynamic spectrin network connectivity can protect against failure in the case of a red cell subjected to an applied strain. The methods presented here could form the basis of disease- and patient-specific computational studies of hereditary diseases affecting the red cell cytoskeleton.

Within reflex circuits, specific anatomical projections allow central neurons to relay sensations to effectors that generate movements. A major challenge is to relate anatomical features of central neural populations -- such as asymmetric connectivity -- to the computations the populations perform. To address this problem, we mapped the anatomy, modeled the function, and discovered a new behavioral role for a genetically-defined population of central vestibular neurons in rhombomeres 5-7 of larval zebrafish. First, we found that neurons within this central population project preferentially to motoneurons that move the eyes downward. Concordantly, when the entire population of asymmetrically-projecting neurons was stimulated collectively, only downward eye rotations were observed, demonstrating a functional correlate of the anatomical bias. When these neurons are ablated, fish failed to rotate their eyes following either nose-up or nose-down body tilts. This asymmetrically-projecting central population thus participates in both up and downward gaze stabilization. In addition to projecting to motoneurons, central vestibular neurons also receive direct sensory input from peripheral afferents. To infer whether asymmetric projections can facilitate sensory encoding or motor output, we modeled differentially-projecting sets of central vestibular neurons. Whereas motor command strength was independent of projection allocation, asymmetric projections enabled more accurate representation of nose-up stimuli. The model shows how asymmetric connectivity could enhance the representation of imbalance during nose-up postures while preserving gaze-stabilization performance. Finally, we found that central vestibular neurons were necessary for a vital behavior requiring maintenance of a nose-up posture: swim bladder inflation. These observations suggest that asymmetric connectivity in the vestibular system facilitates representation of ethologically-relevant stimuli without compromising reflexive behavior.SIGNIFICANCE STATEMENTInterneuron populations use specific anatomical projections to transform sensations into reflexive actions. Here we examined how the anatomical composition of a genetically-defined population of balance interneurons in the larval zebrafish relates to the computations it performs. First, we found that the population of interneurons that stabilize gaze preferentially project to motoneurons that move the eyes downward. Next, we discovered through modeling that such projection patterns can enhance the encoding of nose-up sensations without compromising gaze stabilization. Finally we found that loss of these interneurons impairs a vital behavior, swim bladder inflation, that relies on maintaining a nose-up posture. These observations suggest that anatomical specialization permits neural circuits to represent relevant features of the environment without compromising behavior.

Multifunctional factor progranulin (PGRN) plays an important role in lysosomes, and its mutations and insufficiency are associated with lysosomal storage diseases, including neuronal ceroid lipofuscinosis and Gaucher disease (GD). The first breakthrough in understanding the molecular mechanisms of PGRN as regulator of lysosomal storage diseases came unexpectedly while investigating the role of PGRN in inflammation. Challenged PGRN null mice displayed typical features of GD. In addition, GRN gene variants were identified in GD patients and the serum levels of PGRN were significantly lower in GD patients. PGRN directly binds to and functions as a chaperone of the lysosomal enzyme beta-glucocerebrosidase (GCaase), whose mutations cause GD. In addition, its C-terminus containing granulin E domain, termed Pcgin (PGRN C-terminus for GCase Interaction), is required for the association between PGRN and GCase. The concept that PGRN acts as a chaperone of lysosomal enzymes was further supported and extended by a recent article showing that PGRN acts as a chaperone molecule of lysosomal enzyme cathepsin D (CSTD), and the association between PGRN and CSTD is also mediated by PGRN's C-terminal granulin E domain. Collectively, these reports suggest that PGRN may act as a shared chaperone and regulates multiple lysosomal enzymes.

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.