Influence of DNA-protein interactions on purification and assembly of virus-like particles

Modular virus-like-particles (VLPs), presenting foreign antigens on their surface, are promising candidates for a wide range of future vaccines. A viable production pathway is the expression and purification of viral structural proteins and their subsequent in vitro assembly into VLPs, in a bioprocess environment. One promising approach is the use of murine polyomavirus major capsid protein VP1 as a carrier of modular epitopes from vaccine targets. This platform technology uses E. coli as an expression system and showed promising results in creating VLP vaccines candidates directed at influenza, Group A Streptococcus and other infectious pathogens. However, like other viral capsomeres and viral structures, purification using chromatography techniques remains a challenge as conventional high capacity ion exchange matrices suffer from low binding capacities. In this work the role of DNA-protein interaction during the purification of VLP precursor capsomeres is investigated. It is found that modular VP1 capsomeres coat DNA molecules forming large DNA-protein complexes that are unable to access the pores of chromatographic resins resulting in inefficient column binding. By increasing the salt concentration of the buffer above 0.3M NaCl, the DNA-protein complexes dissociate. At intermediate salt concentrations salt-tolerant ion-exchange resins can be used to efficiently capture and purify VP1 capsomeres, as the salt breaks the aggregates but is insufficient to interfere with binding to the salt-tolerant matrix. This approach increases the binding capacity of VLP precursor proteins by at least a magnitude over published laboratory-based methods. Please click Download on the upper right corner to see the full abstract

Micro-RNA and mRNA Profiles Associated with Ectopic Germinal Center Formation in Thymus Samples of Patients with Autoimmune Myas

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder caused by antibodies directed against proteins present at the post-synaptic surface of neuromuscular junction (NMJ). A characteristic pathology of patients with early onset MG is thymic hyperplasia with ectopic germinal centers (GC). However, mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Micro-RNAs (miRNA) are small, non-coding RNAs that are increasingly appreciated to be involved in the pathology of several autoimmune diseases. In order to determine the central mechanisms involved in the pathology, thymus samples from MG patients were assessed by histology and grouped based on appearance of GC compared to samples without them. MiRNA and mRNA were evaluated using GeneChip® miRNA 4.0 Array and GeneChip® Human Transcriptome Array 2.0, respectively. Partek Genomic Suite 6.6 and Transcript Analysis Console 2.0 programs were used for further analysis. Thirty-four mature miRNA and forty eight annotated mRNA transcripts were identified that were differentially expressed between the two groups with greater than 1.5 fold difference in expression (ANOVA p Our study shows that there is a distinct mRNA and miRNA expression pattern in the thymus and maintenance of autoimmunity is supported by regulatory pathways known to be involved in neoplasia

Differential mRNA expression in ectopic germinal centers of myasthenia gravis thymus

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder resulting in weakness of voluntary muscles. It is caused by antibodies directed against proteins present at the post-synaptic surface of neuromuscular junction (NMJ). A characteristic pathology of patients with early onset MG is thymic hyperplasia with ectopic germinal centers (GC). However, mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. In order to determine the central mechanisms involved in the pathology, thymus samples from MG patients were assessed by histology and grouped based on appearance of GC compared to samples without them. We assessed the differential mRNA expression profiles between the two groups by GeneChip® Human Transcriptome Array 2.0. Partek Genomic Suite 6.6 and Transcript Analysis Console 2.0 programs were used for further analysis. Forty eight annotated mRNA transcripts were identified that were differentially expressed between the two groups with greater than 1.5 fold difference in expression (ANOVA p\u3c0.05). We verified their expression by RT-PCR. We identified Regulator of G protein Signaling 13 or RGS13 that is known to be expressed in GC B-cells and regulate responsiveness to chemokine signaling. Upregulation of RGS13 was found to be associated with specimens having GC. We verified its expression in GC by immunohistochemistry. Gene ontology (GO) enrichment analysis and Ingenuity Pathway Analysis (IPA) core analysis of differentially expressed genes indicate involvement of immune response regulation and cell proliferation pathways, indicating their importance in GC formation and regulation

Increased expression of programmed death ligand 1 (PD-L1) in human pituitary tumors

PURPOSE: Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas. METHODS: PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry. RESULTS: Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes. CONCLUSIONS: Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management

Association of rs780094 in \u3ci\u3eGCKR\u3c/i\u3e with Metabolic Traits and Incident Diabetes and Cardiovascular Disease: The ARIC Study

Objective: The minor T-allele of rs780094 in the glucokinase regulator gene (GCKR) associates with a number of metabolic traits including higher triglyceride levels and improved glycemic regulation in study populations of mostly European ancestry. Using data from the Atherosclerosis Risk in Communities (ARIC) Study, we sought to replicate these findings, examine them in a large population-based sample of African American study participants, and to investigate independent associations with other metabolic traits in order to determine if variation in GKCR contributes to their observed clustering. In addition, we examined the association of rs780094 with incident diabetes, coronary heart disease (CHD), and stroke over up mean follow-up times of 8, 15, and 15 years, respectively. Research Design and Methods: Race-stratified analyses were conducted among 10,929 white and 3,960 black participants aged 45–64 at baseline assuming an additive genetic model and using linear and logistic regression and Cox proportional hazards models. Results: Previous findings replicated among white participants in multivariable adjusted models: the T-allele of rs780094 was associated with lower fasting glucose (p = 10-7) and insulin levels (p = 10-6), lower insulin resistance (HOMA-IR, p=10-9), less prevalent diabetes (p = 10-6), and higher CRP (p = 10-8), 2-h postprandial glucose (OGTT, p = 10-6), and triglyceride levels (p = 10-31). Moreover, the T-allele was independently associated with higher HDL cholesterol levels (p = 0.022), metabolic syndrome prevalence (p = 0.043), and lower beta-cell function measured as HOMA-B (p = 0.011). Among black participants, the T-allele was associated only with higher triglyceride levels (p = 0.004) and lower insulin levels (p = 0.002) and HOMA-IR (p = 0.013). Prospectively, the T-allele was associated with reduced incidence of diabetes (p = 10-4) among white participants, but not with incidence of CHD or stroke. Conclusions: Our findings indicate rs780094 has independent associations with multiple metabolic traits as well as incident diabetes, but not incident CHD or stroke. The magnitude of association between the SNP and most traits was of lower magnitude among African American compared to white participants

Meningioma Genomics: Diagnostic, Prognostic, and Therapeutic Applications

There has been a recent revolution in our understanding of the genetic factors that drive meningioma, punctuating an equilibrium that has existed since Cushing’s germinal studies nearly a century ago. A growing appreciation that meningiomas share similar biologic features with other malignancies has allowed extrapolation of management strategies and lessons from intra-axial central nervous system neoplasms and systemic cancers to meningiomas. These features include a natural proclivity for invasion, frequent intratumoral heterogeneity, and correlation between biologic profile and clinical behavior. Next-generation sequencing has characterized recurrent somatic mutations in NF2, TRAF7, KLF4, AKT1, SMO, and PIK3CA, which are collectively present in ~80% of sporadic meningiomas. Genomic features of meningioma further associate with tumor location, histologic subtype, and possibly clinical behavior. Such genomic decryption, along with advances in targeted pharmacotherapy, provides a maturing integrated view of meningiomas. We review recent advances in meningioma genomics and probe their potential applications in diagnostic, therapeutic, and prognostic frontiers

The Epigenomics of Pituitary Adenoma

Background: The vast majority of pituitary tumors are benign and behave accordingly; however, a fraction are invasive and are more aggressive, with a very small fraction being frankly malignant. The cellular pathways that drive transformation in pituitary neoplasms are poorly characterized, and current classification methods are not reliable correlates of clinical behavior. Novel techniques in epigenetics, the study of alterations in gene expression without changes to the genetic code, provide a new dimension to characterize tumors, and may hold implications for prognostication and management.Methods: We conducted a review of primary epigenetic studies of pituitary tumors with a focus on histone modification, DNA methylation, and transcript modification.Results: High levels of methylation have been identified in invasive and large pituitary tumors. DNA methyltransferase overexpression has been detected in pituitary tumors, especially in macroadenomas. Methylation differences at CpG sites in promoter regions may distinguish several types of tumors from normal pituitary tissue. Histone modifications have been linked to increased p53 expression and longer progression-free survival in pituitary tumors; sirtuins are expressed at higher values in GH-expressing compared to nonfunctional adenomas and correlate inversely with size in somatotrophs. Upregulation in citrullinating enzymes may be an early pathogenic marker of prolactinomas. Numerous genes involved with cell growth and signaling show altered methylation status for pituitary tumors, including cell cycle regulators, components of signal transduction pathways, apoptotic regulators, and pituitary developmental signals.Conclusions: The limited clinical predictive capacity of the current pituitary tumor classification system suggests that tumor subclasses likely remain to be discovered. Ongoing epigenetic studies could provide a basis for adding methylation and/or acetylation screening to standard pituitary tumor workups. Identifying robust correlations between tumor epigenetics and corresponding histological, radiographic, and clinical course information could ultimately inform clinical decision-making

A tuberculosis nationwide prevalence survey in Gambia, 2012.

OBJECTIVE: To estimate the population prevalence of active pulmonary tuberculosis in Gambia. METHODS: Between December 2011 and January 2013, people aged ≥ 15 years participating in a nationwide, multistage cluster survey were screened for active pulmonary tuberculosis with chest radiography and for tuberculosis symptoms. For diagnostic confirmation, sputum samples were collected from those whose screening were positive and subjected to fluorescence microscopy and liquid tuberculosis cultures. Multiple imputation and inverse probability weighting were used to estimate tuberculosis prevalence. FINDINGS: Of 100 678 people enumerated, 55 832 were eligible to participate and 43 100 (77.2%) of those participated. A majority of participants (42 942; 99.6%) were successfully screened for symptoms and by chest X-ray. Only 5948 (13.8%) were eligible for sputum examination, yielding 43 bacteriologically confirmed, 28 definite smear-positive and six probable smear-positive tuberculosis cases. Chest X-ray identified more tuberculosis cases (58/69) than did symptoms alone (43/71). The estimated prevalence of smear-positive and bacteriologically confirmed pulmonary tuberculosis were 90 (95% confidence interval, CI: 53-127) and 212 (95% CI: 152-272) per 100 000 population, respectively. Tuberculosis prevalence was higher in males (333; 95% CI: 233-433) and in the 35-54 year age group (355; 95% CI: 219-490). CONCLUSION: The burden of tuberculosis remains high in Gambia but lower than earlier estimates of 490 per 100 000 population in 2010. Less than half of all cases would have been identified based on smear microscopy results alone. Successful control efforts will require interventions targeting men, increased access to radiography and more accurate, rapid diagnostic tests