Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.     

Genetic polymorphism of vitamin D receptor gene affects the phenotype of PCOS

Aims

Polycystic ovary syndrome (PCOS), a common female endocrine disorder, represents a wide range of clinical manifestations and disease severity. Recent studies suggest an association between gene variants involved in vitamin D metabolism and common metabolic disturbances in PCOS. We aimed to examine the association of vitamin D receptor (VDR) gene variant with PCOS susceptibility and the severity of disease phenotype.

Methods

All participants, including 260 PCOS women (cases) and 221 normoovulatory women (controls), were recruited from a reproductive endocrinology clinic. Cases were divided into the severe and mild PCOS phenotype groups, based on their clinical and paraclinical features. An adenosine to guanine single nucleotide polymorphism of VDR gene (rs757343) was genotyped using the PCR–RFLP method.

Results

Distributions of genotypes and alleles did not differ between cases and controls, indicating that this SNP is not associated with increased risk for PCOS. However, this SNP was found to be associated with the severity of the PCOS phenotype. In particular, presence of the A allele is associated with a 74% increased risk of severe phenotype development (OR, 1.74; 95% CI, 1.07–2.82).

Conclusion

The genetic variant of the VDR was found to have an association with severity of clinical features of PCOS, but none with disease risk.     

HIV‐1 ENV gp120 structural determinants for peptide triazole dual receptor site antagonism

Despite advances in HIV therapy, viral resistance and side‐effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV‐1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co‐receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide's affinity and its ability to inhibit binding of CD4 and the co‐receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single‐round cell infection assays. Two mutations, D474A and T257A, caused large‐scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co‐receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Efficient removal of detergents from proteins and peptides in a spin column format

Detergents are commonly used in protein–chemistry protocols and may be necessary for protein extraction, solubilization, and denaturation; however, their presence interferes with many downstream analysis techniques, including mass spectrometry (MS). To enable downstream analysis, it is critical to remove unbound detergents from protein and peptide samples. In this study, we describe a high-performance resin that offers exceptional detergent removal for proteins and peptides. When used in a spin column format, this resin dramatically improves protein and peptide MS results by more than 95% removal of 1–5% detergents, including sodium dodecyl sulfate (SDS), sodium deoxycholate, Chaps, Triton X-100, Triton X-114, NP-40, Brij-35, octyl glucoside, octyl thioglucoside, and lauryl maltoside, with high recovery of proteins and peptides. Postcolumn liquid chromatography–tandem MS (LC–MS/MS) analysis of trypsin digests of bovine serum albumin (BSA) and HeLa cell lysate revealed excellent sequence coverage, indicating successful removal of detergent from the peptides. Matrix-assisted laser desorption/ionization (MALDI)–MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (<15 min), efficient detergent removal, and high recovery of proteins and peptides.     

Coccolithophorid algae culture in closed photobioreactors

The feasibility of growth, calcium carbonate and lipid production of the coccolithophorid algae (Prymnesiophyceae), Pleurochrysis carterae, Emiliania huxleyi, and Gephyrocapsa oceanica, was investigated in plate, carboy, airlift, and tubular photobioreactors. The plate photobioreactor was the most promising closed cultivation system. All species could be grown in the carboy photobioreactor. However, P. carterae was the only species which grew in an airlift photobioreactor. Despite several attempts to grow these coccolithophorid species in the tubular photobioreactor (Biocoil), including modification of the airlift and sparger design, no net growth could be achieved. The shear produced by turbulence and bubble effects are the most likely reasons for this failure to grow in the Biocoil. The highest total dry weight, lipid and calcium carbonate productivities achieved by P. carterae in the plate photobioreactors were 0.54, 0.12, and 0.06 g L⁻¹ day⁻¹ respectively. Irrespective of the type of photobioreactor, the productivities were P. carterae > E. huxleyi > G. oceanica. Pleurochrysis carterae lipid (20–25% of dry weight) and calcium carbonate (11–12% of dry weight) contents were also the highest of all species tested. Biotechnol. Bioeng. 2011;108:2078–2087. © 2011 Wiley Periodicals, Inc.     

In Silico Designing a Candidate Vaccine Against Breast Cancer

International Journal of Peptide Research and Therapeutics - Breast cancer (BC) is the most common type of women’s cancer with a prevalence of about 25%, although it is rare in men...     

Hybrid of niosomes and bio-synthesized selenium nanoparticles as a novel approach in drug delivery for cancer treatment

Molecular Biology Reports - The current&nbsp;study&nbsp;intends to investigate a novel drug delivery system (DDS) based on niosomes structure (NISM) and bovine serum albumin (BSA) which was...     

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation

Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 degrees C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications.