Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency

<p>Abstract</p> <p>Background</p> <p>Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for <it>in vitro </it>studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for <it>in vitro </it>approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for <it>in vitro </it>investigation of oxygen deficiency.</p> <p>Methods</p> <p>Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. ¹⁴C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system.</p> <p>Results</p> <p>The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. ¹⁴C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation.</p> <p>Conclusion</p> <p>The results of our study indicate that the enzymatic model can find application for <it>in vitro </it>investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.</p

Treatment of proximal humerus fractures. Relative position of different locking plates to the axillary nerve

Objectives. Placement of a proximal humerus locking plate through a percutaneous transdeltoid approach bears the advantages of a minimally invasive approach but may compromise the anterior branches of the axillary nerve. This anatomic study aimed to develop a risk profile for 6 types of modern proximal humerus locking plates as to their interference with the axillary nerve. Materials and methods. In this study six different implants (Arthrex (R), DePuy (R), Knigsee (R), Smith & Nephew (R), Stryker (R) and Synthes (R)) were placed on the intact proximal humerus of 33 embalmed cadaveric upper extremities and the relative positioning between the axillary nerve and the screw holes was determined. Results. All locking plates displayed an area of risk which concerned 3 out of 7 (Arthrex (R)), 4 out of 10 (DePuy (R)), 2 out of 9 (Konigsee (R)), 3 out of 11 (Smith & Nephew (R)), 3 out of 11 (Stryker (R)) and 6 out of 12 (Synthes (R)) screw holes of the plate. Conclusions. Using the anterolateral percutaneous deltoid splitting approach the relative position of the axillary nerve to the holes of a specific implant is of relevance for avoidance of iatrogenic lesions to the nerve

Neurovascular coupling in the human visual cortex studied by diffusing-Wave spectroscopy (dws) and electroencephalography

Biomedical Optics, BIOMED 2008BMD21

Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana.

Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α-Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose-dependent. Activity-based protein profiling (ABPP) revealed that the activities of papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming

Activity-based protein profiling for the identification of novel carbohydrate-active enzymes involved in xylan degradation in the hyperthermophilic euryarchaeon thermococcus sp. Strain 2319x1E

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6-7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.</p

The Role of Social Cognitive Theory in Understanding Firm Innovation: A Literature Review

Past studies have shown the intimacy between social cognitive theory, knowledge management and organizational learning. The latter two are also frequently inquired in the context of firm innovation while the former one is the fundamental theoretical underpinning that provides explanation to many issues associated with the latter two. This paper reviews 19 selected articles that encompass the above three areas relating to firm innovation in an integrating manner, and appreciates the commonalities and differences between different approaches with respect to different units of analysis. The review constitutes of four building blocks: brain physiology, creativity, knowledge and learning, and towards a formal theory. Suggestions direct to future research of the social cognitive theory of firm innovation are made