Human synthetic lethal inference as potential anti-cancer target gene detection

Background: Two genes are called synthetic lethal (SL) if mutation of either alone is not lethal, but mutation of both leads to death or a significant decrease in organism's fitness. The detection of SL gene pairs constitutes a promising alternative for anti-cancer therapy. As cancer cells exhibit a large number of mutations, the identification of these mutated genes' SL partners may provide specific anti-cancer drug candidates, with minor perturbations to the healthy cells. Since existent SL data is mainly restricted to yeast screenings, the road towards human SL candidates is limited to inference methods. Results: In the present work, we use phylogenetic analysis and database manipulation (BioGRID for interactions, Ensembl and NCBI for homology, Gene Ontology for GO attributes) in order to reconstruct the phylogenetically-inferred SL gene network for human. In addition, available data on cancer mutated genes (COSMIC and Cancer Gene Census databases) as well as on existent approved drugs (DrugBank database) supports our selection of cancer-therapy candidates.Conclusions: Our work provides a complementary alternative to the current methods for drug discovering and gene target identification in anti-cancer research. Novel SL screening analysis and the use of highly curated databases would contribute to improve the results of this methodology.

Synthesis, binding affinity and SAR of new benzolactam derivatives as dopamine D3 receptor ligands

A series of new benzolactam derivatives was synthesized and the derivatives were evaluated for theiraffinities at the dopamine D1, D2, and D3 receptors. Some of these compounds showed high D2 and/orD3 affinity and selectivity over the D1 receptor. The SAR study of these compounds revealed structuralcharacteristics that decisively influenced their D2 and D3 affinities. Structural models of the complexesbetween some of the most representative compounds of this series and the D2 and D3 receptors wereobtained with the aim of rationalizing the observed experimental results. Moreover, selected compoundsshowed moderate binding affinity on 5-HT2A which could contribute to reducing the occurrence of extrapyramidalside effects as potential antipsychotics.

Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Oxidative folding of synthetic polypeptides S-protected as tert-butylthio derivatives.

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 degrees C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product.

Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct.

BACKGROUND: The circumsporozoite (CS) protein is a major malaria sporozoite surface antigen currently being considered as vaccine candidate. Plasmodium vivax CS (PvCS) protein comprises a dimorphic central repeat fragment flanked by conserved regions that contain functional domains involved in parasite invasion of host cells. The protein amino (N-terminal) flank has a cleavage region (region I), essential for proteolytic processing prior to parasite invasion of liver cells. METHODS: We have developed a 131-mer long synthetic polypeptide (LSP) named PvNR1R2 that includes the N-terminal flank and the two natural repeat variant regions known as VK210 and VK247. We studied the natural immune response to this region in human sera from different malaria-endemic areas and its immunogenicity in mice. RESULTS: PvNR1R2 was more frequently recognized by sera from Papua New Guinea (PNG) (83%) than by samples from Colombia (24%) when tested by ELISA. The polypeptide formulated in Montanide ISA51 adjuvant elicited strong antibody responses in both C3H and CB6F1 mice strains. Antibodies from immunized mice as well as affinity-purified human IgG reacted with native protein by IFA test. Moreover, mouse immune sera induced strong (90%) in vitro inhibition of sporozoite invasion (ISI) of hepatoma cell lines. CONCLUSIONS: These results encourage further studies in non-human primates to confirm the elicitation of sporozoite invasion blocking antibodies, to assess cell mediated immune responses and the protective efficacy of this polypeptide.

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Boosting of Replication Competent NYVAC Primed HIV-1-Specific T-Cell Responses by HIV Synthetic Long Peptides (SLP)

Background: To enhance the induction of insert specific immune responses, a new generation of replication competent poxvirus vectors was designed and evaluated against non-replicating poxvirus vectors in a HIV vaccine study in non human primates.Methods: Rhesus macaques were immunized with either the non-replicating variant NYVAC-GagPolNef HIV-1 clade C or the replicating NYVAC-GagPolNef-C-KC, boosted with HIVGag- PolEnv-SLP and immune responses were monitored.Results: Gag-specific T-cell responses were only detected in animals immunized with the replicating NYVAC-GagPolNef-C-KC variant. Further enhancement and broadening of the immune response was studied by boosting the animals with novel T-cell immunogens HIVconsv synthetic long peptides (SLP), which direct vaccine-induced responses to the most conserved regions of HIV and contain both CD4 T-helper and CD8 CTL epitopes. The adjuvanted (Montanide ISA-720) SLP divided into subpools and delivered into anatomically separate sites enhanced the Gag-specific T-cell responses in 4 out of 6 animals, to more than 1000 SFC/106 PBMC in some animals. Furthermore, the SLP immunization broadened the immune response in 4 out of 6 animals to multiple Pol epitopes. Even Env-specific responses, to which the animals had not been primed, were induced by SLP in 2 out of 6 animals.Conclusion: This new immunization strategy of priming with replicating competent poxvirus NYVAC-HIVGagPolNef and boosting with HIVGagPolEnv-SLP, induced strong and broad Tcell responses and provides a promising new HIV vaccine approach. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Structural joint inversion of time-lapse crosshole ERT and GPR traveltime data

Time-lapse geophysical monitoring and inversion are valuable tools in hydrogeology for monitoring changes in the subsurface due to natural and forced (tracer) dynamics. However, the resulting models may suffer from insufficient resolution, which leads to underestimated variability and poor mass recovery. Structural joint inversion using cross-gradient constraints can provide higher-resolution models compared with individual inversions and we present the first application to time-lapse data. The results from a synthetic and field vadose zone water tracer injection experiment show that joint 3-D time-lapse inversion of crosshole electrical resistance tomography (ERT) and ground penetrating radar (GPR) traveltime data significantly improve the imaged characteristics of the point injected plume, such as lateral spreading and center of mass, as well as the overall consistency between models. The joint inversion method appears to work well for cases when one hydrological state variable (in this case moisture content) controls the time-lapse response of both geophysical methods. Citation: Doetsch, J., N. Linde, and A. Binley (2010), Structural joint inversion of time-lapse crosshole ERT and GPR traveltime data, Geophys. Res. Lett., 37, L24404, doi: 10.1029/2010GL045482.

Integration of high-resolution geophysical data for the characterization of alluvial aquifers : new approaches and implications for hydrological predictions

Abstract Accurate characterization of the spatial distribution of hydrological properties in heterogeneous aquifers at a range of scales is a key prerequisite for reliable modeling of subsurface contaminant transport, and is essential for designing effective and cost-efficient groundwater management and remediation strategies. To this end, high-resolution geophysical methods have shown significant potential to bridge a critical gap in subsurface resolution and coverage between traditional hydrological measurement techniques such as borehole log/core analyses and tracer or pumping tests. An important and still largely unresolved issue, however, is how to best quantitatively integrate geophysical data into a characterization study in order to estimate the spatial distribution of one or more pertinent hydrological parameters, thus improving hydrological predictions. Recognizing the importance of this issue, the aim of the research presented in this thesis was to first develop a strategy for the assimilation of several types of hydrogeophysical data having varying degrees of resolution, subsurface coverage, and sensitivity to the hydrologic parameter of interest. In this regard a novel simulated annealing (SA)-based conditional simulation approach was developed and then tested in its ability to generate realizations of porosity given crosshole ground-penetrating radar (GPR) and neutron porosity log data. This was done successfully for both synthetic and field data sets. A subsequent issue that needed to be addressed involved assessing the potential benefits and implications of the resulting porosity realizations in terms of groundwater flow and contaminant transport. This was investigated synthetically assuming first that the relationship between porosity and hydraulic conductivity was well-defined. Then, the relationship was itself investigated in the context of a calibration procedure using hypothetical tracer test data. Essentially, the relationship best predicting the observed tracer test measurements was determined given the geophysically derived porosity structure. Both of these investigations showed that the SA-based approach, in general, allows much more reliable hydrological predictions than other more elementary techniques considered. Further, the developed calibration procedure was seen to be very effective, even at the scale of tomographic resolution, for predictions of transport. This also held true at locations within the aquifer where only geophysical data were available. This is significant because the acquisition of hydrological tracer test measurements is clearly more complicated and expensive than the acquisition of geophysical measurements. Although the above methodologies were tested using porosity logs and GPR data, the findings are expected to remain valid for a large number of pertinent combinations of geophysical and borehole log data of comparable resolution and sensitivity to the hydrological target parameter. Moreover, the obtained results allow us to have confidence for future developments in integration methodologies for geophysical and hydrological data to improve the 3-D estimation of hydrological properties.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Optimum Redundant Array Configurations for Earth Observation Aperture Synthesis Microwave Radiometers

Two-dimensional aperture synthesis radiometry is the technologyselected for ESA's SMOS mission to provide high resolution L-bandradiometric imagery. The array topology is a Y-shaped structure. Theposition and number of redundant elements to minimise instrumentdegradation in case of element failure(s) are studied.