Anti-Heparanase Aptamers as Potential Diagnostic and Therapeutic Agents for Oral Cancer

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.     

Process technology for production and recovery of heterologous proteins with Pichia pastoris

Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.     

Xyloglucan and xyloglucan endo-transglycosylases (XET): Tools for ex vivo cellulose surface modification

Wood fibres constitute a renewable raw material for the production of novel biomaterials. The development of efficient methods for cellulose surface modification is essential for expanding the properties of wood fibres for increased reactivity and compatibility with other materials. By combining the high affinity between xyloglucan and cellulose, the unique mechanistic property of xyloglucan endo-transglycosylases (XET, EC 2.4.1.207) to catalyze polysaccharide-oligosaccharide coupling reactions, and traditional carbohydrate synthesis, a new system for the attachment of a wide variety of functional groups to wood pulps has been generated. An overview of recent developments is presented in the context of the structure, physical properties, and historical applications of xyloglucan.     

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.     

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.     

Pattern of xylem phenology in conifers of cold ecosystems at the Northern Hemisphere

The interaction between xylem phenology and climate assesses forest growth and productivity and carbon storage across biomes under changing environmental conditions. We tested the hypothesis that patterns of wood formation are maintained unaltered despite the temperature changes across cold ecosystems. Wood microcores were collected weekly or biweekly throughout the growing season for periods varying between 1 and 13 years during 1998–2014 and cut in transverse sections for assessing the onset and ending of the phases of xylem differentiation. The data set represented 1321 trees belonging to 10 conifer species from 39 sites in the Northern Hemisphere and covering an interval of mean annual temperature exceeding 14 K. The phenological events and mean annual temperature of the sites were related linearly, with spring and autumnal events being separated by constant intervals across the range of temperature analysed. At increasing temperature, first enlarging, wall‐thickening and mature tracheids appeared earlier, and last enlarging and wall‐thickening tracheids occurred later. Overall, the period of wood formation lengthened linearly with the mean annual temperature, from 83.7 days at ?2 °C to 178.1 days at 12 °C, at a rate of 6.5 days °C^?1. April–May temperatures produced the best models predicting the dates of wood formation. Our findings demonstrated the uniformity of the process of wood formation and the importance of the environmental conditions occurring at the time of growth resumption. Under warming scenarios, the period of wood formation might lengthen synchronously in the cold biomes of the Northern Hemisphere.     

Salmonella in animal slurry can be destroyed by aeration at low temperatures

Cattle and other animals infected by Salmonella can emit high numbers of these bacteria. To determine an effective means for reducing this bacterial group in animal slurry, samples were subjected to aeration in laboratory experiments and in farm-scale slurry tanks. A clear reduction in Salmonella levels was found in laboratory experiments at temperatures from 4 to 40 °C. Aeration in farm-scale slurry tanks increased the temperature above the ambient temperatures (often less than 0 °C) to maxima ranging between 19 and 40 °C. Farm-scale aeration resulted in similar reductions in Salmonella as those achieved in laboratory experiments. Thus, reductions, ranging from greater than 99% of the initial number to no detectable Salmonella , could be reached after 2–5 weeks using aeration processes with cattle slurries contaminated by Salm. infantis or pig slurry contaminated by Salm. typhimurium . These results suggest that farmers can control the spread of Salmonella from slurry to agricultural fields. The reduction mechanisms remain unknown, though the increase in pH (to 7·6–9·0) found in slurries after aeration might exert a decreasing effect on these bacteria.     

Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

Summary A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect as little as 2–5 ng enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labeled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin.