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Selected AbstractsIn vitro degradation of articular cartilage: does trypsin treatment produce consistent results?JOURNAL OF ANATOMY, Issue 2 2006H. R. Moody Abstract It is common practice in laboratories to create models of degraded articular cartilage in vitro and use these to study the effects of degeneration on cartilage responses to external stimuli such as mechanical loading. However, there are inconsistencies in the reported action of trypsin, and there is no guide on the concentration of trypsin or the time to which a given sample can be treated so that a specific level of proteoglycan depletion is achieved. This paper argues that before any level of confidence can be established in comparative analysis it is necessary to first obtain samples with similar properties. Consequently, we examine the consistency of the outcome of the artificial modification of cartilage relative to the effects of the common enzyme, trypsin, used in the process of in vitro proteoglycan depletion. The results demonstrate that for a given time and enzyme concentration, the action of trypsin on proteoglycans is highly variable and is dependent on the initial distribution and concentration of proteoglycans at different depths, the intrinsic sample depth, the location in the joint space and the medium type, thereby sounding a note of caution to researchers attempting to model a proteoglycan-based degeneration of articular cartilage in their experimental studies. [source] Growth of pure cultures of Verocytotoxin-producing Escherichia coli in a range of enrichment mediaJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008C.L. Baylis Abstract Aims:, This study compared the growth of different strains of Verocytotoxin-producing Escherichia coli (VTEC) in a range of selective enrichment media. Methods and Results:, Turbidometric and impedance methods were used to determine the growth of VTEC in pure culture in different enrichment media. Ten strains failed to grow in buffered peptone water + vancomycin, cefsulodin, cefixime at 42°C and some failed to grow, or grew poorly in E. coli (EC) medium supplemented with 20 mg l,1 novobiocin and modified EC supplemented with 20 mg l,1 novobiocin at 37°C and 42°C. Individual VTEC strains were sensitive to the selective agents in some media. Statistical analysis of the conductance detection times of 10 strains showed no overall effect of temperature alone (P = 0·66) but there were significant (P < 0·001) effects as a result of the combination of medium and temperature and these two factors were influenced by strain. Conclusions:, Growth of VTEC during enrichment is dependent on different factors alone or in combination. These include medium type, presence of certain selective agents or antibiotics, incubation temperature and the initial population of VTEC. Sensitivity to these conditions can be strain related. Significance and Impact of the Study:, This study highlighted differences in the ability of some enrichment media to support the growth of VTEC, making them unsuitable for the isolation of VTEC, especially low numbers of non-O157 strains. [source] Framework for the Rapid Optimization of Soluble Protein Expression in Escherichia coli Combining Microscale Experiments and Statistical Experimental DesignBIOTECHNOLOGY PROGRESS, Issue 4 2007R. S. Islam A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g·L,1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost. [source] Leaf fungi of two wild plants in Assiut, EgyptFEDDES REPERTORIUM, Issue 7-8 2004S. K. Hemida Leaves of two wild species of the flora of Egypt: Calotropis procera (Ait.) Ait., Asclepiadaceae and Chrozophora plicata (Vahl) A.Juss. ex Spreng., Euphorbiaceae have been studied morphologically and mycologically, in addtion to air borne fungi. Fifty five species and two varieties belonging to 26 genera of phyllosphere and phylloplane fungi were isolated from both plant species on glucose- and cellulose-agar media. Mycological analysis was done monthly over six months (July to December, 2003). Alternariaalternata, Aspergillus fumigatus, A. flavus and A. niger were the basic fungal species found on leaf surfaces. Phylloplane of C. plicata caught specifically Chaetomiumglobosum, C.,spirale, Cochliobolus lunatus, Drechslera halodes, Fusarium incarnatum, F. oxysporum, Memnoniella echinata and Papulaspora sepedonioides. The total counts of phyllosphere fungi of C.,plicata were nearly twice as much as those of C.,procera regardless medium's type. Forty species and one variety belonging to 22 genera of air borne fungi were recovered all over the experimental period (six months). Alternaria, Aspergillus, Cladosporium and Penicillium were the most frequently isolated species. The presented results revealed that, leaf shape and density (hair density and type) may be the most important factors of the biodiversity of the fungal species on the studied taxa. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Die Blätter zweier Wildarten aus der Flora Ägyptens: Calotropis procera (Ait.) Ait., Asclepiadeceae, und Chrozophora plicata(Vahl) A.Juss. ex Spreng., Euphorbiaceae, wurden in Bezug auf ihre Morphologie und Mykologie untersucht sowie zusätzlich auf ihre "luftgeborenen" Pilze. Auf beiden Arten wurden auf einem Glukose- bzw. Cellulose-Medium insgesamt 55 Arten und zwei Varietäten aus 26 Gattungen phyllosphärer und phylloplaner Pilze nachgewiesen. Die mykologischen Analysen wurden über einen Zeitraum von sechs Monaten (Juli bis Dezember 2003) durchgeführt. Alternariaalternata, Aspergillus fumigatus, A. flavus und A. niger waren die Basis-Pilzarten, die auf den Blattoberflächen ermittelt wurden. Auf C. plicata waren Chaetomiumglobosum, C. spirale, Cochliobolus lunatus, Drechslera halodes, Fusarium incarnatum, F. oxysporum, Memnoniella echinata und Papulaspora sepedonioides die häufigsten phylloplanen Arten. Ungeachtet des Mediumtyps war die Anzahl phyllosphärischer Pilze auf C. plicata etwa zweimal so hoch wie auf C. procera. Über die gesamte Versuchszeit von sechs Monaten wurden 40 Arten und eine Varietät aus 22 Gattungen "luftgeborener" Pilze beobachtet. Mit Alternaria, Aspergillus, Cladosporium und Penicillium fanden sich die am häufigsten isolierten Arten. Aus den erzielten Ergebnissen kann man ableiten, dass Blattform und Haare (Dichte und Typ) die wichtigsten Faktoren für die Biodiversität der untersuchten Pilzarten sind. [source] | ||||||||||