Molecular Confirmation of Intraspecific Tomato (<Emphasis Type="Italic">Solanum lycopersicum)</Emphasis> Hybrids and Their Evaluation Against Late Blight and Cucumber Mosaic Virus

Tomato is one of the most consumed vegetables in the world. Diseases are the number one concern in the development of high-yield and disease-resistant tomato hybrids which is the foremost priority of breeders. Present study was conducted (1) to develop DNA-based markers for genetic confirmation of tomato F₁ hybrids, (2) to utilize sequenced characterized amplified region (SCAR) marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato and (3) to evaluate male and female parental genotypes and their F₁ hybrids against late blight (LB) and cucumber mosaic virus (CMV). For molecular studies, 58 previously reported markers including RAPDs (10), SCAR (01), EST-SSR (01) and SSR (46) were applied. The SCAR marker clearly differentiated the LB3 and LB4 from Roma and T-1359 and provided evidence for Ph-3 gene. The SCAR marker was able to confirm the Ph-3 gene in the hybrids Roma × LB4, Roma × LB3, Riogrande × LB2, Riogrande × LB3 and Roma × LB7. Out of several tested primers, SSR-22 proved useful for genetic confirmation of F₁ hybrid TMS1 × Money Maker (MM). For LB, tested hybrids/genotypes were ranked as susceptible to highly susceptible with different infection percentage (IP). However, the pace of symptom development was slower in hybrid Rio × LB2, 45% IP after 10 days of inoculation compared with 85% disease in one of the parent genotypes (Riogrande). None of the tested genotypes was found resistant; however, TMS1 responded as tolerant against CMV using mechanical inoculation. Under natural field conditions, TMS1 was found resistant while hybrids TMS1 × Naqeeb and TMS1 × MM were tolerant where as others were found to be susceptible. In conclusion, all tomato hybrids were genetically confirmed using DNA-based markers. SCAR marker was useful for marker-assisted confirmation of the Ph-3 gene in parental lines and hybrids; however, this gene was unable to provide protection against the local population of P. infestans.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon?+?and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~?25.5% and ~?6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.     

Fluid–structure interaction in aortic cross-clamping: Implications for vessel injury

Vascular cross-clamping is applied in many cardiovascular surgeries such as coronary bypass, aorta repair and valve procedures. Experimental studies have found that clamping of various degrees caused damage to arteries. This study examines the effects of popular clamps on vessel wall. Models of the aorta and clamp were created in Computer Assisted Design and Finite Element Analysis packages. The vessel wall was considered as a non-linear anisotropic material while the fluid was simulated as Newtonian with pulsatile flow. The clamp was applied through displacement time function. Fully coupled two-way solid–fluid interaction models were developed. It was found that the clamp design significantly affected the stresses in vessel wall. The clamp with a protrusion feature increased the overall Von Mises stress by about 60% and the compressive stress by more than 200%. Interestingly, when the protrusion clamp was applied, the Von Mises stress at the lumen (endothelium) side of artery wall was about twice that of the outer wall. This ratio was much higher than that of the plate-like clamp which was about 1.3. The flow reversal process was demonstrated during clamping. Vibrations, flow and wall shear stress oscillations were detected immediately before total vessel occlusion. The commonly used protrusion clamp increased stresses in vessel wall, especially the compressive stress. This design also significantly increased the stresses on endothelium, detrimental to vessel health. The present findings are relevant to surgical clamp design as well as the transient mechanical loading on the endothelium and potential injury. The deformation and stress analysis may provide valuable insights into the mode of tissue injury during cross-clamping.     

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae.     

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman–Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P4_1, with R_work/R_free values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme–inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.     

Mixed Convection Flow of Viscoelastic Fluid by a Stretching Cylinder with Heat Transfer

Flow of viscoelastic fluid due to an impermeable stretching cylinder is discussed. Effects of mixed convection and variable thermal conductivity are present. Thermal conductivity is taken temperature dependent. Nonlinear partial differential system is reduced into the nonlinear ordinary differential system. Resulting nonlinear system is computed for the convergent series solutions. Numerical values of skin friction coefficient and Nusselt number are computed and discussed. The results obtained with the current method are in agreement with previous studies using other methods as well as theoretical ideas. Physical interpretation reflecting the contribution of influential parameters in the present flow is presented. It is hoped that present study serves as a stimulus for modeling further stretching flows especially in polymeric and paper production processes.