A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1 replication

Background

Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.

Results

When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)₂R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.

Conclusions

This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.

     

Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotic hemoglobin: evidence for a role in oxygen transport

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O(2) , we were able to collect O(2) -binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O(2) noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O(2) transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana.     

Analysis of IL-17<Superscript>+</Superscript>cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response

Introduction  

In this study, we analysed the number of IL-17⁺ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.     

Influence of tyrosine-derived moieties and drying conditions on the formation of helices in gelatin

The single and triple helical organization of protein chains strongly influences the mechanical properties of gelatin-based materials. A chemical method for obtaining different degrees of helical organization in gelatin is covalent functionalization, while a physical method for achieving the same goal is the variation of the drying conditions of gelatin solutions. Here we explored how the introduction of desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT) linked to lysine residues of gelatin influenced the kinetics and thermodynamic equilibrium of the helicalization process of single and triple helices following different drying conditions. Drying at a temperature above the helix-to-coil transition temperature of gelatin (T > T(c), called v(short)) generally resulted in gelatins with relatively lower triple helical content (X(c,t) = 1-2%) than lower temperature drying (T < T(c), called v(long)) (X(c,t) = 8-10%), where the DAT(T) functional groups generally disrupted helix formation. While different helical contents affected the thermal transition temperatures only slightly, the mechanical properties were strongly affected for swollen hydrogels (E = 4-13 kPa for samples treated by v(long) and E = 120-700 kPa for samples treated by v(short)). This study shows that side group functionalization and different drying conditions are viable options to control the helicalization and macroscopic properties of gelatin-based materials.     

Magnetic and fluorescent glycopolymer hybrid nanoparticles for intranuclear optical imaging

The synthesis of galactose-displaying core-shell nanospheres exhibiting both fluorescent and magnetic properties by grafting a glycocopolymer consisting of 6-O-methacryloylgalactopyranose (MAGal) and 4-(pyrenyl)butyl methacrylate (PyMA) onto magnetic silica particles via thiol-ene chemistry is reported. Magnetization measurements indicated that neither the encapsulation of the iron oxide particles into silica nor the grafting of the glycocopolymer chains had a significant influence on the superparamagnetic properties. This not only simplifies the purification of the particles but may facilitate the use of the particles in applications such as hyperthermia or magnetic resonance imaging (MRI). Furthermore, the hydrophilic glycopolymer shell provided solubility of the particles in aqueous medium and enabled the uptake of the particles into the cytoplasm and nucleus of lung cancer cells via carbohydrate-lectin recognition effects.     

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH ^−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH ^−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH ^−/− iPS cell lines, we aggregated FAH ^−/−-iPS cells with tetraploid embryos and obtained entirely FAH ^−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH ^−/− mice. Then, we transduced FAH cDNA into the FAH ^−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.     

Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry

In metastasis, the cancer cells that travel through the body are capable of establishing new tumors in locations remote from the site of the original disease. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. Here, we have used the "in vivo flow cytometer" to study if there is any relationship between metastatic potential and depletion kinetics of circulating tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labelled cells in vivo. We have improved the counting algorithm and measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC-3 prostate cancer cells are depleted faster from the circulation than LNCaP cells. In addition, we have measured the depletion kinetics of two related human hepatocellular carcinoma (liver cancer) cell lines, high-metastatic HCCLM3 cells, and low-metastatic HepG2 cells. More than 60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.     

Transcatheter aortic valve implantation and hybrid coronary artery revascularization: time to combine them

We report on simultaneous off-pump coronary artery bypass grafting to the left anterior descending artery, modified transapical aortic valve implantation, and stenting of the circumflex and right coronary arteries in an 84-year-old patient. The poly-morbid patient with a logistic EuroSCORE of 85% experienced recent myocardial infarction; the left ventricular ejection fraction was reduced to 20%. Postoperative recovery was fast and short. The strategy described is the next logical step in broadening the indication for transcatheter aortic valve interventions.