microRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection.

miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels.

Profiling of MicroRNA Targets Using Activity-Based Protein Profiling: Linking Enzyme Activity to MicroRNA-185 Function.

MicroRNAs (miRNAs) act as cellular signal transducers through repression of protein translation. Elucidating targets using bioinformatics and traditional quantitation methods is often insufficient to uncover global miRNA function. Herein, alteration of protein function caused by miRNA-185 (miR-185), an immunometabolic miRNA, was determined using activity-based protein profiling, transcriptomics, and lipidomics. Fluorophosphonate-based activity-based protein profiling of miR-185-induced changes to human liver cells revealed that exclusively metabolic serine hydrolase enzymes were regulated in activity, some with roles in lipid and endocannabinoid metabolism. Lipidomic analysis linked enzymatic changes to levels of cellular lipid species, such as components of very-low-density lipoprotein particles. Additionally, inhibition of one miR-185 target, monoglyceride lipase, led to decreased hepatitis C virus levels in an infectious model. Overall, the approaches used here were able to identify key functional changes in serine hydrolases caused by miR-185 that are targetable pharmacologically, such that a small molecule inhibitor can recapitulate the miRNA phenotype.

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

ABPP and Host-Virus Interactions.

Successful viral infection, as well as any resultant antiviral response, relies on numerous sequential interactions between host and viral factors. These interactions can take the form of affinity-based interactions between viral and host macromolecules or active, enzyme-based interactions, consisting both of direct enzyme activity performed by viral enzymes and indirect modulation of the activity of the host cell's enzymes via viral interference. This activity has the potential to transform the local microenvironment to the benefit or detriment of both the virus and the host, favouring either the continuation of the viral life cycle or the host's antiviral response. Comprehensive characterisation of enzymatic activity during viral infection is therefore necessary for the understanding of virally induced diseases. Activity-based protein profiling techniques have been established as effective and practicable tools with which to interrogate the regulation of enzymes' catalytic activity and the roles played by these enzymes in various cell processes. This paper will review the contributions of these techniques in characterising the roles of both host and viral enzymes during viral infection in humans.

Activity-Based Phosphatidylinositol Kinase Probes Detect Changes to Protein-Protein Interactions During Hepatitis C Virus Replication.

Protein-protein interactions are integral to host-virus interactions and can contribute significantly to enzyme regulation by changing the localization of both host and viral enzymes within the cell, inducing conformational change relevant to enzyme activity or recruiting other additional proteins to form functional complexes. Identifying the interactors of active enzymes using an activity-based protein profiling probe has allowed us to characterize both normal enzyme activation mechanisms and the manner by which these mechanisms are hijacked and altered by the hepatitis C virus (HCV). Here, we report use of a novel activity-based probe, PIKBPyne, which labels phosphatidylinositol kinases (PIKs) in an activity-based manner, to investigate HCV-dependent changes in protein-protein interactions for PI4KB. Herein, we report the synthesis of new variations on PIKBPyne, compare their ability to label the interacting partners of PI4KB, and demonstrate the utility of our approach in characterizing virus-mediated changes to host function.

Chemical Methods for Probing Virus-Host Proteomic Interactions.

Interactions between host and pathogen proteins constitute an important aspect of both infectivity and the host immune response. Different viruses have evolved complex mechanisms to hijack host-cell machinery and metabolic pathways to redirect resources and energy flow toward viral propagation. These interactions are often critical to the virus, and thus understanding these interactions at a molecular level gives rise to opportunities to develop novel antiviral strategies for therapeutic intervention. This review summarizes current advances in chemoproteomic methods for studying these molecular altercations between different viruses and their hosts.

The role of microRNAs in metabolic interactions between viruses and their hosts.

Productive viral infection requires changes to the cellular metabolic landscape in order to obtain the building blocks and create the microenvironments necessary for the viral life cycle. In mammals, these alterations of metabolic pathways have been shown to be mediated in part by host and virus-encoded microRNAs. To counteract virally-induced changes in the cellular metabolic profile, the interferon-regulated antiviral response restricts viral access to key metabolites by altering cellular metabolism, mediated through induction of specific microRNAs regulating key lipid biosynthetic processes. In this review, we examine recent studies demonstrating the important role of microRNAs in the regulation of metabolic flux during viral infection.

Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe.

To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome.

Soraphen A: A Probe for Investigating the Role of de Novo Lipogenesis during Viral Infection.

Many viruses including the hepatitis C virus (HCV) induce changes to the infected host cell metabolism that include the up-regulation of lipogenesis to create a favorable environment for the virus to propagate. The enzyme acetyl-CoA carboxylase (ACC) polymerizes to form a supramolecular complex that catalyzes the rate-limiting step of de novo lipogenesis. The small molecule natural product Soraphen A (SorA) acts as a nanomolar inhibitor of acetyl-CoA carboxylase activity through disruption of the formation of long highly active ACC polymers from less active ACC dimers. We have shown that SorA inhibits HCV replication in HCV cell culture models expressing subgenomic and full-length replicons (IC50 = 5 nM) as well as a cell culture adapted virus. Using coherent anti-Stokes Raman scattering (CARS) microscopy, we have shown that SorA lowers the total cellular lipid volume in hepatoma cells, consistent with a reduction in de novo lipogenesis. Furthermore, SorA treatment was found to depolymerize the ACC complexes into less active dimers. Taken together, our results suggest that SorA treatment reverses HCV-induced lipid accumulation and demonstrate that SorA is a valuable probe to study the roles of ACC polymerization and enzymatic activity in viral pathogenesis.

Attributions about pain as predictors of psychological symptomatology, sexual function, and dyadic adjustment in women with vestibulodynia.

The present study examined whether attributions for vulvo-vaginal pain predicted pain intensity, sexual function, as well as psychological and dyadic adjustment in women with vestibulodynia. Women with vestibulodynia (N = 77) completed measures of attributions, pain, psychological distress, sexual functioning, and dyadic adjustment. They also took part in a structured interview and a gynaecological examination for diagnostic purposes. Attributions are represented by: (1) internality (personal responsibility) or externality (cause lies in an external situation); (2) globality (entire life affected by the problem) or specificity (problem affecting only a specific situation); (3) stability (problem will still remain in the future) or instability (weak probability that the problem will be maintained with time); and (4) partner responsibility (partner responsible or not for the problem). Results indicated that attributions were not significantly correlated with pain outcomes. However, after controlling for pain intensity and relationship duration, internal attributions predicted higher dyadic adjustment, both global and stable attributions predicted lower dyadic adjustment and higher psychological distress, whereas global attributions also predicted increased sexual impairment. Findings suggest that cognitive factors, such as attributions, may be related to psychological distress, sexual functioning, and dyadic adjustment in women with vestibulodynia. Results also highlight the importance of adhering to a biopsychosocial perspective focusing on pain reduction, sexual rehabilitation, and relationship enhancement in the treatment of dyspareunia.

Provoked vestibulodynia: psychological predictors of topical and cognitive-behavioral treatment outcome.

Psychological factors have been found to impact the pain experience and associated sexual impairment of women suffering from provoked vestibulodynia (PV). Despite a lack of randomized treatment outcome studies, particularly concerning psychological predictors of outcome, recent studies have shown that topical applications and cognitive-behavioral therapy (CBT) are among the most popular first-line interventions for PV. The present study aimed to determine the extent to which baseline fear-avoidance variables and pain self-efficacy were differentially associated with topical application and CBT outcomes at six-month follow-up. Data were obtained from 97 women who completed a randomized trial comparing these two treatments. Regression analyses revealed that for topical treatment, higher levels of baseline avoidance predicted worse pain and sexual functioning outcomes, whereas higher levels of pain self-efficacy predicted better outcomes. For CBT, higher levels of baseline fear of pain and catastrophizing contributed to higher pain intensity at follow-up, whereas higher levels of pain self-efficacy were associated with less pain. Psychological factors did not predict sexual functioning outcomes for CBT. Consistent with biopsychosocial models of pain and sexual dysfunction, results indicate that psychological factors contribute to pain and sexual impairment following treatment for PV. Specifically, findings suggest that fear-avoidance variables and pain self-efficacy are significant predictors of topical and CBT treatment outcomes in women with PV.

Fear avoidance and self-efficacy in relation to pain and sexual impairment in women with provoked vestibulodynia.

BACKGROUND: Provoked vestibulodynia is believed to be the most frequent cause of vulvodynia in women of childbearing age, with prevalence rates of up to 12% in the general population. Despite this high prevalence and the fact that vestibulodynia impacts negatively on quality of life, in particular sexual functioning, there has been a paucity of sound research to elucidate the condition's etiology. More specifically, few studies have focused on the role of psychologic factors in the experience of vulvo-vaginal pain and associated sexual impairment. OBJECTIVES: The present study aimed to determine the extent to which fear avoidance variables (catastrophizing, anxiety, fear of pain, hypervigilance) and self-efficacy differentially influenced changes in levels of induced and intercourse pain and also associated sexual dysfunction in these women. METHODS: Data were obtained from 75 vestibulodynia participants who completed a gynecologic examination, structured interview, and standardized questionnaires. RESULTS: The results of regression analyses revealed that higher catastrophizing, fear of pain, and hypervigilance in addition to lower self-efficacy together accounted for 15% of the variation in increased intercourse pain intensity. Among these, only catastrophizing contributed unique variance to intercourse pain. Results also showed that higher state anxiety and fear of pain (escape/avoidance) and also lower self-efficacy explained 22% of the variation in women's sexual impairment. However, only self-efficacy was found to be an independent correlate of sexual impairment. CONCLUSION: Findings support a theoretical model of vestibulodynia as a pain disorder influenced among others by cognitive and affective factors.

Male partners of women with provoked vestibulodynia: attributions for pain and their implications for dyadic adjustment, sexual satisfaction, and psychological distress.

INTRODUCTION: Provoked vestibulodynia is a female genital pain condition that results in sexual dysfunction and impacts negatively on the couple. Although patients' causal attributions have been linked to worse psychosexual outcomes, no study has documented the male partners' perspective of this distressing problem and its potential influence on their psychosexual adaptation. AIM: To identify whether male partners' attributions for vestibulodynia are possible predictors of their dyadic adjustment, sexual functioning, sexual satisfaction, and psychological distress, as well as of women's pain and sexual functioning. METHODS: Thirty-eight women with vestibulodynia first completed measures of pain intensity and sexual functioning. Male partners responded to mailed questionnaires assessing their own attributions for genital pain as well as their psychological distress, relationship adjustment, sexual functioning, and sexual satisfaction. MAIN OUTCOME MEASURES: Women completed the McGill-Melzack Pain Questionnaire (MPQ) and the Female Sexual Function Index (FSFI). Attributions of male partners were measured using an adapted version of the Attributional Style Questionnaire (ASQ)-Partner Version. Men also filled out the Brief Symptom Inventory (BSI), the Dyadic Adjustment Scale (DAS), the Sexual History Form (SHF), and the Global Measure of Sexual Satisfaction (GMSEX). RESULTS: All four negative attribution dimensions and higher levels of women's pain intensity successfully predicted increased psychological distress in male partners. Higher levels of both internal and global attributions were associated with men's poorer dyadic adjustment, whereas global and stable attributions were related to their lower sexual satisfaction. Attributions failed to significantly predict sexual functioning in male partners and women's pain and sexual functioning. CONCLUSIONS: Evaluation and treatment of sexual pain problems should involve both partners and should explore the role of negative attributions.

Do psychosexual factors play a role in the etiology of provoked vestibulodynia? A critical review.

The aim of this review was to critically examine published studies concerning the psychosexual aspects of provoked vestibulodynia. Despite the presence of several methodological limitations, some findings were consistently replicated. Overall, women with vestibulodynia demonstrate impaired sexual functioning, namely, lower levels of sexual desire, arousal, and frequency of intercourse. Childhood physical and sexual abuse represent potential risk factors for the development of this condition. Additionally, specific psychological states such as anxiety, fear of pain, hypervigilance, catastrophizing, and depression, are more frequently reported by these women. More rigorous studies are needed to establish which psychosexual variables may exacerbate and/or maintain vestibulodynia.

The treatment of provoked vestibulodynia: a critical review.

OBJECTIVE: To carry out a critical review of published studies concerning the treatment of provoked vestibulodynia. METHODS: MEDLINE, PsycINFO, and Cochrane were used to identify treatment studies published between January 1996 and December 2006. All studies published in English that dealt specifically with the treatment of provoked vestibulodynia were included in the review regardless of their methodological quality. Thirty-eight treatment studies were thus examined in the present paper. RESULTS: Since 1996, surgical treatment has received somewhat less empirical attention. Nevertheless, it still boasts the best success rates, which range from 61% to 94%. More studies have focused on medical treatments, yielding success rates varying between 13% and 67%. Behavioral treatments have been the least studied, although 35% to 83% of patients benefit from them. Despite these interesting results, only 5 of the 38 treatment studies reviewed are randomized clinical trials. Furthermore, the majority of studies have several methodological weaknesses, such as the absence of (1) control or placebo group, (2) double-blind evaluation, (3) pretreatment pain evaluation, and (4) validated measures of pain and sexual functioning. DISCUSSION: On the basis of the results of the reviewed prospective studies and the randomized clinical trials, vestibulectomy is the most efficacious treatment to date. Though some medical treatments seem little effective, others appear promising and should be investigated further, as is the case with behavioral treatments. Additional randomized clinical trials are necessary to confirm the efficacy of surgery and validate nonsurgical treatments for provoked vestibulodynia.