The grapefruit flavonoid naringenin as a Hepatitis C virus therapy : efficacy, mechanism and delivery

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 143-144).Hepatitis C virus (HCV) infection accounts for approximately 40% of chronic liver disease in the United States and results in an estimated 8,000-10,000 deaths annually. Simulations suggest that in the next decade morbidity and mortality associated with HCV infections will result in approximately 200,000 deaths and direct medical expenditures of over $10 billion. Furthermore, recent WHO estimates of worldwide prevalence suggest that up to 2% of the world population is infected with HCV, representing between 120 and 200 million people. For reasons that are still poorly understood, the current standard of care is effective only in a subset of patients, and depends on both patient-related and disease-related characteristics. Sustained virological response (SVR) - HCV RNA in patient plasma drops below detectable levels at week 24 following completion of treatment, which is thought to be indicative of curing the disease - is attainable in only ~50% of patients. In recent years, HCV production has been shown to be inextricably linked to lipid metabolism and to the secretion of very low density lipoproteins (VLDL) from hepatocytes. This suggests that by modulating lipid metabolism in the cell, viral production may be reduced in a clinically relevant manner.(cont.) This work begins by characterizing the link between VLDL secretion and HCV production in the Huh7.5.1/JFH-1 system. We proceed to examine the effects of naringenin, a grapefruit flavonoid, on the production of HCV. Naringenin has been shown previously to reduce VLDL secretion from hepatocytes, and we demonstrate its ability to block HCV production, as well. We explore the mechanism of naringenin's effect on HCV, and show that the flavonoid prevents the assembly of infectious viruses in the cell. Despite previous success by several groups in describing the mechanisms involved in naringenin's effect on VLDL secretion, these mechanisms are thought to account only for ~50% of the observed inhibition, suggesting a deeper understanding of the underlying principals is still lacking. We suggest that naringenin exerts its metabolic, and consequently, antiviral effects through modulation of nuclear recepetor (NR) activity. NRs are a superfamily of ligand-regulated transcription factors known to have an important role in maintaining the homeostasis of metabolites. We show that naringenin activates peroxisome proliferator activated receptors (PPARs), NRs known to drive [beta]-oxidation; and inhibits the liver X receptor (LXR), known to drive lipogenesis and cholesterol synthesis. Despite naringenin's promise as a possible treatment for HCV, its low bioavailability, limits its clinical potential. We conclude this work by showing that naringenin's solubility and bioavailability - and thus, clinical relevance - can be greatly enhanced by complexation with [beta]-cyclodextrins.by Jonathan Goldwasser.Ph.D

Interactive Proofs for Verifying Machine Learning

We consider the following question: using a source of labeled data and interaction with an untrusted prover, what is the complexity of verifying that a given hypothesis is "approximately correct"? We study interactive proof systems for PAC verification, where a verifier that interacts with a prover is required to accept good hypotheses, and reject bad hypotheses. Both the verifier and the prover are efficient and have access to labeled data samples from an unknown distribution. We are interested in cases where the verifier can use significantly less data than is required for (agnostic) PAC learning, or use a substantially cheaper data source (e.g., using only random samples for verification, even though learning requires membership queries). We believe that today, when data and data-driven algorithms are quickly gaining prominence, the question of verifying purported outcomes of data analyses is very well-motivated. We show three main results. First, we prove that for a specific hypothesis class, verification is significantly cheaper than learning in terms of sample complexity, even if the verifier engages with the prover only in a single-round (NP-like) protocol. Moreover, for this class we prove that single-round verification is also significantly cheaper than testing closeness to the class. Second, for the broad class of Fourier-sparse boolean functions, we show a multi-round (IP-like) verification protocol, where the prover uses membership queries, and the verifier is able to assess the result while only using random samples. Third, we show that verification is not always more efficient. Namely, we show a class of functions where verification requires as many samples as learning does, up to a logarithmic factor

Moloney murine leukemia virus decay mediated by retroviral reverse transcriptase degradation of genomic RNA

AbstractRetroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 °C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme

Transcriptional Regulation of Human and Rat Hepatic Lipid Metabolism by the Grapefruit Flavonoid Naringenin: Role of PPARα, PPARγ and LXRα

Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARα, PPARγ, and LXRα. We show it activates the ligand-binding domain of both PPARα and PPARγ, while inhibiting LXRα in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRα, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARα co-activator, PGC1α. The flavonoid activates PPAR response element (PPRE) while suppressing LXRα response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRα-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRα inhibitor

AUDIT: Practical Accountability of Secret Processes

The US federal court system is exploring ways to improve the accountability of electronic surveillance, an opaque process often involving cases sealed from public view and tech companies subject to gag orders against informing surveilled users. One judge has proposed publicly releasing some metadata about each case on a paper cover sheet as a way to balance the competing goals of (1) secrecy, so the target of an investigation does not discover and sabotage it, and (2) accountability, to assure the public that surveillance powers are not misused or abused. Inspired by the courts\u27 accountability challenge, we illustrate how accountability and secrecy are simultaneously achievable when modern cryptography is brought to bear. Our system improves configurability while preserving secrecy, offering new tradeoffs potentially more palatable to the risk-averse court system. Judges, law enforcement, and companies publish commitments to surveillance actions, argue in zero-knowledge that their behavior is consistent, and compute aggregate surveillance statistics by multi-party computation (MPC). We demonstrate that these primitives perform efficiently at the scale of the federal judiciary. To do so, we implement a hierarchical form of MPC that mirrors the hierarchy of the court system. We also develop statements in succinct zero-knowledge (SNARKs) whose specificity can be tuned to calibrate the amount of information released. All told, our proposal not only offers the court system a flexible range of options for enhancing accountability in the face of necessary secrecy, but also yields a general framework for accountability in a broader class of secret information processes

Cognitive Information Processing

Contains reports on seven research projects.National Science Foundation (Grant SED76-81985)Graphic Arts Research Foundation (Grant)Providence Gravure, Inc. (Grant)Associated Press (Grant)National Institutes of Health (Grant 1 RO1 GM22547-01)National Institutes of Health (Grant 1 PO1 AG00354-01)Health Sciences Fund (Grant 76-11

Enhancement of Naringenin Bioavailability by Complexation with Hydroxypropoyl-β-Cyclodextrin

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and Cmax increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (K01DK080241)Harvard Clinical Nutrition Research Center (P30-DK040561)European Research Council (Starting Grant (TMIHCV 242699))Massachusetts General Hospital (BioMEMS Resource Center (P41 EB-002503))Alexander Silberman Institute of Life Science

Record breaking Rubik's Cube robot

The world's fastest Rubik's Cube-solving robot has been developed by students at Swinburne University of Technology. The robot, named Ruby, can solve the scrambled puzzle in just over 10 seconds, including the time taken to scan the initial status of the cube. It was built from scratch by six students as their final year project for the double degree in Bachelor of Engineering (Robotics and Mechatronics)/Bachelor of Science (Computer Science and Software Engineering)