Impact of antibody aggregation on a flowthrough anion‐exchange membrane process

The impact of typical anion‐exchange flowthrough conditions on the IgG mass loading of an anion‐exchange membrane scale‐down unit (Mustang® Q coin) was investigated. High performance size‐exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion‐exchange flowthrough conditions. The small filtration area presented by the 0.35 mL membrane volume Mustang® Q coin limited the membrane throughput due to fouling from the aggregates at higher antibody loading. Data in this report indicated that a 0.2 μm hybrid polyethersulfone and polyvinylidene fluoride membrane in‐line prefilter with a minimum filtration area of 20 sq cm alleviated the Mustang® Q coin fouling. The combined cake filtration and intermediate blocking model was proposed as the most likely membrane pore blocking mechanism. Increasing the filtration area in the in‐line prefilter resulted in higher IgG mass throughput. Thus, using an appropriately sized in‐line prefilter could provide more robust antibody throughput performance on scale‐down membrane anion‐exchange units. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

Ovalbumin-related Protein X Is a Heparin-binding Ov-Serpin Exhibiting Antimicrobial Activities

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys³⁶⁷-His³⁶⁸ is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.     

Human Herpesvirus 6A Infection and Immunopathogenesis in Humanized Rag2?/?γc?/? Mice

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2^−/−γc^−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.     

Use of Agent-Based Modeling To Explore the Mechanisms of Intracellular Phosphorus Heterogeneity in Cultured Phytoplankton

There can be significant intraspecific individual-level heterogeneity in the intracellular P of phytoplankton, which can affect the population-level growth rate. Several mechanisms can create this heterogeneity, including phenotypic variability in various physiological functions (e.g., nutrient uptake rate). Here, we use modeling to explore the contribution of various mechanisms to the heterogeneity in phytoplankton grown in a laboratory culture. An agent-based model simulates individual cells and their intracellular P. Heterogeneity is introduced by randomizing parameters (e.g., maximum uptake rate) of daughter cells at division. The model was calibrated to observations of the P quota of individual cells of the centric diatom Thalassiosira pseudonana, which were obtained using synchrotron X-ray fluorescence (SXRF). A number of simulations, with individual mechanisms of heterogeneity turned off, then were performed. Comparison of the coefficient of variation (CV) of these and the baseline simulation (i.e., all mechanisms turned on) provides an estimate of the relative contribution of these mechanisms. The results show that the mechanism with the largest contribution to variability is the parameter characterizing the maximum intracellular P, which, when removed, results in a CV of 0.21 compared to a CV of 0.37 with all mechanisms turned on. This suggests that nutrient/element storage capabilities/mechanisms are important determinants of intrapopulation heterogeneity.     

The velvet complex governs mycotoxin production and virulence of Fusarium oxysporum on plant and mammalian hosts

Fungal pathogens provoke devastating losses in agricultural production, contaminate food with mycotoxins and give rise to life‐threatening infections in humans. The soil‐borne ascomycete Fusarium oxysporum attacks over 100 different crops and can cause systemic fusariosis in immunocompromised individuals. Here we functionally characterized VeA, VelB, VelC and LaeA, four components of the velvet protein complex which regulates fungal development and secondary metabolism. Deletion of veA, velB and to a minor extent velC caused a derepression of conidiation as well as alterations in the shape and size of microconidia. VeA and LaeA were required for full virulence of F. oxysporum on tomato plants and on immunodepressed mice. A critical contribution of velvet consists in promoting chromatin accessibility and expression of the biosynthetic gene cluster for beauvericin, a depsipeptide mycotoxin that functions as a virulence determinant. These results reveal a conserved role of the velvet complex during fungal infection on plants and mammals.