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.     

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.     

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.     

Characterization of Growth Suppressive Functions of a Splice Variant of Cyclin D2

We have recently cloned a novel splice variant of cyclin D2 termed as cycD2SV. CycD2SV overexpression in several immortalized cell lines led to formation of ubiquitinated protein aggregates accompanied by a significant decrease in cell proliferation. Based on immuno co-localization and ultrastructural analysis experiments, cycD2SV protein aggregates were frequently found in various subcellular compartments such as endosomes, autophagosomes, lysosomes and the microtubule organizing centre. Secondary structure analysis revealed that the amino terminal α-helix in cycD2SV is not tightly packed with the cyclin box suggesting a misfolded conformation compared to other cyclins. Deletion analysis suggests that 1–53 amino acid region of cycD2SV may be required for protein aggregation and 54–136 amino acid region may mediate cell cycle inhibition. Based on co-immunoprecipitation experiments, we have shown that cycD2SV binds to cycD2 as well as CDK4. In addition, gene expression analysis demonstrated an upregulation in GADD45α and dynamin 2 mRNA levels in cycD2SV overexpressing cells. These two proteins are known to play critical roles in the DNA damage response and apoptosis pathways. TUNEL experiments were negative for apoptosis, however, cycD2SV expressing cells were more sensitive to cell death induced by external stressors such as trypsinization. Collectively our results suggest that cycD2SV mediates cell cycle inhibition by sequestering endogenous cell cycle proteins, such as cycD2 and CDK4, and possibly targeting them for ubiquitin mediated protein degradation.     

A Major Outer Membrane Protein of Rahnella aquatilis Functions as a Porin and Root Adhesin

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60°C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand β-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.     

CT-based semi-automatic quantification of vertebral fracture restoration

Minimally invasive surgeries aiming to restore fractured vertebral body are increasing; therefore, our goals were to create a 3D vertebra reconstruction process and design clinical indices to assess the vertebral restoration in terms of heights, angles and volumes. Based on computed tomography (CT)-scan of the vertebral spine, a 3D reconstruction method as well as relevant clinical indices were developed. First, a vertebra initial solution requiring 5 min of manual adjustments is built. Then an image processing algorithm places this solution in the CT-scan images volume to adjust the model's nodes. On the vertebral body's anterior and posterior parts, nine robust heights, volume and endplate angle measurement methods were developed. These parameters were evaluated by reproducibility and accuracy studies. The vertebral body reconstruction accuracy was 1.0 mm; heights and volume accuracy were, respectively, 1.2 and 179 mm³. In conclusion, a 3D vertebra reconstruction process requiring little user time was proposed as well as 3D clinical indices assessing fractured and restored vertebra.