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Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice

Mon, 03/21/2016 - 4:03pm

BACKGROUND and AIMS: The inflammasome is a well-characterized inducer of inflammation in alcoholic steatohepatitis (ASH). Inflammasome activation requires two signals for mature interleukin (IL)-1beta production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH.

METHODS: Wild-type mice, ATP receptor 2x7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol.

RESULTS: The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1beta. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells.

CONCLUSIONS: Our data indicate that the second signal in inflammasome activation and IL-1beta production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

Bone Mineral Density as a Predictor of Subsequent Wrist Fractures: Findings From the Women's Health Initiative Study

Mon, 03/21/2016 - 4:03pm

CONTEXT: Wrist fractures are common among postmenopausal women. Associations of bone mineral density (BMD) and 10-year predicted risk of major osteoporotic fracture (MOF) with wrist fractures are poorly characterized.

OBJECTIVE: The objective was to examine associations between the Fracture Risk Assessment Tool (FRAX)-predicted risk of MOF, BMD, BMD change, and wrist fracture.

DESIGN: This was a prospective observational study with a mean follow-up of 8.5 years.

SETTING: This study included 40 US centers.

PARTICIPANTS: A total of 11 392 participants from the Women's Health Initiative BMD Cohort aged 50-79 years at baseline were included in this study.

INTERVENTIONS: None.

MAIN OUTCOME: The goal was to measure incident wrist fracture.

RESULTS: A FRAX-predicted MOF risk > /=9.3% identified 17% of the women aged < 65 years who subsequently experienced wrist fracture. Each one standard deviation lower BMD was associated with higher wrist fracture risk, with adjusted hazard ratio (95% confidence interval) of 1.66 (1.42-1.93) for femoral neck (FN) BMD and 1.45 (1.28-1.64) for lumbar spine BMD. Compared with FN BMD T score > /= -1.0, wrist fracture adjusted hazard ratios (95% confidence interval) were: 1.51 (1.06-2.16) for a T score between -1.01 and -1.49; 1.93 (1.36-2.72) for T score between -1.50 and -1.99; 2.52 (1.77-3.60) for a T score between -2.00 and -2.49; and 2.65 (1.78-3.95) for a T score < /= -2.5. Decrease in FN BMD between baseline and year 3 was associated with increased risk of subsequent wrist fracture; however, change in lumbar spine BMD was not.

CONCLUSIONS: Lumbar spine and femoral neck BMDs were associated with incident wrist fracture, but the FRAX threshold recommended to identify screening candidates did not identify the majority of women who subsequently experienced wrist fracture. Improved understanding of determinants of wrist fractures is warranted.

Wrist Fracture and Risk of Subsequent Fracture: Findings from the Women's Health Initiative Study

Mon, 03/21/2016 - 4:02pm

Wrist fractures are common in postmenopausal women and are associated with functional decline. Fracture patterns after wrist fracture are unclear. The goal of this study was to determine the frequency and types of fractures that occur after a wrist fracture among postmenopausal women. We carried out a post hoc analysis of data from the Women's Health Initiative Observational Study and Clinical Trials (1993-2010) carried out at 40 US clinical centers. Participants were postmenopausal women aged 50 to 79 years at baseline. Mean follow-up duration was 11.8 years. Main measures included incident wrist, clinical spine, humerus, upper extremity, lower extremity, hip, and total non-wrist fractures and bone mineral density (BMD) in a subset. Among women who experienced wrist fracture, 15.5% subsequently experienced non-wrist fracture. The hazard for non-wrist fractures was higher among women who had experienced previous wrist fracture than among women who had not experienced wrist fracture: non-wrist fracture overall (hazard ratio [HR] = 1.40, 95% confidence interval [CI] 1.33-1.48), spine (HR = 1.48, 95% CI 1.32-1.66), humerus (HR = 1.78, 95% CI 1.57-2.02), upper extremity (non-wrist) (HR = 1.88, 95% CI 1.70-2.07), lower extremity (non-hip) (HR = 1.36, 95% CI 1.26-1.48), and hip (HR = 1.50, 95% CI 1.32-1.71) fracture. Associations persisted after adjustment for BMD, physical activity, and other risk factors. Risk of non-wrist fracture was higher in women who were younger when they experienced wrist fracture (interaction p value 0.02). Associations between incident wrist fracture and subsequent non-wrist fracture did not vary by baseline BMD category (normal, low bone density, osteoporosis). A wrist fracture is associated with increased risk of subsequent hip, vertebral, upper extremity, and lower extremity fractures. There may be substantial missed opportunity for intervention in the large number of women who present with wrist fractures.

The Montreal Cognitive Assessment: Creating a Crosswalk with the Mini-Mental State Examination

Mon, 03/21/2016 - 4:02pm

OBJECTIVES: To establish Montreal Cognitive Assessment (MoCA) scores that correspond to well-established cut-points on the Mini-Mental State Examination (MMSE).

DESIGN: Cross-sectional observational study.

SETTING: General medical service of a large teaching hospital.

PARTICIPANTS: Individuals aged 75 and older (N = 199; mean age 84, 63% female).

MEASUREMENTS: The MoCA (range 0-30) and the MMSE (range 0-30) were administered within 2 hours of each other. The Abbreviated MoCA (A-MoCA; range 0-22) was calculated from the full MoCA. Scores from the three tests were analyzed using equipercentile equating, a statistical method for determining comparable scores on different tests of a similar construct by estimating percentile equivalents.

RESULTS: MoCA scores were lower (mean 19.3 +/- 5.8) than MMSE scored (mean 24.1 +/- 6.6). Traditional MMSE cut-points of 27 for mild cognitive impairment and 23 for dementia corresponded to MoCA scores of 23 and 17, respectively.

CONCLUSION: Scores on the full and abbreviated versions of the MoCA can be linked directly to the MMSE. The MoCA may be more sensitive to changes in cognitive performance at higher levels of functioning.

Formative Clinical Geriatrics Experiences for First- and Second-Year Medical Students: An Elusive Goal

Mon, 03/21/2016 - 4:02pm

Providing first- and second-year (preclerkship) medical students with a formative clinical experience that effectively teaches clinical skills poses significant challenges for medical educators.

Memantine improves buprenorphine/naloxone treatment for opioid dependent young adults

Mon, 03/21/2016 - 4:02pm

BACKGROUND: Opioid use disorders are considered a serious public health problem among young adults. Current treatment is limited to long-term opioid substitution therapy, with high relapse rates after discontinuation. This study evaluated the co-administration of memantine to brief buprenorphine pharmacotherapy as a treatment alternative.

METHODS: 13-week double-blind placebo-controlled trial evaluating 80 young adult opioid dependent participants treated with buprenorphine/naloxone 16-4mg/day and randomized to memantine (15mg or 30mg) or placebo. Primary outcomes were a change in the weekly mean proportion of opioid use, and cumulative abstinence rates after rapid buprenorphine discontinuation on week 9.

RESULTS: Treatment retention was not significantly different between groups. The memantine 30mg group was significantly less likely to relapse and to use opioids after buprenorphine discontinuation. Among participants abstinent on week 8, those in the memantine 30mg group (81.9%) were significantly less likely to relapse after buprenorphine was discontinued compared to the placebo group (30%) (p < 0.025). Also, the memantine 30mg group had significantly reduced opioid use (mean=0, SEM+/-0.00) compared to the placebo group (mean=0.33, SEM+/-0.35; p < 0.004) during the last 2 weeks of study participation.

CONCLUSIONS: Memantine 30mg significantly improved short-term treatment with buprenorphine/naloxone for opioid dependent young adults by reducing relapse and opioid use after buprenorphine discontinuation. Combined short-term treatment with buprenorphine/naloxone may be an effective alternative treatment to long-term methadone or buprenorphine maintenance in young adults.

Principles of Medical Oncology

Mon, 03/21/2016 - 3:15pm

Medical oncologists are internists specializing in the care of the adult cancer patient. They frequently function as a primary care physician for patients with cancer. This chapter in Cancer Concepts: A Guidebook for the Non-Oncologist will review the practice of medical oncology and some general principles followed in the use of chemotherapy. Concepts including goals of chemotherapy treatment, evaluation of the effects of chemotherapy, integration of chemotherapy with other cancer treatments, and use of combinations of chemotherapy drugs will be covered.

Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: experience of the FSHD Italian National Registry

Mon, 03/21/2016 - 10:31am

OBJECTIVES: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1-3 repeats (1-3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1-3 DRA.

SETTING: Italy.

PARTICIPANTS: 66 index cases and 33 relatives carrying 1-3 DRA.

OUTCOMES: The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk.

RESULTS: No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1-3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment.

CONCLUSIONS: The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity.

Transgenic Drosophila for Investigating DUX4 and FRG1, Two Genes Associated with Facioscapulohumeral Muscular Dystrophy (FSHD)

Mon, 03/21/2016 - 10:10am

Facioscapulohumeral muscular dystrophy (FSHD) is typically an adult onset dominant myopathy. Epigenetic changes in the chromosome 4q35 region linked to both forms of FSHD lead to a relaxation of repression and increased somatic expression of DUX4-fl (DUX4-full length), the pathogenic alternative splicing isoform of the DUX4 gene. DUX4-fl encodes a transcription factor expressed in healthy testis and pluripotent stem cells; however, in FSHD, increased levels of DUX4-fl in myogenic cells lead to aberrant regulation of target genes. DUX4-fl has proven difficult to study in vivo; thus, little is known about its normal and pathogenic roles. The endogenous expression of DUX4-fl in FSHD-derived human muscle and myogenic cells is extremely low, exogenous expression of DUX4-fl in somatic cells rapidly induces cytotoxicity, and, due in part to the lack of conservation beyond primate lineages, viable animal models based on DUX4-fl have been difficult to generate. By contrast, the FRG1 (FSHD region gene 1), which is linked to FSHD, is evolutionarily conserved from invertebrates to humans, and has been studied in several model organisms. FRG1 expression is critical for the development of musculature and vasculature, and overexpression of FRG1 produces a myopathic phenotype, yet the normal and pathological functions of FRG1 are not well understood. Interestingly, DUX4 and FRG1 were recently linked when the latter was identified as a direct transcriptional target of DUX4-FL. To better understand the pathways affected in FSHD by DUX4-fl and FRG1, we generated transgenic lines of Drosophila expressing either gene under control of the UAS/GAL4 binary system. Utilizing these lines, we generated screenable phenotypes recapitulating certain known consequences of DUX4-fl or FRG1 overexpression. These transgenic Drosophila lines provide resources to dissect the pathways affected by DUX4-fl or FRG1 in a genetically tractable organism and may provide insight into both muscle development and pathogenic mechanisms in FSHD.

Scalpel or Straitjacket: CRISPR/Cas9 Approaches for Muscular Dystrophies

Mon, 03/21/2016 - 10:10am

Versatility of CRISPR/Cas9-based platforms makes them promising tools for the correction of diverse genetic/epigenetic disorders. Here we contrast the use of these genome editing tools in two myopathies with very different molecular origins: Duchenne muscular dystrophy, a monogenetic disease, and facioscapulohumeral muscular dystrophy, an epigenetic disorder with unique therapeutic challenges.

Oncologic Imaging

Wed, 03/16/2016 - 12:20pm

Imaging is an integral part of the multidisciplinary management of cancer. Radiographic techniques are indispensable for proper staging of cancers and evaluation of the response of tumors to treatment. A wide variety of imaging modalities is available to clinicians. This chapter in Cancer Concepts: A Guidebook for the Non-Oncologist will introduce the role of radiology in the diagnosis and treatment of cancer.

The Epithelial Transmembrane Protein PERP Is Required for Inflammatory Responses to S. typhimurium Infection: A Dissertation

Mon, 03/14/2016 - 11:19am

Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea.

Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity.

In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation.

Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.

Gender Differences in Choice of Procedure and Case Fatality Rate for Elderly Patients with Acute Cholecystitis: A Masters Thesis

Mon, 03/14/2016 - 10:53am

Background: Treatment decisions for elderly patients with gallbladder pathology are complex. Little is known about what factors go into treatment decisions in this population. We used Medicare data to examine gender-based differences in the use of cholecystectomy vs. cholecystostomy tube placement in elderly patients with acute cholecystitis.

Methods: We queried a 5% random sample of Medicare data (2009-2011) for patients >65 admitted for acute cholecystitis (by ICD-9 code) who subsequently underwent a cholecystectomy and/or cholecystostomy tube placement. Demographic information (age, race), clinical characteristics (Elixhauser index, presence of biliary pathology), and hospital outcomes (case fatality rate, length of stay, need for ICU care) were compared by gender. A multivariable model was used to examine predictors of cholecystectomy vs. cholecystostomy tube placement.

Results: Of 4063 patients admitted with cholecystitis undergoing the procedures of interest just over half (58%) were women. The majority of patients (93%) underwent cholecystectomy. Compared to women, men were younger (average age 76 vs. 78, p value < 0.01) and had few comorbidities (average Elixhauser 1.2 vs. 1.4 p value < 0.01). Case fatality rate was similar between men (2.5%) and women (2.4% p value 0.48). A higher percentage of men spent time in the ICU (36%) compared to women (31% p value < 0.01). On multivariable analysis men were 30% less likely to undergo cholecystectomy (OR 0.69, 95% CI 0.53-0.91).

Conclusion: Elderly men are less likely than elderly women to undergo cholecystectomy for acute cholecystitis despite being younger with less co morbidity and are more likely to spend time in the ICU. More research is needed to determine whether a difference in treatment is contributing to the higher rate of ICU utilization in elderly men with acute cholecystitis.

Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation

Thu, 03/10/2016 - 1:09pm

PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance.

By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila.

How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.

The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.

Single-Molecule Imaging Reveals that Argonaute Re-Shapes the Properties of its Nucleic Acid Guides: A Dissertation

Thu, 03/10/2016 - 1:09pm

Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Counter to the rules of nucleic acid hybridization alone, we find that mouse AGO2 and its guide bind to microRNA targets 17,000 times tighter than the guide without Argonaute. Moreover, AGO2 can distinguish between microRNA-like targets that make seven base pairs with the guide and the products of cleavage, which bind via nine base pairs: AGO2 leaves the cleavage products faster, even though they pair more extensively.

This thesis presents a detailed kinetic interrogation of microRNA and RNA interference pathways. We discovered sub-domains within the previously defined functional domains created by Argonaute and its bound DNA or RNA guide. These sub-domains have features that no longer conform to the well-established properties of unbound oligonucleotides. It is by re-writing the rules for nucleic acid hybridization that Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than that of RNA or DNA. Taken altogether, these studies further our understanding about the biology of small RNA silencing pathways and may serve to guide future work related to all RNA-guided endonucleases.

Hepatitis C Virus: Structural Insights into Protease Inhibitor Efficacy and Drug Resistance: A Dissertation

Thu, 03/10/2016 - 1:09pm

The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity.

My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier.

The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.

Diet Quality and Evening Snacking in Relation to Sleep Duration and Quality among Women with Young Children: A Dissertation

Thu, 03/10/2016 - 1:09pm

Background: Mothers’ diets impact their health and the health of their children, but diet quality is suboptimal among women with young children. Evening snacking among women with young children, especially consumption of high-calorie, high-carbohydrate snacks, may impact overall diet quality and glucose metabolism. Short sleep duration and poor sleep quality may be potential risk factors. We examined whether sleep duration and poor sleep quality were associated with diet quality and evening snacking among women with young children.

Methods: Data were from the National Health and Nutrition Examination Survey (NHANES) 2005-2012, nationally representative cross-sectional surveys of noninstitutionalized U.S. population. Eligible participants were non-pregnant women aged 20-44 years within 5 years of childbirth who completed two 24-hour dietary recalls and completed questions on sleep duration and quality.

Results: Among US women with young children, sleep duration was not associated with diet quality. However, overall sleep quality was associated with poorer diet quality. Short sleep duration was not associated with the consumption of neither evening snacks, nor energy intake from or nutrient consumption of evening snacks.

Conclusion: The findings of this dissertation provide information useful for informing the direction of future research of dietary quality and eating behaviors of U.S. women with young children. Studies are needed to explore whether improvement in sleep quality may improve diet quality among women with young children, which has the potential to improve both maternal and children’s health. Research may be better focused on identifying other psychosocial and behavioral risk factors for unhealthy dietary behaviors among US women with young children.

JNK and PTEN cooperatively control the development of invasive adenocarcinoma of the prostate

Wed, 03/09/2016 - 1:06pm

The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway is implicated in cancer, but the role of JNK in tumorigenesis is poorly understood. Here, we demonstrate that the JNK signaling pathway reduces the development of invasive adenocarcinoma in the phosphatase and tensin homolog (Pten) conditional deletion model of prostate cancer. Mice with JNK deficiency in the prostate epithelium (DeltaJnk DeltaPten mice) develop androgen-independent metastatic prostate cancer more rapidly than control (DeltaPten) mice. Similarly, prevention of JNK activation in the prostate epithelium (DeltaMkk4 DeltaMkk7 DeltaPten mice) causes rapid development of invasive adenocarcinoma. We found that JNK signaling defects cause an androgen-independent expansion of the immature progenitor cell population in the primary tumor. The JNK-deficient progenitor cells display increased proliferation and tumorigenic potential compared with progenitor cells from control prostate tumors. These data demonstrate that the JNK and PTEN signaling pathways can cooperate to regulate the progression of prostate neoplasia to invasive adenocarcinoma.

Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism

Wed, 03/09/2016 - 1:06pm

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.

Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo

Wed, 03/09/2016 - 1:06pm

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).