Selection of catalytically active biotin ligase and trypsin mutants by phage display

Phage display has been shown to facilitate greatly the selectionof polypeptides with desired properties by establishing a directlink between the polypeptide and the gene that encodes it. However,selection for catalytic activities displayed on phage remainsa challenge, since reaction products diffuse away from the enzymeand make it difficult to recover catalytically active phage–enzymes.We have recently described a selection methodology in whichthe reaction substrate (and eventually the reaction product)is anchored on calmodulin-tagged phage–enzymes by meansof a calmodulin binding peptide. Phage displaying a catalyticactivity are physically isolated by means of affinity reagentsspecific for the product of reaction. In this study, we investigatedthe efficiency of selection for catalysis by phage display,using a ligase (the Escherichia coli biotin ligase BirA) andan endopeptidase (the rat trypsin His57     

Candida albicans sensitization in patients with atopic bronchial asthma and atopic dermatitis

Candida albicans, a component of normal human microflora, can induce synthesis of specific IgE-antibodies in patients with atopic bronchial asthma and atopic dermatitis. The study included 25 patients with atopic dermatitis sensitized to C.albicans and 23 patients with atopic dermatitis non-sensitized to C.albicans. The sensitization was determined by the skin test and enzyme immunoassay. The patients had the history of atopic dermatitis exacerbation after taking food containing baking yeasts. Atopic dermatitis with sensitization to C.albicans is characterized by severe course correlating with the following indices: high total IgE (r = 0.6), level of IgE antibodies to C.albicans (r = 0.6), level of serum IgG (r = 0.46) and IgA (r = 0.33). Contrary to adults, children with sensitization to C.albicans had decreased relative number of CD4+, CD8+ and CD72+ of lymphocyte subpopulations. Thus, sensitization to C.albicans manifests in severe atopic dermatitis which in children is often associated with immune deficiency.     

Optimal Portfolio Hedging with Nonlinear Derivatives and Transaction Costs

We consider the problem of dynamically hedging a fixed portfolio of assets in the presence of non-linear instruments and transaction costs, as well as constraints on feasible hedging positions. We assume an investor maximizing the expected utility of his terminal wealth over a finite holding period, and analyse the dynamic portfolio optimization problem when the trading interval is fixed. An approximate solution is obtained from a two-stage numerical procedure. The problem is first transformed into a nonlinear programming problem which utilizes simulated coefficient matrices. The nonlinear programming problem is then solved numerically using standard constrained optimization techniques.     

Transient Receptor Potential Ankyrin 1 (TRPA1)—An Inflammation-Induced Factor in Human HaCaT Keratinocytes

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel mainly studied in sensory neurons where it mediates itch, pain and neurogenic inflammation. Recently, some nonneuronal cells have also been shown to express TRPA1 to support inflammatory responses. To address the role of TRPA1 in skin inflammation, we aimed to investigate TRPA1 expression in keratinocytes. HaCaT cells (a model of human keratinocytes) and skin biopses from wild-type and TRPA1 deficient mice were used in the studies. TRPA1 expression in nonstimulated keratinocytes was very low but significantly inducible by the proinflammatory cytokine tumor necrosis factor (TNF) in an nuclear factor kappa B (NF-κB), and mitogen-activated protein (MAP) kinase (p38 and c-Jun N-terminal kinase, JNK)-dependent manner. Interestingly, drugs widely used to treat skin inflammation, the calcineurin inhibitors tacrolimus and cyclosporine and the glucocorticoid dexamethasone, significantly decreased TRPA1 expression. Furthermore, pharmacological inhibition and genetic deletion of TRPA1 reduced the synthesis of TNF-induced monocyte chemoattractant protein 1 (MCP-1) in keratinocytes and mouse skin biopsies. In conclusion, these findings point to an inflammatory role for TRPA1 in keratinocytes and present TRPA1 as a potential drug target in inflammatory skin diseases.     

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive alpha-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.     

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating (3)H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating (3)H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.     

Integration of microfluidic sample delivery system on silicon nanowire-based biosensor

Silicon nanowire-based (SiNW) biosensors have gained a lot of attention during recent years. However, studies often totally neglect, or only briefly describe, the incorporation of microfluidic channel into the sensor architecture, although it is a crucial step towards a real lab-on-chip device. This paper proposes a process that can be applied to integration of microfluidic sample delivery system onto different SiNW biosensors. The sample delivery system includes a hydrophilic channel that enables the use of capillary action in delivering sample directly onto the sensor array, which leads to reduced sample loss, faster detection process, and frees from the use of external pumps. In addition, the microfluidic channel system protects the fragile SiNWs from mechanical shocks, chemical spatters, and dust. The sample delivery system was fabricated of surface treated polydimethylsiloxane (PDMS), using a four-step approach, as follows: (1) master molds for soft lithography were etched onto Si. (2) PDMS replicas of the molds were fabricated and (3) bonded onto example sensor chips using oxygen plasma. (4) Oxygen plasma treatment also enabled the attachment of polyvinylpyrrolidone (PVP) to the sample channel surfaces to synthesize hydrophilic polymer coating. A contact angle for the PVP treated PDMS was 21 after 17 days, indicating the formation of a long-term hydrophilic PDMS surface. Finally, the example SiNW sensor is modified to allow direct real-time detection of thyroid-stimulating hormone (TSH). The sensor was able to detect as low TSH concentration values as 0.5 mIU/l, which indicates a successfully integrated sample delivery system.