TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

Disturbance-dependent Yellow-legged Gull (Larus michahellis) predation on Larid chicks decreases with chick age

Predation is one of the key factors shaping the dynamics of animal populations. In birds, nest loss due to predation can be a significant cause of low reproductive success. Ground-nesting birds are among the bird groups most susceptible to predation, mainly because their nests are easily accessible to a broad suite of potential predators. For these birds, anthropogenic disturbances can generate changes in nest predation risk by altering their antipredator behaviour and also by altering the behaviour of the predator species, i.e. the predator becoming much more aware of predation opportunities due to frequent disturbances and/or motivated to repeat predation attempts when some are successful. To date, most previous studies investigating this have focused on a single effect, either predation or disturbance, on chick survival. It remains unknown how the risk of predation with and without disturbance varies with chick age. In this study, we used behavioural observations to assess how the interaction between predators and disturbance affects predation risk in chicks and how this interacts with chick age. Specifically, we investigated the effect of disturbance caused by humans and stray dogs on the predation of Slender-billed Gull Chroicocephalus genei chicks by Yellow-legged Gulls Larus michahellis, and whether this depended on the age of the chicks. Our results revealed that disturbance had a significant positive effect on predation measures of Slender-billed Gull chicks by Yellow-legged Gulls, but that this effect was mediated both by disturbance type and the age of chicks. Stray dogs entering the colony had a stronger disturbance effect on chicks than passing humans, increasing predation risk by Yellow-legged Gulls. Our results also showed that chick age interacts with disturbance type to determine the predation risk. This is probably mediated by chicks' capacity to escape predation by gathering in a single large crèche that runs into the water when disturbed. To preserve Slender-billed Gull colonies in one of its few remaining breeding sites in Tunisia, and as gulls tend to react even when the disturbance occurs relatively far from the colonies, it is crucial to (1) restrict human access to dikes and islets where large colonies breed and (2) construct artificial islets attractive to gulls and inaccessible to stray dogs.     

Physiological and biochemical changes in different sugar beet genotypes infected with root-knot nematode

Ten genotypes of sugar beet plant either monogerm or multigerm seeds were screened under greenhouse conditions for both susceptibility and biochemical reaction to root-knot nematode (RKN) Meloidogyne incognita. All the tested genotypes were susceptible to nematode infection according to the number of root galls and gall indices. All infected genotypes exhibited significant reduction in chlorophyll a, b and carotenoids compared to non-infected ones. The total indole acetic acid and total phenolic compounds contents (mean of both shoot and root) increased significantly in most infected genotypes compared to non-infected genotypes except Disk-01-99 and Monte Rosa as well as LP16 and LP15 genotypes, respectively. Also, total polyamine contents (putrescine, spermidine and spermine) showed significant increases in response to infection with nematodes in all genotypes. The same trend was observed in lipid peroxidation expressed with malondialdehyde content in all tested genotypes. Activities of polyphenol oxidase, peroxidase, superoxide dismutase and catalase enzymes were also induced in most infected genotypes compared with non-infected genotypes. Generally, infection with RKNs induced the appearance of new protein bands at molecular masses 303, 288, 42 and 37?KDa in all infected genotypes. The differentiation in the appearance and/or disappearance of protein bands according to susceptibility to infection reflects the variation between genotypes in defense against infection.     

Inactivation of template-directed misfolding of infectious prion protein by ozone

Misfolded prions (PrP(Sc)) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrP(Sc)). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrP(Sc), as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log(10)) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter(-1) min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

Comparative phenology and growth of four Cenchrus ciliaris L. accessions established under arid bioclimate in Tunisia

A phenological study is one of the first steps in understanding the function of ecosystems. This is because phenological events reflect the way in which the species exploit the so‐called favourable periods in order to gain carbon and to allocate photosynthetic products for growth and reproduction. The objective of the present work was to examine the phenology of reproduction and the above‐ground growth of several Cenchrus ciliaris accessions, growing under the arid bioclimate in Tunisia. These accessions collected in the south of Tunisia are: Bou Hedma (P1), Tozeur (P2), Raas Jedir (P3) and Sidi Toui (P4). It was demonstrated that an important phenological variability exists within the different accessions studied. The statistical analysis (correlation and principal component analysis) showed that the accession from Bou Hedma (P1) was the most precocious and vigorous one.     

BIOSYSTEMATIC STUDIES IN THE BOUTELOUA CURTIPENDULA COMPLEX. V. MEGASPOROGENESIS AND EMBRYO SAC DEVELOPMENT

Megasporogenesis and embryo sac development in the sexually reproducing taxa Bouteloua warnockii (2n = 22), B. media (2n = 20), B. uniflora Vasey var. uniflora (2n = 20), B. uniflora var. coahuilensis Gould and Kapadia (2n = 20), and B. curtipendula var. curlipendula (2n = 40) all were found to be of the Adoxa type, in which all 4 megaspores persist and divide once to form an 8-nucleate embryo sac. On the other hand, evidence indicated that plants of B. curtipendula var. caespitosa with high ancuploid chromosome numbers reproduce by pseudogamous fertilization of an aposporous embryo sac. In this taxon the megaspore mother cell did not go beyond the first anaphase of meiosis and the functional embryo sac developed from a nucellar cell. Although the 8-nucleate embryo sac was typical, a 3-nucleate embryo sac was observed to develop in some cases.     

Characterization of a cysteine protease from wheat Triticum aestivum (cv. Giza 164)

Enzymes, especially proteases, have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. A cysteine protease, used extensively in the food industry, was purified from germinated wheat Triticum aestivum (cv. Giza 164) grains through a simple reproducible method consisting of extraction, ion exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 61000+/-1200-62000+/-1500 by SDS-PAGE and gel filtration. The cysteine protease had an isoelectric point and pH optimum at 4.4 and 4.0, respectively. The enzyme exhibited more activity toward azocasein than the other examined substrates with K(m) 2.8+/-0.15 mg azocasein/ml. In addition, it had a temperature optimum of 50 degrees C and based on a heat stability study 55% of its initial activity remained after preincubation of the enzyme at 50 degrees C for 30 min prior to substrate addition. All the examined metal cations inhibited the enzyme except Co(2+), Mg(2+), Mn(2+) and Li(+). The proteolytic activity of the enzyme was inhibited by thiol-specific inhibitors, whereas iodoacetate and p-hydroxymercuribenzoate caused a competitive inhibition with Ki values 6+/-0.3 mM and 21+/-1.2 microM, respectively. Soybean trypsin inhibitor had no effect on the enzyme. The enzyme activity remained almost constant for 150 days of storage at -20 degrees C. The properties of this enzyme, temperature and pH optima, substrate specificity, stability and sensitivity to inhibitors or activators, meet the prerequisites needed for food industries.     

WAMI: a web server for the analysis of minisatellite maps

Background  

Minisatellites are genomic loci composed of tandem arrays of short repetitive DNA segments. A minisatellite map is a sequence of symbols that represents the tandem repeat array such that the set of symbols is in one-to-one correspondence with the set of distinct repeats. Due to variations in repeat type and organization as well as copy number, the minisatellite maps have been widely used in forensic and population studies. In either domain, researchers need to compare the set of maps to each other, to build phylogenetic trees, to spot structural variations, and to study duplication dynamics. Efficient algorithms for these tasks are required to carry them out reliably and in reasonable time.