Comparison of expression optimization of new derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK

Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut⁺ strain and KM71H as a Mut^s strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure.     

Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.     

In vivo lung morphometry with hyperpolarized 3He diffusion MRI in canines with induced emphysema: disease progression and comparison with computed tomography.

Despite a long history of development, diagnostic tools for in vivo regional assessment of lungs in patients with pulmonary emphysema are not yet readily available. Recently, a new imaging technique, in vivo lung morphometry, was introduced by our group. This technique is based on MRI measurements of diffusion of hyperpolarized (3)He gas in lung air spaces and provides quantitative in vivo tomographic information on lung microstructure at the level of the acinar airways. Compared with standard diffusivity measurements that strongly depend on pulse sequence parameters (mainly diffusion time), our approach evaluates a "hard number," the average acinar airway radius. For healthy dogs, we find here a mean acinar airway radius of approximately 0.3 mm compared with 0.36 mm in healthy humans. The purpose of the present study is the application of this technique for quantification of emphysema progression in dogs with experimentally induced disease. The diffusivity measurements and resulting acinar airway geometrical characteristics were correlated with the local lung density and local lung-specific air volume calculated from quantitative computed tomography data obtained on the same dogs. The results establish an important association between the two modalities. The observed sensitivity of our method to emphysema progression suggests that this technique has potential for the diagnosis of emphysema and tracking of disease progression or improvement via a pharmaceutical intervention.     

Applying neural networks to predict HPC-I/O bandwidth over seismic data on lustre file system for ExSeisDat

Cluster Computing - HPC or super-computing clusters are designed for executing computationally intensive operations that typically involve large scale I/O operations. This most commonly involves...     

Epigenetics in osteoarthritis: Novel spotlight

Osteoarthritis (OA) is the most common type of arthritis and no longer is considered as an absolute consequence of joint mechanical use (wear and tear); rather recent data demonstrate the pivotal role of inflammatory mediators in the development and progression of this disease. This multifactorial disease results from several environmental and inherited factors. Genetic cannot solely explain all the contribution share of inheritance and, this way, it is speculated that epigenetics can play a role, too. Moreover, environmental factors can induce local epigenetic changes. The epigenetic contribution to OA pathogenesis occurs at all of its levels, DNA methylation, histone modification, microRNA, and long noncoding RNA. In fact, during early phases of OA pathogenesis, environmental factors employ epigenetic mechanisms to provide a positive feedback for the OA-related pathogenic mechanisms and pathways with an ultimate outcome of a well-established clinical OA. These epigenetic changes stay during clinical disease and prevent the body natural healing and regenerative processes to work properly, resulting in an incurable disease condition. In this review article, we aimed to have an overview on the studies performed with regard to understanding the role of epigenetics in the etiopathogenesis of OA and highlighted the importance of such kind of regulatory mechanisms within this context.     

Magnetoelectric nanocomposite scaffold for high yield differentiation of mesenchymal stem cells to neural-like cells

While the differentiation factors have been widely used to differentiate mesenchymal stem cells (MSCs) into various cell types, they can cause harm at the same time. Therefore, it is beneficial to propose methods to differentiate MSCs without factors. Herein, magnetoelectric (ME) nanofibers were synthesized as the scaffold for the growth of MSCs and their differentiation into neural cells without factors. This nanocomposite takes the advantage of the synergies of the magnetostrictive filler, CoFe₂O ₄ nanoparticles (CFO), and piezoelectric polymer, polyvinylidene difluoride (PVDF). Graphene oxide nanosheets were decorated with CFO nanoparticles for a proper dispersion in the polymer through a hydrothermal process. After that, the piezoelectric PVDF polymer, which contained the magnetic nanoparticles, underwent the electrospun process to form ME nanofibers, the ME property of which has the potential to be used in areas such as tissue engineering, biosensors, and actuators.     

Wnt lipidation: Roles in trafficking,modulation, and function

The Wnt signaling pathway consists of various downstream target proteins that have substantial roles in mammalian cell proliferation, differentiation, and development. Its aberrant activity can lead to uncontrolled proliferation and tumorigenesis. The posttranslational connection of fatty acyl chains to Wnt proteins provides the unique capacity for regulation of Wnt activity. In spite of the past belief that Wnt molecules are subject to dual acylation, it has been shown that these proteins have only one acylation site and undergo monounsaturated fatty acylation. The Wnt monounsaturated fatty acyl chain is more than just a hydrophobic coating and appears to be critical for Wnt signaling, transport, and receptor activation. Here, we provide an overview of recent findings in Wnt monounsaturated fatty acylation and the mechanism by which this lipid moiety regulates Wnt activity from the site of production to its receptor interactions.