Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic β-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of β-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol–gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 μM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.     

A Cost Effectiveness Analysis of Salt Reduction Policies to Reduce Coronary Heart Disease in Four Eastern Mediterranean Countries

Background

Coronary Heart Disease (CHD) is rising in middle income countries. Population based strategies to reduce specific CHD risk factors have an important role to play in reducing overall CHD mortality. Reducing dietary salt consumption is a potentially cost-effective way to reduce CHD events. This paper presents an economic evaluation of population based salt reduction policies in Tunisia, Syria, Palestine and Turkey.

Methods and Findings

Three policies to reduce dietary salt intake were evaluated: a health promotion campaign, labelling of food packaging and mandatory reformulation of salt content in processed food. These were evaluated separately and in combination. Estimates of the effectiveness of salt reduction on blood pressure were based on a literature review. The reduction in mortality was estimated using the IMPACT CHD model specific to that country. Cumulative population health effects were quantified as life years gained (LYG) over a 10 year time frame. The costs of each policy were estimated using evidence from comparable policies and expert opinion including public sector costs and costs to the food industry. Health care costs associated with CHDs were estimated using standardized unit costs. The total cost of implementing each policy was compared against the current baseline (no policy). All costs were calculated using 2010 PPP exchange rates. In all four countries most policies were cost saving compared with the baseline. The combination of all three policies (reducing salt consumption by 30%) resulted in estimated cost savings of $235,000,000 and 6455 LYG in Tunisia; $39,000,000 and 31674 LYG in Syria; $6,000,000 and 2682 LYG in Palestine and $1,3000,000,000 and 378439 LYG in Turkey.

Conclusion

Decreasing dietary salt intake will reduce coronary heart disease deaths in the four countries. A comprehensive strategy of health education and food industry actions to label and reduce salt content would save both money and lives.     

Morphological and biochemical changes in maize under drought and salinity stresses in a semi-arid environment

Abstract

This study investigated some morphological and biochemical responses of maize to drought and salinity in open field in Tunisia with the aim of gaining insights into tolerance mechanisms. After seedling emergence, five treatments were applied until maturity: optimal water supply (control, 100% of maximum evapotranspiration - ETM); irrigation at 70% ETM (moderate drought) and at 35% ETM (severe drought); optimal supply of water containing 3?g NaCl L^?1 (moderate salinity) and 6?g NaCl L^?1 (severe salinity). Here, we demonstrated that extreme drought and salinity severely decreased the leaf area (?74% and ?55%, respectively) and the above-ground biomass (?35% and ?31%, respectively) at silking stage, indicating that the photosynthetic leaf apparatus is highly sensitive and that drought has a greater effect than salinity. Grain yield losses were also exacerbated under extreme stress conditions, viz. severe drought (?85% versus controls) and severe salinity (?73%), while productivity under moderate salinity approximated that of moderate drought, possibly due to increases in leaf chlorophyll and carotenoid content and K/Na ratio. The leaf area and its relative water content were positively correlated with grain yield under both salinity and drought stresses, and may therefore be used as markers for effective screening of maize genotypes for better stress tolerance.     

Update of the spectrum of GJB2 gene mutations in Tunisian families with autosomal recessive nonsyndromic hearing loss

Hearing loss is the most frequent sensory disorder. It affects 3 in 1000 newborns. It is genetically heterogeneous with 60 causally-related genes identified to date. Mutations in GJB2 gene account for half of all cases of non-syndromic deafness. The aim of this study was to determine the relative frequency of GJB2 allele variants in Tunisia. In this study, we screened 138 patients with congenital hearing loss belonging to 131 families originating from different parts of Tunisia for mutations in GJB2 gene. GJB2 mutations were found in 39% of families (51/131). The most common mutation was c.35delG accounting for 35% of all cases (46/131). The second most frequent mutation was p.E47X present in 3.8% of families. Four identified mutations in our cohort have not been reported in Tunisia; p.V37I, c.235delC, p.G130A and the splice site mutation IVS1+1G>A (0.76%). These previously described mutations were detected only in families originating from Northern and not from other geographical regions in Tunisia. In conclusion we have confirmed the high frequency of c.35delG in Tunisia which represents 85.4% of all GJB2 mutant alleles. We have also extended the mutational spectrum of GJB2 gene in Tunisia and revealed a more pronounced allelic heterogeneity in the North compared to the rest of the country.     

Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III

Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon–intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.     

Model for fitting longitudinal traits subject to threshold response applied to genetic evaluation for heat tolerance

A semi-parametric non-linear longitudinal hierarchical model is presented. The model assumes that individual variation exists both in the degree of the linear change of performance (slope) beyond a particular threshold of the independent variable scale and in the magnitude of the threshold itself; these individual variations are attributed to genetic and environmental components. During implementation via a Bayesian MCMC approach, threshold levels were sampled using a Metropolis step because their fully conditional posterior distributions do not have a closed form. The model was tested by simulation following designs similar to previous studies on genetics of heat stress. Posterior means of parameters of interest, under all simulation scenarios, were close to their true values with the latter always being included in the uncertain regions, indicating an absence of bias. The proposed models provide flexible tools for studying genotype by environmental interaction as well as for fitting other longitudinal traits subject to abrupt changes in the performance at particular points on the independent variable scale.     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

Adh 1 first intron polymorphisms in Argentinean populations of Ceratitis capitata

DNA size polymorphisms were utilized in a study of 24 natural populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae) from Argentina. The first intron of alcohol dehydrogenase 1 gene (Adh₁) was amplified using exon priming intron crossing‐polymerase chain reaction. Three size variants were detected among the 307 samples analyzed. To better differentiate the size variants, further digestion of PCR products with the EcoRI restriction enzyme was carried out. Complete nucleotide sequences of the three‐allele variants were obtained and single changes, insertions, deletions, and EcoRI recognition sites were located. Population allele frequencies were analyzed and a global mean heterozygosity (He) of 0.33 was obtained. In most populations, observed allelic frequencies conformed to Hardy–Weinberg expectations. Significant differences between provinces and sampling sites within these provinces, and among some populations were found. The average number of insects exchanged among populations (Nm) was estimated and high values were observed between Argentina and populations from two African countries (Morocco and Kenya), Australia, and Hawaii (Kauai). Pest introduction sources and dispersion patterns in Argentina are discussed based on these results as well as on available bibliographical data.