Faculty Bibliography

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

Background: Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. Accordingly, numerous small molecules have been developed to modulate immune cell function, many of which are promising immunotherapy candidates. However, effective immunotherapies require precise effects only on pathological immune cells without causing side effects on the healthy tissues or other immune cells. Results: We here propose a radically different approach that implement and evaluate in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein (HDL), which can preferentially deliver therapeutic compounds to pathological immune cells in atherosclerotic plaques^1,2. Using the Apoe ^-/- mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining nanomaterial characterization, in vitro assays, optical imaging, and immunological techniques (a). These distinct physiochemical properties among the library nanoparticles resulted in an approximate 6-fold difference in promoting cholesterol efflux from macrophages, 10-fold difference among blood half-lives, 3.35-fold difference in relative aorta-to-liver accumulation, and 3.84-fold difference in relative aortic-to-splenic macrophage accumulation. In a proof-of-concept study, we identified an ideal drug-delivery nanoparticle with a long blood half-life, low liver retention, and high specificity to atherosclerotic macrophages. We formulated into the nanoparticle (Rx-HDL) a liver receptor X agonist (GW3965), whose high liver toxicity failed its clinical translation. Compared to an undesirable nanoparticle with poor properties for drug delivery (Rx-PLGA-HDL), Rx-HDL minimally retained in the liver while still efficiently delivered GW3965 to atherosclerotic plaques, revealed by in vivo PET imaging and ex vivo flow cytometry. In a one-week intensive treatment regimen in atherosclerotic mice, Rx-HDL totally abolished GW3965's liver toxicity (b ). Finally, a 6-week long-term treatment with Rx-HDL produced significant therapeutic effects on atherosclerotic plaques (c). Conclusion: In this study, for the first time, we demonstrate a systemic in vivo immune cell screening of a nanoparticle library can produce effective immunotherapy for atherosclerosis. Screening the immune cell specificity of nanoparticles can be employed to develop tailored therapies for atherosclerosis and other inflammatory diseases. [IMAGE PRESENTED]

Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degradation. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metabolism and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson Disease and Alzheimer Disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal proteolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clinical and experimental studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum.

AIMS: To evaluate whether an ultra-low-dose CT protocol can diagnose selected limb fractures as well as conventional CT (C-CT). PATIENTS AND METHODS: We prospectively studied 40 consecutive patients with a limb fracture in whom a CT scan was indicated. These were scanned using an ultra-low-dose CT Reduced Effective Dose Using Computed Tomography In Orthopaedic Injury (REDUCTION) protocol. Studies from 16 selected cases were compared with 16 C-CT scans matched for age, gender and type of fracture. Studies were assessed for diagnosis and image quality. Descriptive and reliability statistics were calculated. The total effective radiation dose for each scanned site was compared. RESULTS: The mean estimated effective dose (ED) for the REDUCTION protocol was 0.03 milliSieverts (mSv) and 0.43 mSv (p < 0.005) for C-CT. The sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the REDUCTION protocol to detect fractures were 0.98, 0.89, 0.98 and 0.89 respectively when two occult fractures were excluded. Inter- and intra-observer reliability for diagnosis using the REDUCTION protocol (kappa = 0.75, kappa = 0.71) were similar to those of C-CT (kappa = 0.85, kappa = 0.82). Using the REDUCTION protocol, 3D CT reconstructions were equivalent in quality and diagnostic information to those generated by C-CT (kappa = 0.87, kappa = 0.94). CONCLUSION: With a near 14-fold reduction in estimated ED compared with C-CT, the REDUCTION protocol reduces the amount of CT radiation substantially without significant diagnostic decay. It produces images that appear to be comparable with those of C-CT for evaluating fractures of the limbs. Cite this article: Bone Joint J 2016;98-B:1668-73.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

PURPOSE OF REVIEW:Better tools are sorely needed for both the prevention and treatment of cardiovascular diseases, which account for more than one-third of the deaths in Western countries. MicroRNAs typically regulate the expression of several mRNAs involved in the same biological process. Therapeutic manipulation of miRNAs could restore the expression of multiple players within the same physiologic pathway, and ideally offer better curative outcomes than conventional approaches that target only one single player within the pathway. This review summarizes available studies on the prospective value of targeting miRNAs to prevent dyslipidemia and atherogenesis. RECENT FINDINGS:Silencing the expression of miRNAs that target key genes involved in lipoprotein metabolism in vivo with antisense oligonucleotides results in the expected de-repression of target mRNAs in liver and atherosclerotic plaques. However, the consequences of long-term antimiRNA treatment on both circulating lipoproteins and athero-protection are yet to be established. SUMMARY:A number of studies have demonstrated the efficacy of miRNA mimics and inhibitors as novel therapeutic tools for treating dyslipidemia and cardiovascular diseases. Nevertheless, concerns over unanticipated side-effects related to de-repression of additional targets should not be overlooked for miRNA-based therapies.

The process of embryonic morphogenesis involves cell shape changes, which are accompanied by constant remodeling of cell junctions. Rho GTPases are signaling proteins that regulate adherens junctions (AJ) by mediating the recycling of junction components, actin polymerization, and myosin activity. We investigated the role of the CDC-42 GTPase during elongation of C. elegans embryo, during which epidermal cells extend along their anterior-posterior axis and shrink along their dorsal-ventral axis, lengthening the embryo four-fold. Depletion of CDC-42 caused embryonic lethality at different stages of elongation. Overactivation of CDC-42 in sensitized hmp-1/alpha-catenin hypomorphic mutants, which have partially compromised junctions, also led to embryonic arrest, suggesting that CDC-42 activity might be regulated at junctions during elongation. Rho GTPases are activated by GEFs and inhibited by RhoGAPs. Looking for a possible regulator of CDC-42 at the junctions, we found that the conserved C. elegans RhoGAP PAC-1/ARHGAP21 co-localizes with CDC-42 at adherens junctions in embryonic epithelial tissues. Genetic interactions tests showed that PICC-1/CCDC85A-C, which encodes a coiled-coil protein that interacts with PAC-1 and with the junction component JAC-1/p120, likely functions with PAC-1 in the same pathway during elongation. pac-1 mutant embryos develop normally, but pac-1 mutations enhance the lethality of hypomorphic hmp-1 mutants, as we observed in the case of CDC-42 overactivation. The RhoGAP activity of PAC-1 is required for its function, but not for its localization, suggesting that PAC-1 regulates the strength of AJs by locally inhibiting CDC-42 GTPase activity. In support of this hypothesis, decreasing CDC-42 levels partially rescues the lethality of hmp-1 pac-1 double mutants, and overexpressing PAC-1 lowers levels of an active CDC-42 biosensor at AJs. In pac-1 mutants and embryos expressing constitutively active CDC-42, levels of AJ proteins were increased at junctions. Consistent with this increase being the cause of elongation defects, overexpression of HMR-1/ E-cadherin enhances the lethality of hmp-1 mutants. Based on these results, we propose that CDC-42 activity must be limited at junctions to ensure that appropriate levels of junction components are present as these structures remodel during morphogenetic events

In the past decade, microRNAs (miRNAs) have emerged as key regulators of circulating levels of lipoproteins. Specifically, recent work has uncovered the role of miRNAs in controlling the levels of atherogenic low-density lipoprotein LDL (LDL)-cholesterol by post-transcriptionally regulating genes involved in very low-density lipoprotein (VLDL) secretion, cholesterol biosynthesis, and hepatic LDL receptor (LDLR) expression. Interestingly, several of these miRNAs are located in genomic loci associated with abnormal levels of circulating lipids in humans. These findings reinforce the interest of targeting this subset of non-coding RNAs as potential therapeutic avenues for regulating plasma cholesterol and triglyceride (TAG) levels. In this review, we will discuss how these new miRNAs represent potential pre-disposition factors for cardiovascular disease (CVD), and putative therapeutic targets in patients with cardiometabolic disorders. This article is part of a Special Issue, entitled: MicroRNAs and lipid/energy metabolism and related diseases, edited by Carlos Fernandez-Hernando and Yajaira Suarez.

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Aged skin heals wounds poorly, increasing susceptibility to infections. Restoring homeostasis after wounding requires the coordinated actions of epidermal and immune cells. Here we find that both intrinsic defects and communication with immune cells are impaired in aged keratinocytes, diminishing their efficiency in restoring the skin barrier after wounding. At the wound-edge, aged keratinocytes display reduced proliferation and migration. They also exhibit a dampened ability to transcriptionally activate epithelial-immune crosstalk regulators, including a failure to properly activate/maintain dendritic epithelial T cells (DETCs), which promote re-epithelialization following injury. Probing mechanism, we find that aged keratinocytes near the wound edge don't efficiently upregulate Skints or activate STAT3. Notably, when epidermal Stat3, Skints, or DETCs are silenced in young skin, re-epithelialization following wounding is perturbed. These findings underscore epithelial-immune crosstalk perturbations in general, and Skints in particular, as critical mediators in the age-related decline in wound-repair.