Identification of Genetically Modified Zebrafish (Danio rerio) by Protein- and DNA-Analysis

The import, sale and possession of fluorescent transgenic zebrafish, offered under the name “GloFish^?” in U.S. aquarium shops, are not permitted in the European Union. A PCR-based method has been developed to detect transgenic zebrafish harbouring the gene (dsRed) coding for the red fluorescent protein, originally isolated from the marine sponge Discosoma striata. Two types of PCR have been performed: (i) PCR to detect amplifiable genomic zebrafish DNA was checked using primers specific for the zebrafish parvalbumin gene; (ii) PCR with primers to specifically amplify the dsRed gene. In both PCR systems, genomic DNA isolated from wild type zebrafish was used as control template, in the second PCR system, the plasmid dsRed2-N1 was used as a positive control. Applying this method to several specimens of presumed GloFish^? from traders in the Netherlands and Germany revealed the presence of transgenic fish. In addition, a rapid method for screening zebrafish suspected to be genetically modified has been developed by measuring the fluorescence of water-soluble protein. Received: March 7, 2007     

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 °C to 35 °C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R² value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (μ) was modeled by square root type equation, and the relationship between μ and lag time (λ) was described by a rule of μ × λ = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (°C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R²), bias factor (B_f) and accuracy factor (A_f), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R², B_f and A_f values were 0.958, 1.019 and 1.035, respectively.     

Life history parameters of Lasioderma serricorne (F.) as influenced by food sources

Fecundity, egg to adult survival rate, developmental time, and adult body weight of the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae) were evaluated on seven food sources at 28 °C. Ground chili, paprika, cayenne pepper, chewing leaf tobacco, cigar tobacco, a commercial insect bait referred to as NOW bait, and wheat flour were used to evaluate mean lifetime fecundity. The highest fecundity (52.4±4.8 eggs/female) was observed in wheat flour, whereas the lowest fecundity (5.8±0.8 eggs/female) was observed in cigar tobacco. Among the seven food sources, beetles reared in wheat flour showed the highest survival rate of 91.0±2.7%. Only 15% of the eggs laid in NOW bait developed to the adult stage. In the three food sources containing Capsicum spp. the survival rate ranged from 30% to 40%. The egg, larval, and pupal development times varied from 3 to 5, 38 to 92 and 4 to 18 d, respectively, among food sources. Body weight and adult longevity studies showed that the heavier adults also had the longest life span. Ovipositing female L. serricorne appear to discriminate among different food sources. Although L. serricorne laid eggs in all food sources evaluated, larval and pupal survival were lowest in NOW bait. Information on the biology and host use pattern of L. serricorne may help to explain how various stored commodities are affected by this species and may lead to develop appropriate pest management strategies for this insect pest.     

Biosynthesis of PP-V, a monascorubramine homologue, by Penicillium sp. AZ

The biosynthetic pathway of PP-V, a new monascorubramine homologue, was elucidated by ¹³C-labeling studies. The [1-¹³C] of acetate was incorporated into 2-, 3a-, 4a-, 6-, 8-, 9-, 11-, 13-, 15-, 17-, and 19-Cs of PP-V, and the [2-¹³C], into 3-, 4-, 5-, 8a-, 9a-, 10-, 12-, 14-, 16-, 18-, and 20-Cs. These incorporation patterns coincide with those reported in the biosynthesis of a Monascus azaphilone pigment, monascorubrin.     

Comparison of antimicrobial resistance in Salmonella and Campylobacter isolated from turkeys in the Midwest USA

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest^®. In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase (int1), class 1 integrons (Salmonella and Campylobacter) and a multidrug efflux pump (Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene (int1), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1, but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.     

Dietary administration of ewe diets with a distillate from rosemary leaves (Rosmarinus officinalis L.): Influence on lamb meat quality

The effect of including distilled rosemary leaf in the diet of pregnant ewes on subsequent lamb meat quality was studied. Thirty-six Segureña ewes were randomly assigned to three homogeneous groups. One group was fed a basal diet (BD) as control while the diet of the other two groups was modified by substituting 10% (R₁) and 20% (R₂) of the BD with a pellet made from 50% barley and 50% of distilled rosemary leaves (DRL). Meat spoilage (TVC, PSY and MYC), TBARS, CIELab coordinates and the sensory characteristics contribution of fresh lamb meat packed in MAP (70% O₂:30% CO₂) were analyzed on days 0, 7, 14 and 21. In general, R₁ and R₂ had higher a^* values, better scores for meat and fat colour (P < 0.05) and lower TBARS and rancid odour (P < 0.05), than the control samples. The total viable count was lower in meat DRL. No statistically significant differences were detected between the two treatments (10–20% DRL).     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Scanner-based color measurement in L * a * b * format with artificial neural networks (ANN)

A computerized inspection system (CIS) that uses a flat-bed scanner, a computer, and an algorithm and graphical user interface coded and designed in Matlab^® 7.0 was developed to determine food color based on CIE L ^* a ^* b ^*, a color format. The USA Federal Color Standard printouts (SP) comprised of 456 different colors were used to train and test the artificial neural network (ANN) integrated CIS. Strong correlations were found between the results estimated from ANN-integrated CIS and those obtained from spectrophotometer (R ², 0.991, 0.989, and 0.995 for L ^*, a ^*, and b ^*, respectively) for test images data set. Various food samples were also evaluated to test the performance of the CIS. A good agreement, R ², 0.958, 0.938, and 0.962 for L ^*, a ^*, and b ^*, respectively, was found between color measurement with CIS and a spectrophotometer. CIS with a mean error of 0.60% and 2.34% for test and various food samples, respectively, has an ability to imitate the results obtained from a spectrophotometer. CIS allows users to store the captured picture for further use and estimate the overall color or the color of selected region of the samples either heterogeneous in color or amorphous in shape.     

Residual efficacy of synergized pyrethrin + methoprene aerosol against larvae of Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae)

Wheat flour and different packaging surfaces (cardboard, flour bag, muslin bag, paper bag, pallet wrap, plastic overwrap, polyethylene) were exposed to aerosol formulations of either 1% active ingredient (AI) pyrethrin (synergized with piperonyl butoxide)+ 33.6% (AI) methoprene or 3% (AI) pyrethrin + 33.6% AI methoprene. The mixture was formulated as specified on the insecticide labels to give a 100 to 1 ratio of active ingredient pyrethrin to methoprene. Residual bioassays were conducted every two weeks for 16 weeks post-exposure to the aerosol by placing four-week-old larvae of the red flour beetle, Tribolium castaneum (Herbst), or the confused flour beetle, Tribolium confusum (Jacqueline duVal), on treated flour or a treated packaging surface with untreated flour added. T. castaneum was clearly the more susceptible of the two species. Less than 2% of T. castaneum larvae exposed to aerosol-treated flour or packaging surfaces emerged as normal adults, regardless of the pyrethrin concentration. Most of the T. castaneum larvae on treated flour did not advance to the pupal stage because they were either developmentally arrested or died as larvae. They were able to develop further on the treated packaging surfaces, but still could not emerge as adults. T. confusum larvae exposed to aerosol-treated flour or packaging surfaces were able to develop to the pupal or adult stage. Emergence of normal-appearing adults from T. confusum larvae exposed on the packaging surfaces treated with 1% pyrethrin + methoprene gradually increased (range of 29.7 ± 2.9 to 49.0 ± 6.7%, depending on the surface), whereas adult emergence of larvae exposed to treated flour peaked at 10 weeks post-exposure. However, when T. confusum was exposed to 3% pyrethrin + methoprene treated flour or packaging surfaces, adult emergence was reduced. Overall there were few significant differences attributable to the individual packaging surfaces.     

Assessment of the microbiological safety of salad vegetables and sauces from kebab take-away restaurants in the United Kingdom

The purpose of this study was to establish the microbiological safety of salad vegetables and sauces served in kebab take-away restaurants. Comparison with published microbiological guidelines revealed that 4.7% of 1213 salad vegetable samples were of unsatisfactory microbiological quality due to Escherichia coli and/or Staphylococcus aureus levels at ≥10² cfu g⁻¹. Another 0.3% of salad samples were of unacceptable quality due to S. aureus at ≥10⁴ cfu g⁻¹ (2 samples) or the presence of Salmonella Kentucky (1 sample). Cucumber was the most contaminated salad vegetable with regards to unsatisfactory levels of E. coli (6.0%) or S. aureus (4.5%). Five percent of 1208 sauce samples were of unsatisfactory microbiological quality due to E. coli, S. aureus at ≥10² cfu g⁻¹ and/or Bacillus cereus and other Bacillus spp. at ≥10⁴ cfu g⁻¹. A further 0.6% of sauce samples were of unacceptable quality due to Bacillus spp. (Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis) at ≥10⁵ cfu g⁻¹ or the presence of Salmonella Agbeni (1 sample). More samples of chilli sauce (8.7%) were of unsatisfactory or unacceptable microbiological quality than any other sauce types. The results emphasize the need for good hygiene practices in kebab take-away restaurants handling these types of ready-to-eat products.     

Radiation-inducible TNF-α gene expression under stress-inducible promoter gadd 153 for cancer therapy

We demonstrated the effectiveness of radiation-inducible expression of the TNF-α gene for cancer therapy in vitro. The TNF-α gene under the control of the stress-inducible promoter, gadd 153, was introduced into the human glioma cell line, U251-SP. Without cobalt-60 gamma irradiation, no cytotoxicity against the transfected cells was observed. When the transfected cells were irradiated with 10 or 20 gray (Gy), the gadd 153 promoter was highly induced and the expression level of TNF-α increased. Five days after the irradiation, the TNF-α productions of each cell irradiated with 10 and 20 Gy were 30 and 100 times higher than the basal level, respectively. The cytotoxicities against the transfected cells 5 d after irradiation with 10 and 20 Gy were 79% or 91%, respectively, which are much higher than those against the nontransfected cells that were irradiated at the same dose (43% and 78%, respectively). These results demonstrate that the gadd 153-TNF-α system may be an effective tool for radiosurgery of malignant brain tumors.     

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×10⁹ colony-forming units per milliliter or higher and pathogen spore suspensions of 5×10⁴ spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.     

The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group

The present studies aimed at an analysis of the expression level of genes PKM2 and CAST in Longissimus lumborum [LL] muscle tissue of pigs differing as regards the glycolytic potential [GP] and drip loss [DL] from the LL muscle, with reference to the genetic group. The studies covered a total of 65 pigs: 20 purebred Landrace [L], 22 crossbreeds of Landrace with the Yorkshire [L × Y] and 23 three-breed crosses (Landrace × Yorkshire) × Duroc [(L × Y) × D]. In the case of gene PKM2 one may observe in (L × Y) × D crossbreds, compared to L × Y crossbreds, an increased expression, closely related with the increase in dry matter content, including intramuscular fat, as well as a more favourable progress of glycolytic and energy metabolism during the early time post mortem (pH₄₅ and R₁). Compared with Landrace animals, the lower expression of the CAST gene observed in (L × Y) × D pigs is manifested by a marked improvement of meat quality (R₁ pH₄₅ pH₂₄, pH₄₈), arising from the rate of glycolytic and energy metabolism, typical for normal meat, that in effect results in its higher culinary and technological value.     

Hyperspectral imaging for the investigation of quality deterioration in sliced mushrooms (Agaricus bisporus) during storage

In this study, the potential application of hyperspectral imaging (HSI) for quality prediction of white mushroom slices during storage at 4 °C and 15 °C was investigated. Mushroom slice quality was measured in terms of moisture content, colour (CIE Lightness, L* and yellowness, b*) and texture (hardness, H and chewiness, Ch). Hyperspectral images were obtained using a pushbroom line-scanning HSI instrument, operating in the wavelength range of 400–1,000 nm with spectroscopic resolution of 5 nm. Multiple linear regression (MLR) and Principal Component Regression (PCR) models were developed to investigate the relationship between reflectance and the various quality parameters measured. 20 optimal wavelengths for quality prediction were selected after performing an exhaustive search for the best subsets of predictor variables on a calibration set of 84 samples. PCR applied to the set of optimal wavelengths gave the best performance as compared to MLR and PCR on the entire wavelength range. When applied to an independent validation set of samples, PCR models developed on the calibration set were capable of predicting moisture content with RMSEP of 0.74% w.b. and R ² of 0.75, L* with RMSEP of 0.47 and R ² of 0.95, b* with RMSEP of 0.66 and R ² of 0.75, H with RMSEP of 0.49 N and R ² of 0.77 and Ch with RMSEP of 0.27 N and R ² of 0.72. Virtual images showing the distribution of moisture content on the mushroom surface were generated from the estimated PCR model. Results from this study could be used for the development of a non-destructive monitoring system for prediction of sliced mushroom quality.     

Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis

Corynebacterium glutamicum is known as a host species for amino acid production. This microorganism was recently noticed as a host that produces secreted proteins. In this study, we performed ¹³C metabolic flux analysis (¹³C-MFA) on a recombinant C. glutamicum strain that secretes a heterologous transglutaminase (TGase) to improve TGase secretion. For the ¹³C-MFA of a TGase-secreting C. glutamicum strain in batch cultivation, a ¹³C-labeling experiment and measurement of mass isotopomer distributions of proteinogenic amino acids were performed, and metabolic fluxes were determined considering the changes in fractional ¹³C-labeling of proteinogenic amino acids with respect to culture time. The TGase yield increased at the stationary phase but decreased toward its end. The results of ¹³C-MFA revealed that the flux from glycolysis to the TCA cycle gradually increased during TGase secretion. We speculate that the NADH/NAD⁺ ratio in the cells increases and that as a result, the specific glucose uptake rate decreases in the stationary phase because of the increased flux of the TCA cycle. Since it is expected that a decrease in the NADH/NAD⁺ ratio would improve the TGase yield, we tried to enhance lactate production in a TGase-secreting C. glutamicum strain to decrease cellular NADH levels by increasing the pH level in the culture. The TGase yield increased in 1.4-fold by increasing the pH from 6.7 to 7.2, indicating that the TGase yield was successfully improved on the basis of the ¹³C-MFA.     

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ^B) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD_ΔsigB, EGD_ΔprfA and EGD_ΔprfAΔsigB with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD_ΔsigB and EGD_ΔprfAΔsigB (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD_ΔsigB and EGD_ΔprfAΔsigB) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3 M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transciption were observed in EGD_ΔsigB and EGD_ΔprfAΔsigB. Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ^B might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.     

Comparative analysis of acid resistance in Listeria monocytogenes and Salmonella enterica strains before and after exposure to poultry decontaminants. Role of the glutamate decarboxylase (GAD) system

Data on the ability of chemical poultry decontaminants to induce an acid stress response in pathogenic bacteria are lacking. This study was undertaken in order to compare the survival rates in acid broths of Listeria monocytogenes and Salmonella enterica strains, both exposed to and not exposed to decontaminants. The contribution of the glutamate decarboxylase (GAD) acid resistance system to the survival of bacteria in acid media was also examined. Four strains (L. monocytogenes serovar 1/2, L. monocytogenes serovar 4b, S. enterica serotype Typhymurium and S. enterica serotype Enteritidis) were tested before (control) and after exposure to trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide and peroxyacids (strains were repeatedly passed through media containing increasing concentrations of a compound). Stationary-phase cells (10⁸ cfu/ml) were inoculated into tryptic soy broth (TSB) acidified with citric acid (pH 2.7 and 5.0) with or without glutamate (10 mM) added, and incubated at 37 °C for 15 min. Survival percentages (calculated from viable colonies) varied from 2.47 ± 0.67% to 91.93 ± 5.83%. L. monocytogenes cells previously exposed to acid decontaminants (citric acid and peroxyacids) showed, when placed in acid TSB, a higher (P < 0.05) percentage of survival (average 38.80 ± 30.52%) than control and pre-exposed to non-acidic decontaminants strains (22.82 ± 23.80%). Similar (P > 0.05) survival percentages were observed in previously exposed to different decontaminants and control Salmonella strains. The GAD acid resistance system did not apparently play any role in the survival of L. monocytogenes or S. enterica at a low pH. This study demonstrates for the first time that prior exposure to acidic poultry decontaminants increases the percentage of survival of L. monocytogenes exposed to severe acid stress. These results have important implications for the meat industry when considering which decontaminant treatment to adopt.     

Biotransformation of (R)-(+)- and (S)-(−)-limonene to α-terpineol by Penicillium digitatum— investigation of the culture conditions

The biotransformation of (R)-(+)- and (S)-(−)-limonene by Penicillium digitatum was investigated. One strain of P. digitatum was able to convert (R)-(+)-limonene to pure (R)-(+)-α-terpineol in 8 h with a yield of up to 93%. It was found that (R)-(+)-limonene was converted much better into α-terpineol than (S)-(−)-limonene, and that no significant chemical conversion of the substrate occurred in control flasks at pH 3.5. The culture conditions involved such as the type and concentration of co-solvent applied and the sequential addition of substrate were investigated, taking into account some findings on the physical behaviour of the system. The highest bioconversion yields were obtained when the substrate was applied as a diluted solution in EtOH.