Capsomer Vaccines Protect Mice from Vaginal Challenge with Human Papillomavirus

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18

Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2

AbstractVaccination with papillomavirus L2 has been shown to induce neutralizing antibodies that protect against homologous type infection and cross-neutralize a limited number of genital HPVs. Surprisingly, we found that antibodies to bovine papillomavirus (BPV1) L2 amino acids 1–88 induced similar titers of neutralizing antibodies against Human papillomavirus (HPV)16 and 18 and BPV1 pseudoviruses and also neutralized HPV11 native virions. These antibodies also neutralized each of the other pseudovirus types tested, HPV31, HPV6 and Cottontail rabbit papillomavirus (CRPV) pseudoviruses, albeit with lower titers. HPV16, HPV18, HPV31, HPV6 and CRPV L2 anti-sera also displayed some cross-neutralization, but the titers were lower and did not encompass all pseudoviruses tested. This study demonstrates the presence of broadly cross-neutralizing epitopes at the N-terminus of L2 that are shared by cutaneous and mucosal types and by types that infect divergent species. BPV1 L2 was exceptionally effective at inducing cross-neutralizing antibodies to these shared epitopes

Clinical and genetic determinants of warfarin pharmacokinetics and pharmacodynamics during treatment initiation

Variable warfarin response during treatment initiation poses a significant challenge to providing optimal anticoagulation therapy. We investigated the determinants of initial warfarin response in a cohort of 167 patients. During the first nine days of treatment with pharmacogenetics-guided dosing, S-warfarin plasma levels and international normalized ratio were obtained to serve as inputs to a pharmacokinetic-pharmacodynamic (PK-PD) model. Individual PK (S-warfarin clearance) and PD (I max) parameter values were estimated. Regression analysis demonstrated that CYP2C9 genotype, kidney function, and gender were independent determinants of S-warfarin clearance. The values for I max were dependent on VKORC1 and CYP4F2 genotypes, vitamin K status (as measured by plasma concentrations of proteins induced by vitamin K absence, PIVKA-II) and weight. Importantly, indication for warfarin was a major independent determinant of I max during initiation, where PD sensitivity was greater in atrial fibrillation than venous thromboembolism. To demonstrate the utility of the global PK-PD model, we compared the predicted initial anticoagulation responses with previously established warfarin dosing algorithms. These insights and modeling approaches have application to personalized warfarin therapy. © 2011 Gong et al

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer

<p>Abstract</p> <p>Background</p> <p>The absence of highly sensitive and specific serum biomarkers makes mass screening for ovarian cancer impossible. The claudin proteins are frequently overexpressed in ovarian cancers, but their potential as prognostic, diagnostic, or detection markers remains unclear. Here, we have explored the possible use of these proteins as screening biomarkers for ovarian cancer detection.</p> <p>Methods</p> <p>Claudin protein shedding from cells was examined by immunoblotting of conditioned culture media. The presence of claudins in exosomes released from ovarian cancer cells was demonstrated by sucrose gradient separation and immunogold electron microscopy experiments. Claudin-4-containing exosomes in the plasma of ovarian cancer patients were evaluated in a pilot panel of 63 ovarian cancer patients and 50 healthy volunteers. The CA125 marker was also assessed in these samples and compared with claudin-4 positivity.</p> <p>Results</p> <p>We show that full-length claudins can be shed from ovarian cancer cells in culture and found in the media as part of small lipid vesicles known as exosomes. Moreover, 32 of 63 plasma samples from ovarian cancer patients exhibited the presence of claudin-4-containing exosomes. In contrast, only one of 50 samples from individuals without cancer exhibited claudin-4-positive exosomes. In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively. The two tests did not appear to be independent and were strongly correlated.</p> <p>Conclusion</p> <p>Our work shows for the first time that claudin-4 can be released from ovarian cancer cells and can be detected in the peripheral circulation of ovarian cancer patients. The development of sensitive assays for the detection of claudin-4 in blood will be crucial in determining whether this approach can be useful, alone or in combination with other screening methods, for the detection of ovarian cancer.</p

Knowledge-Driven Multi-Locus Analysis Reveals Gene-Gene Interactions Influencing HDL Cholesterol Level in Two Independent EMR-Linked Biobanks

Genome-wide association studies (GWAS) are routinely being used to examine the genetic contribution to complex human traits, such as high-density lipoprotein cholesterol (HDL-C). Although HDL-C levels are highly heritable (h2∼0.7), the genetic determinants identified through GWAS contribute to a small fraction of the variance in this trait. Reasons for this discrepancy may include rare variants, structural variants, gene-environment (GxE) interactions, and gene-gene (GxG) interactions. Clinical practice-based biobanks now allow investigators to address these challenges by conducting GWAS in the context of comprehensive electronic medical records (EMRs). Here we apply an EMR-based phenotyping approach, within the context of routine care, to replicate several known associations between HDL-C and previously characterized genetic variants: CETP (rs3764261, p = 1.22e-25), LIPC (rs11855284, p = 3.92e-14), LPL (rs12678919, p = 1.99e-7), and the APOA1/C3/A4/A5 locus (rs964184, p = 1.06e-5), all adjusted for age, gender, body mass index (BMI), and smoking status. By using a novel approach which censors data based on relevant co-morbidities and lipid modifying medications to construct a more rigorous HDL-C phenotype, we identified an association between HDL-C and TRIB1, a gene which previously resisted identification in studies with larger sample sizes. Through the application of additional analytical strategies incorporating biological knowledge, we further identified 11 significant GxG interaction models in our discovery cohort, 8 of which show evidence of replication in a second biobank cohort. The strongest predictive model included a pairwise interaction between LPL (which modulates the incorporation of triglyceride into HDL) and ABCA1 (which modulates the incorporation of free cholesterol into HDL). These results demonstrate that gene-gene interactions modulate complex human traits, including HDL cholesterol

Topiramate-Induced Modulation of Hepatic Molecular Mechanisms: An Aspect for Its Anti-Insulin Resistant Effect

Topiramate is an antiepileptic drug known to ameliorate insulin resistance besides reducing body weight. Albeit liver plays a fundamental role in regulation of overall insulin resistance, yet the effect of topiramate on this organ is controversial and is not fully investigated. The current work aimed to study the potential hepatic molecular mechanistic cassette of the anti-insulin resistance effect of topiramate. To this end, male Wistar rats were fed high fat/high fructose diet (HFFD) for 10 weeks to induce obese, insulin resistant, hyperglycemic animals, but with no overt diabetes. Two HFFD-groups received oral topiramate, 40 or 100 mg/kg, for two weeks. Topiramate, on the hepatic molecular level, has opposed the high fat/high fructose diet effect, where it significantly increased adiponectin receptors, GLUT2, and tyrosine kinase activity, while decreased insulin receptor isoforms. Besides, it improved the altered glucose homeostasis and lipid profile, lowered the ALT level, caused subtle, yet significant decrease in TNF-α, and boosted adiponectin in a dose dependent manner. Moreover, topiramate decreased liver weight/, visceral fat weight/, and epididymal fat weight/body weight ratios. The study proved that insulin-resistance has an effect on hepatic molecular level and that the topiramate-mediated insulin sensitivity is ensued partly by modulation of hepatic insulin receptor isoforms, activation of tyrosine kinase, induction of GLUT2 and elevation of adiponectin receptors, as well as their ligand, adiponectin, besides its known improving effect on glucose tolerance and lipid homeostasis

Identification and functional characterization of G6PC2 coding variants influencing glycemic traits define an effector transcript at the G6PC2-ABCB11 locus.

Genome wide association studies (GWAS) for fasting glucose (FG) and insulin (FI) have identified common variant signals which explain 4.8% and 1.2% of trait variance, respectively. It is hypothesized that low-frequency and rare variants could contribute substantially to unexplained genetic variance. To test this, we analyzed exome-array data from up to 33,231 non-diabetic individuals of European ancestry. We found exome-wide significant (P<5×10-7) evidence for two loci not previously highlighted by common variant GWAS: GLP1R (p.Ala316Thr, minor allele frequency (MAF)=1.5%) influencing FG levels, and URB2 (p.Glu594Val, MAF = 0.1%) influencing FI levels. Coding variant associations can highlight potential effector genes at (non-coding) GWAS signals. At the G6PC2/ABCB11 locus, we identified multiple coding variants in G6PC2 (p.Val219Leu, p.His177Tyr, and p.Tyr207Ser) influencing FG levels, conditionally independent of each other and the non-coding GWAS signal. In vitro assays demonstrate that these associated coding alleles result in reduced protein abundance via proteasomal degradation, establishing G6PC2 as an effector gene at this locus. Reconciliation of single-variant associations and functional effects was only possible when haplotype phase was considered. In contrast to earlier reports suggesting that, paradoxically, glucose-raising alleles at this locus are protective against type 2 diabetes (T2D), the p.Val219Leu G6PC2 variant displayed a modest but directionally consistent association with T2D risk. Coding variant associations for glycemic traits in GWAS signals highlight PCSK1, RREB1, and ZHX3 as likely effector transcripts. These coding variant association signals do not have a major impact on the trait variance explained, but they do provide valuable biological insights