Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.     

Viability analysis of multi-fishery

This work is about the viability domain corresponding to a model of fisheries management. The dynamic is subject of two constraints. The biological constraint ensures the stock perennity where as the economic one ensures a minimum income for the fleets. Using the mathematical concept of viability kernel, we find out a viability domain which simultaneously enables the fleets to exploit the resource, to ensure a minimum income and stock perennity.     

Hsp70-2 gene polymorphism: susceptibility implication in Tunisian patients with coronary artery disease

ABSTRACT: Coronary artery disease (CAD) is a multifactorial disease where genetic and environmental factors interact in complex ways to cause the disease. Heat shock protein genes are involved in the progress of CAD. This implies that genetic variants of Hsp70-2 genes might contribute to the development of the disease. Aim of study The aim of this study was to characterize statistical correlation of linkage between lipid profiles, polymorphism PstI site of Hsp70-2 gene and coronary artery diseases. Patients and methods This study was carried out on Tunisian patients with CAD recruited from Hospital of Fattouma Bourguiba of Monastir-Tunisia. Polymerase chain reaction and restriction enzymes were used to determine the genotypic distributions in 252 unrelated patients and 151 healthy control subjects. Further, ApoA-I and ApoB as well as the serum total of cholesterol, HDL, triglyceride, and hs-CRP levels were measured. RESULTS: We showed a decreased level of ApoA-I, whereas the levels of each of ApoB and hs-CRP were increased in patients with CAD compared with control group. In addition our studies of a polymorphic PstI site of Hsp70-2 gene lying in the coding region at position 1267 of the Hsp70-2 gene have revealed that the frequency of P1/P2 heterozygote was 0.484 in patient group compared with control group (0.476, p = 0.046). Whereas, the frequency of the P2/P2 homozygote was 0.190 in patient group and only 0.099 in controls (P = 0.006). These results indicate that the odds ratio of CAD associated with the Hsp70-2 polymorphism is confined the P2/P2 homozygotes (OR 2.498; P = 0.006). CONCLUSION: Taken together, our results indicate that the high frequency of P2/P2 genotype is associated with elevated levels of biochemical parameters (LDL cholesterol, hs-CRP) in Tunisian patient group. The Hsp70-2 polymorphism has susceptibly implication in CAD. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1118340895703689.     

3D reconstruction of the ribs from lateral and frontal X-rays in comparison to 3D CT-scan reconstruction

Subject-specific three-dimensional (3D) reconstructions of the ribs can be obtained from biplanar X-rays. The goal of this study was to evaluate the accuracy and the inter-observer reproducibility of this technique in comparison to CT-scan reconstructions. CT scans and biplanar X-rays were obtained from 50 ribs (from three cadaveric rib cages). Three experienced experimenters reconstructed each rib from biplanar X-rays. Morphometric parameters were then computed from the rib midlines. Differences were computed between parameters obtained from the 3D reconstructions based on biplanar X-rays and from CT scans. The accuracy was computed as the mean of this difference for the 50 ribs from all three experimenters. The inter-observer variability was assessed using the coefficient of variation (CV) between the three observers. The CT-scan reconstructions were considered to be the gold standard in spite of their limitations for rib reconstructions. According to the different linear parameters, the accuracy of the reconstructions was found to be between -6mm (-2%) and 3mm, (4%). The accuracy of the current method was close to that of CT-scan reconstructions. The inter-observer variability was between 3% and 6%. Frontal and lateral X-rays are commonly obtained clinically, so 3D reconstructions can be used without increased radiation exposure to the patient.     

3D Evaluation of the acetabular coverage assessed by biplanar X-rays or single anteroposterior X-ray compared with CT-scan

Considering the increasing development of three dimensional (3D) imaging, the 3D assessment of the acetabular coverage is to become the most interesting tool for the detection of acetabular pathologies. Biplanar X-rays based methods allow a 3D reconstruction of the hip with a reduced radiation dose. This study proposes a 3D assessment method of the acetabular coverage from biplanar X-rays or from an anteroposterior X-ray (conventional clinical imaging). An in vitro evaluation of the method was performed on six hip joints in comparison with computed tomography. The global coverage, the local coverage and the acetabular rim orientation were estimated in 3D. The mean global acetabular coverage was 40% with an estimated mean accuracy of 1.3% for the biplanar X-rays based method. This study evaluated a 3D assessment method of the acetabular coverage from biplanar X-rays or anteroposterior X-ray and open the way for clinical in vivo applications.     

Effect of hexachlorobenzene on some male reproductive parameters in Meriones unguiculatus

Organochlorine pesticides are known to cause disturbances in many physiological functions. The effects of in vivo administered hexachlorobenzene (HCB) on the male testicular function were studied in Meriones unguiculatus. Three groups of sexually mature meriones were orally exposed to 1.6, 4, and 16 mg/kg of body weight in olive oil for 30 days. Morphological and morphometrical estimations were applied to quantify some structural constituents of the testes. Testicular weight was significantly decreased in all treated groups, while no change was noted in seminal vesicle weight. A decrease in the spermatozoid content of the seminiferous tube was noted and appeared correlated with a modification of the process of spermatogenesis. Spermatic activity in HCB-treated animals testes decreased significantly, particularly in the group treated with the higher dose (60+/-3.16% vs. 88+/-4.89% in controls). Plasma testosterone levels were decreased significantly in the groups treated with 4 and 16 mg[HCB]/kg BW (0.48+/-0.08 ng/ml and 0.54+/-0.07 ng/ml) comparatively to controls (1.08+/-0.1 ng/ml) p<0.01.     

The exopolysaccharide of Rhizobium sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus roots but contributes to root colonization

Microbial exopolysaccharides (EPSs) play key roles in plant–microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots ( Arabidopsis thaliana and Brassica napus ). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene ( gta ). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root–soil interface, root colonization, but not in biofilm formation.     

Biochemical characterization,cloning and molecular modeling of a digestive lipase from red seabream (Pagrus major): Structural explanation of the interaction deficiency with colipase and lipidic interface

Red seabream digestive lipase (RsDL) was purified from fresh pyloric caeca. Pure RsDL has an apparent molecular mass of 50 kDa. The RsDL is more active on short‐chain triacylglycerols (TC₄), and enzymatic activity decreases when medium (TC₈) or long‐chain (olive oil) triacylglycerols were used as substrates. The specific activities of RsDL are very weak as compared to those obtained with classical pancreatic lipases. No colipase was detected in the red seabream pyloric caeca. Furthermore, the RsDL was not activated by a mammal colipase. Similar results were reported for annular seabream lipase. In order to explain structurally the discrepancies between sparidae and mammal lipases, genes encoding mature RsDL and five other lipases from sparidae fish species were cloned and sequenced. Phylogenetic studies indicated the closest homology of sparidae lipases to bird pancreatic ones. Structural models were built for annular seabream and RsDL under their closed and open forms using mammal pancreatic lipases as templates. Several differences were noticed when analyzing the amino acids corresponding to those involved in HPL binding to colipase. This is likely to prevent interaction between the fish lipase and the mammalian colipase and may explain the fact that mammalian colipase is not effective in activating sparidae lipases. In addition, several hydrophobic residues, playing a key role in anchoring pancreatic lipase onto the lipid interface, are replaced by polar residues in fish lipases. This might explain the reason why the latter enzymes display weak activity levels when compared to mammalian pancreatic lipases.     

New 1,2,3-triazole linked ciprofloxacin-chalcones induce DNA damage by inhibiting human topoisomerase I& II and tubulin polymerization

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC₅₀s ranged from 2.53-8.67 µM, 8.67–62.47 µM, and 4.19–24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC₅₀ values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC₅₀ values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC₅₀ = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.