Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.     

A model ensemble approach to determine the humus building efficiency of organic amendments in incubation experiments

Organic amendments are important to sustain soil organic matter (SOM) and soil functions in agricultural soils. Information about the contribution of organic amendments to SOM can be derived from incubation experiments. In this study, data from 72 incubated organic amendments including plant residues, digestates and manure were analysed. The incubation data was compiled from three experimental setups with varying incubation times, soils and incubation temperatures, in which CO₂ release was measured continuously. The analysis of the incubation data was performed with an approach relying on conceptual parts of C-TOOL, CCB, Century, ICBM, RothC and Yasso which are all well-approved first-order carbon models that differ in structure and abstraction level. All models are an approximation of reality, whereby each model differs in understanding of the processes involved in soil carbon dynamics. To accumulate the advantages from each model a model ensemble was performed for each substrate. With the ability of each carbon model to compute the distribution of carbon into specific SOM pools a new approach for evaluating organic amendments in terms of humus building efficiency is presented that, depends on the weighted model fit of each ensemble member. Depending on the organic substrate added to the soil, the time course of CO₂ release in the incubation studies was predicted with different accuracy by the individual model concepts. Averaging the output of the individual models leads to more robust prediction of SOM dynamics. The E_HUM value is easy to interpret and the results are in accordance with the literature.     

Prevalence of Campylobacter jejuni and Salmonella during pig slaughtering

It was found that 79% of healthy pigs, slaughtered in three different slaughterhouses in the Netherlands, were intestinal carriers of Campylobacter jejuni (mean number 4000 cfu per g), and 21% of the same pigs had Salmonella in the intestinal tract (mean number 10 cfu per g). Immediately after slaughter, Campylobacter was swabbed from 9% of the carcasses and Salmonella from 13%. It is concluded from these data that most of the contamination on carcasses does not originate directly from the intestinal tracts of the animals but rather from surfaces, equipment, and utensils in the slaughter hall. It was demonstrated that Salmonella could survive in the slaughter hall, whereas Campylobacter died off, probably due to its vulnerability to drying conditions and its inability to grow at temperatures below 30 degrees C. Campylobacter was not isolated from the carcasses after cooling. It had been shown earlier that this again was caused by dry conditions, brought about by the use of forced ventilation in the cooling rooms. In an additional investigation, Campylobacter was not isolated from 248 samples of minced pork (10 g each), whereas Salmonella was found in 13% of these samples.     

Influence of oil type on the amounts of acrylamide generated in a model system and in French fries

Acrylamide formation was studied by use of a new heating methodology, based on a closed stainless steel tubular reactor. Different artificial potato powder mixtures were homogenized and subsequently heated in the reactor. This procedure was first tested for its repeatability. By use of this experimental setup, it was possible to study the acrylamide formation mechanism in the different mixtures, eliminating some variable physical and chemical factors during the frying process, such as heat flux and water evaporation from and oil ingress into the food. As a first application of this optimized heating concept, the influence on acrylamide formation of the type of deep-frying oil was investigated. The results obtained from the experiments with the tubular reactor were compared with standardized French fry preparation tests. In both cases, no significant difference in acrylamide formation could be found between the various heating oils applied. Consequently, the origin of the deep-frying vegetable oils did not seem to affect the acrylamide formation in potatoes during frying. Surprisingly however, when artificial mixtures did not contain vegetable oil, significantly lower concentrations of acrylamide were detected, compared to oil-containing mixtures.     

Competition for electron donors among nitrate reducers,ferric iron reducers,sulfate reducers,and methanogens in anoxic paddy soil

Slurries of anoxic paddy soil were either freshly prepared or were partially depleted in endogenous electron donors by prolonged incubation under anaerobic conditions. Endogenous NO ₃ ^– was reduced within 4 h, followed by reduction of Fe³⁺ and SO ₄ ^2– , and later by production of CH₄. Addition of NO ₃ ^– slightly inhibited the production of Fe²⁺ in the depleted but not in the fresh paddy soil. Inhibition was overcome by the addition of H₂, acetate, or a mixture of fatty acids (and other compounds), indicating that these compounds served as electron donors for the bacteria reducing NO ₃ ^– and/or ferric iron. Addition on NO ₃ ^– also inhibited the reduction of SO ₄ ^2– in the depleted paddy soil. This inhibition was only overcome by H₂, but not by acetate or a mixture of compounds, indicating that H₂ was the predominant electron donor for the bacteria involved in NO ₃ ^– and/or SO ₄ ^2– reduction. SO ₄ ^2– reduction was also inhibited by exogenous Fe³⁺, but only in the depleted paddy soil. This inhibition was overcome by either H₂, acetate, or a mixture of compounds, suggesting that they served as electron donors for reduction of Fe³⁺ and/or SO ₄ ²⁺ . CH₄ production was inhibited by NO ₃ ^– both in depleted and in fresh paddy soil. Fe³⁺ and SO ₄ ^2– also inhibited methanogenesis, but the inhibition was stronger in the depleted than in the fresh paddy soil. Inhibition of CH₄ production was paralleled by a decrease in the steady state concentration of H₂ to a level which provided a free enthalpy of less than G=–17 kJ mol^-1 CH₄ compared to more than G=–32 kJ mol^-1 CH₄ in the control. The results indicate that in the presence of exogenous fe³⁺ or SO ₄ ²⁺ , methanogenic bacteria were outcompeted for H₂ by bacteria reducing Fe³⁺ or SO ₄ ²⁺ .Deceased on 27 December 1992     

Chemical conversion of alpha-keto acids in relation to flavor formation in fermented foods

Formation of flavor compounds from branched-chain alpha-keto acids in fermented foods such as cheese is believed to be mainly an enzymatic process, while the conversion of phenyl pyruvic acid, which is derived from phenylalanine, also proceeds chemically. In this research, the chemical conversion of alpha-keto acids to aldehydes with strong flavor characteristics was studied, with the main focus on the conversion of alpha-ketoisocaproic acid to the aldehyde 2-methylpropanal, and a manganese-catalyzed reaction mechanism is proposed for this conversion. The mechanism involves keto-enol tautomerism, enabling molecular oxygen to react with the beta-carbon atom of the alpha-keto acid, resulting in a peroxide. This peroxide can react in several ways, leading to unstable dioxylactone or noncyclic intermediates. These intermediates will break down into an aldehyde and oxalate or carbon oxides (CO and CO(2)). All the alpha-keto acids tested were converted at pH 5.5 and in the presence of manganese, although their conversion rates were rather diverse. This chemical reaction might provide new ways for controlling cheese flavor formation with the aim of acceleration of the ripening process or diversification of the flavor characteristics.