High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.     

Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system

Summary A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency (42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots. Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the full-length genes in some of the transgenics.     

Localization of carboxypeptidase A-like enzyme in rat kidney

In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.     

Field observations on nitrogen catch crops

Nitrogen catch crops help to reduce the loss of nitrogen from arable cropping systems during autumn and winter. The ability of catch crops to absorb nitrogen from the soil profile is affected by rate and depth of colonization of the soil by roots. The aim of the current work was to analyze total root length and root length density of catch crops in relation to above ground growth, nitrogen supply and crop species. In two field experiments roots were sampled with an auger. Experimental factors included crop species (winter rye, Secale cereale and forage rape, Brassica napus ssp. oleifera (Metzg.) Sinsk., or oil radish, Raphanus sativus spp. oleiferus (DC.) Metzg.), two sowing dates S1 and S2 (end of August and three weeks later) and two nitrogen treatments: N0, no nitrogen applied, and N1, nitrogen applied at non-limiting rate.The natural logarithm of the total root length, measured in the top 40 cm, L_0–40 (km m^-2), was linearly related to natural logarithm of the dry weight of the shoot, W (g m^-2). There was no effect of species or sowing date on this relation. For a given W, N1 treatments showed lower values of L_0–40 than N0 treatments. The decline in root length density, D (cm cm^-3), with depth, X (cm), was described with the function ln D = ln D₀ – qX, where D₀ is the value of D at zero depth and q the linear coefficient. D₀ was linearly related to L_0–40, without effect of species, time of observation or N supply. The ratio D₀/q, an estimate of the absolute root length, was 1.24 × L_0–40.Together the relations enable estimates to be made of total root length and of root length distribution with depth using shoot dry weight of catch crops and its change with time as input. The generation of such estimates of root distribution is necessary for model studies in which the efficacy of catch crops to prevent N losses is evaluated in relation to sowing dates, distribution of N in the soil profile and the distribution of rainfall in the season.     

Functional constraints on nest characteristics of pebble mounds of breeding male hornyhead chub Nocomis biguttatus

Breeding male hornyhead chub Nocomis biguttatus constructed nests in areas with relatively high but less than maximum flow rate and greater than average water depth. Nests comprised c. 3000 pebbles for a total mass of 11 kg. Males selected pebbles of smaller diameter but higher density than pebbles in the immediate vicinity. Thus, nests balanced the risk of mound erosion and energetic cost of nest construction with the benefits of protection from egg predators and a stable internal flow rate for oxygenation. These data help establish environmental management goals for the conservation of N. biguttatus and the lotic ecosystems dependent upon them.     

On‐line yeast propagation process monitoring and control using an intelligent automatic control system

The monitoring and control of bioprocesses is a challenging task. This applies particularly if the actions to the process have to be carried out in real‐time. This work presents a system for on‐line monitoring and control of batch yeast propagation under limiting conditions based on a virtual plant operator, which uses the concept of intelligent control algorithms by means of fuzzy logic theory. Process information is provided on‐line using a sensor array comprising the measurement of OD, operating temperature, pressure, density, dissolved oxygen, and pH value. In this context practical problems arising through on‐line sensing and signal processing are addressed. The preprocessed sensor data are fed to a neural network for on‐line biomass estimation. The root mean squared error of prediction is 4 × 10⁶ cells/mL. The proposed system then triggers temperature and aeration by usage of a temperature dependent metabolic growth model and sensor data. The deviation of the predicted biomass from that of the reference trajectory as modeled by the metabolic growth model and its temporal derivative are used as inputs for the fuzzy temperature controller. The inputs used by the fuzzy aeration controller are the deviation of measured extract from that of the reference trajectory, the predicted cell count, and the dissolved oxygen concentration. The fuzzy‐based expert system allows to provide the desired yeast cell concentration of 100–120 × 10⁶ cells/mL at a minimum residual extract limit of 6.0 g/100 g at the required point of time. Thus, a dynamic adjustment of the propagation process to the overall production schedule is possible in order to produce the required amount of biomass at the right time.     

Lovastatin and (+)‐geodin production by Aspergillus terreus from crude glycerol

The use of pure substrate represents a significant proportion of the cost of manufacturing a drug such as lovastatin. This study explores the production of lovastatin and (+)‐geodin by Aspergillus terreus ATCC 20542 using biodiesel‐derived crude glycerol (CG) as a feedstock. Shake flask experiments showed reduced lovastatin production and glycerol consumption in the presence of 10–50 g/L CG with respect to pure glycerol controls. At 50 g/L, lovastatin and (+)‐geodin production was significantly reduced by 82 and 73%, respectively. The lowest lovastatin inhibition was detected in 30 g/L of CG (48%), which was accompanied by a significant rise in (+)‐geodin production (338%). Further investigation was performed on three major impurities found in CG, namely methanol (MeOH), sodium chloride (NaCl), and fatty acids (oleic acid and palmitic acid (PA), soap). None was particularly inhibitory for lovastatin, except soap and PAs, which reduced its production by more than 50% at all concentrations tested. In contrast, (+)‐geodin was inhibited in the presence of MeOH and PA by up to 46 and 91%, respectively. These observations indicate that partial purification of CG would be potentially useful in improving production of lovastatin and (+)‐geodin by A. terreus.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Modeling and Analysis of Unsteady Axisymmetric Squeezing Fluid Flow through Porous Medium Channel with Slip Boundary

The aim of this article is to model and analyze an unsteady axisymmetric flow of non-conducting, Newtonian fluid squeezed between two circular plates passing through porous medium channel with slip boundary condition. A single fourth order nonlinear ordinary differential equation is obtained using similarity transformation. The resulting boundary value problem is solved using Homotopy Perturbation Method (HPM) and fourth order Explicit Runge Kutta Method (RK4). Convergence of HPM solution is verified by obtaining various order approximate solutions along with absolute residuals. Validity of HPM solution is confirmed by comparing analytical and numerical solutions. Furthermore, the effects of various dimensionless parameters on the longitudinal and normal velocity profiles are studied graphically.