Faculty Bibliography

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. METHODS: To analyze the evolution of the buoyant density of in vivo-derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. RESULTS: Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. CONCLUSIONS: Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet.

Neural stem cells (NSCs) are multipotent, self-renewing progenitors that generate progeny that differentiate into neurons and glia. NSCs in the adult mammalian brain are generally quiescent. Environmental stimuli such as learning or exercise can activate quiescent NSCs, inducing them to proliferate and produce new neurons and glia. How are these behaviours coordinated? The neurovasculature, the circulatory system of the brain, is a key component of the NSC microenvironment, or 'niche'. Instructive signals from the neurovasculature direct NSC quiescence, proliferation, self-renewal and differentiation. During ageing, a breakdown in the niche accompanies NSC dysfunction and cognitive decline. There is much interest in reversing these changes and enhancing NSC activity by targeting the neurovasculature therapeutically. Here we discuss principles of neurovasculature-NSC crosstalk, and the implications for the design of NSC-based therapies. We also consider the emerging contributions to this field of the model organism Drosophila melanogaster.

The majority of medical students complete the United States Medical Licensing Examination Step 1 after their foundational sciences; however, there are compelling reasons to examine this practice. This article provides the perspectives of eight MD-granting medical schools that have moved Step 1 after the core clerkships, describing their rationale, logistics of the change, outcomes, and lessons learned. The primary reasons these institutions cite for moving Step 1 after clerkships are to foster more enduring and integrated basic science learning connected to clinical care and to better prepare students for the increasingly clinical focus of Step 1. Each school provides key features of the preclerkship and clinical curricula and details concerning taking Steps 1 and 2, to allow other schools contemplating change to understand the landscape. Most schools report an increase in aggregate Step 1 scores after the change. Despite early positive outcomes, there may be unintended consequences to later scheduling of Step 1, including relatively late student reevaluations of their career choice if Step 1 scores are not competitive in the specialty area of their choice. The score increases should be interpreted with caution: These schools may not be representative with regard to mean Step 1 scores and failure rates. Other aspects of curricular transformation and rising national Step 1 scores confound the data. Although the optimal timing of Step 1 has yet to be determined, this article summarizes the perspectives of eight schools that changed Step 1 timing, filling a gap in the literature on this important topic.

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr^-/- mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

The function of protein products generated from intramembraneous cleavage by the gamma-secretase complex is not well defined. The gamma-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein which results in Abeta, a transmembrane (TM) peptide. Another protein that undergoes a very similar gamma-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of the tyrosine kinase receptor B (TrkB). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time- dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Since this residue is immediately after the gamma-secretase cleavage site, we then examined if the p75(alphagamma) peptide, which is a product of both alpha- and gamma- cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether our results suggest that TrkB activation by p75(alphagamma) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimers disease, and epilepsy.

The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

High throughput technologies opened a new era in biomedicine by enabling massive analysis of gene expression at both RNA and protein levels. Unfortunately, expression data obtained in different experiments are often poorly compatible, even for the same biologic samples. Here, using experimental and bioinformatic investigation of major experimental platforms, we show that aggregation of gene expression data at the level of molecular pathways helps to diminish cross- and intra-platform bias otherwise clearly seen at the level of individual genes. We created a mathematical model of cumulative suppression of data variation that predicts the ideal parameters and the optimal size of a molecular pathway. We compared the abilities to aggregate experimental molecular data for the 5 alternative methods, also evaluated by their capacity to retain meaningful features of biologic samples. The bioinformatic method OncoFinder showed optimal performance in both tests and should be very useful for future cross-platform data analyses.