Faculty Bibliography

Constitutive activation of canonical WNT-TCF signaling is implicated in multiple diseases, including intestine and lung cancers, but there are no WNT-TCF antagonists in clinical use. We have performed a repositioning screen for WNT-TCF response blockers aiming to recapitulate the genetic blockade afforded by dominant-negative TCF. We report that Ivermectin inhibits the expression of WNT-TCF targets, mimicking dnTCF, and that its low concentration effects are rescued by direct activation by TCFVP 16. Ivermectin inhibits the proliferation and increases apoptosis of various human cancer types. It represses the levels of C-terminal beta-CATENIN phosphoforms and of CYCLIN D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases. In vivo, Ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without obvious side effects. Analysis of single semi-synthetic derivatives highlights Selamectin, urging its clinical testing and the exploration of the macrocyclic lactone chemical space. Given that Ivermectin is a safe anti-parasitic agent used by > 200 million people against river blindness, our results suggest its additional use as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.

AIMS: It is well-known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant "Cx43D378stop") led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: a) the morphology of clusters formed by the microtubule plus-end tracking protein "end binding 1" (EB1), b) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin rich sites, c) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. METHODS AND RESULTS: Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa and microtubules. CONCLUSIONS: NaV1.5 and EB1 localization at cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Solid lipid nanoparticles (SLNs) have emerged as an excellent substitute over polymeric nanoparticles and, when incorporated with chitosan which activates the macrophage to impart an immune response, produce excellent results to fight against deleterious diseases like leishmaniasis where its parasite diminishes the immunity of the host to induce resistance. Based upon this hypothesis, chitosan-coated SLNs were developed and loaded with amphotericin B (AmB) for immunoadjuvant chemotherapy of Leishmania infection. Both uncoated and chitosan-coated AmB-loaded SLNs (AmB-SLNs) were fabricated using solvent emulsification and evaporation method. The various processes and formulation parameters involved in AmB-SLN preparation were optimized with respect to particle size and stability of the particles. In vitro hemolytic test credited the formulations to be safe when injected in the veins. The cellular uptake analysis demonstrated that the chitosan-coated AmB-SLN was more efficiently internalized into the J774A.1 cells. The in vitro antileishmanial activity revealed their high potency against Leishmania-infected cells in which chitosan-coated AmB-SLNs were distinguishedly efficacious over commercial formulations (AmBisome and Fungizone). An in vitro cytokine estimation study revealed that chitosan-coated AmB-SLNs activated the macrophages to impart a specific immune response through enhanced production of TNF-alpha and IL-12 with respect to normal control. Furthermore, cytotoxic studies in macrophages and acute toxicity studies in mice evidenced the better safety profile of developed formulation in comparison to marketed formulations. This study indicates that the AmB-SLNs are a safe and efficacious drug delivery system which promises strong competence in antileishmanial chemotherapy and immunotherapy.

The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.

One of the most powerful aspects of biological inquiry using model organisms is the ability to control gene expression. A holy grail is both temporal and spatial control of the expression of specific gene products - that is, the ability to express or withhold the activity of genes or their products in specific cells at specific times. Ideally such a method would also regulate the precise levels of gene activity, and alterations would be reversible. The related goal of controlled or purposefully randomized expression of visible markers is also tremendously powerful. While not all of these feats have been accomplished in Caenorhabditis elegans to date, much progress has been made, and recent technologies put these goals within closer reach. Here, I present published examples of successful two-component site-specific recombination in C. elegans. These technologies are based on the principle of controlled intra-molecular excision or inversion of DNA sequences between defined sites, as driven by FLP or Cre recombinases. I discuss several prospects for future applications of this technology.

This protocol describes how to prepare rough microsomes from dog pancreas. These microsomes can be used to analyze mechanisms of protein translocation and membrane insertion.

This protocol describes how to prepare rat liver rough microsomes that contain undegraded membrane-bound polysomes and can function very well in an in vitro translation system. It uses endogenous ribonuclease inhibitor in all steps, avoiding pelleting rough microsomes in all steps and sacrificing good recovery.

BACKGROUND: Animal spinal cord injury (SCI) models have proved invaluable in better understanding the mechanisms involved in traumatic SCI and evaluating the effectiveness of experimental therapeutic interventions. Over the past 25 years, substantial gains have been made in developing consistent, reproducible and reliable animal SCI models. STUDY DESIGN: Review. OBJECTIVE: The objective of this review was to consolidate current knowledge on SCI models and introduce newer paradigms that are currently being developed. RESULTS: SCI models are categorized based on the mechanism of injury into contusion, compression, distraction, dislocation, transection or chemical models. Contusion devices inflict a transient, acute injury to the spinal cord using a weight-drop technique, electromagnetic impactor or air pressure. Compression devices compress the cord at specific force and duration to cause SCI. Distraction SCI devices inflict graded injury by controlled stretching of the cord. Mechanical displacement of the vertebrae is utilized to produce dislocation-type SCI. Surgical transection of the cord, partial or complete, is particularly useful in regenerative medicine. Finally, chemically induced SCI replicates select components of the secondary injury cascade. Although rodents remain the most commonly used species and are best suited for preliminary SCI studies, large animal and nonhuman primate experiments better approximate human SCI. CONCLUSION: All SCI models aim to replicate SCI in humans as closely as possible. Given the recent improvements in commonly used models and development of newer paradigms, much progress is anticipated in the coming years.