Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production by Lactococcus lactis

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach

Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of di¿erent Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes speci¿c for STEC (vt1,vt2, eae, andehxA) and the gene forE. coli16S (hui). Optimization was performed using a Box Behnken design, andthe limit of detection for each virulence factor was established. Finally,this NAMIA using CNPs was tested with DNA from 48 ¿eld strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and speci¿city values higher than 0.80 and e¿ciency values higher than 0.92. Kappa coe¿cients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used forvt1, eae, and ehxA, and a perfect agreement (k = 1) forvt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed ¿t for purpose, i.e., it is valuable for fast screening of ampli¿ed genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.This work was partially supported by the Generalitat Valenciana (BEST/2009/026), the Universidad Politecnica de Valencia (PAID-00-09-2837), and the Dutch Ministry of Agriculture, Nature and Food Quality (Strategic research program Food Safety, Monitoring and Detection KB-06-005). The authors thank Dr. Eva Moller Nielsen at the Danish Veterinary Institute (Copenhagen, Denmark) for providing E. coli control strains and Dr. Lutz Geue (Friedrich-Loeffler-Institut, Wusterhausen, Germany) and Dr. Dorte Dopfer (School of Veterinary Medicine, University of Wisconsin, Madison, WI) for field isolates.Noguera Murray, PS.; Posthuma-Trumpie, GA.; Van Tuil, M.; Van Der Wal, FJ.; De Boer, A.; Moers, APHA.; Van Amerongen, A. (2011). Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Analytical Chemistry. 83:8531-8536. https://doi.org/10.1021/ac201823v853185368

Ergodicity of Traffic Flow with Constant Penetration Rate for Traffic Monitoring via Floating Vehicle Technique

Traffic congestion in Asian megacities has become extremely worse, and any means to lessen the congestion level is urgently needed. Building an efficient mass transportation system is clearly necessary. However, implementing Intelligent Transportation Systems (ITS) have also been demonstrated effective in various advanced countries. Recently, the floating vehicle technique (FVT), an ITS implementation, has become cost effective to provide real-time traffic information with proliferation of the smartphones. Although many publications have discussed various issues related to the technique, none of them elaborates the discrepancy of a single floating car data (FCD) and the associated fleet data. This work addresses the issue based on an analysis of Sugiyama et al's experimental data. The results indicate that there is an optimum averaging time interval such that the estimated velocity by the FVT reasonably representing the traffic velocity