Development of a BRET2 screening assay using beta-arrestin 2 mutants

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.     

Translational research in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are receiving unusual attention recently as great strides have been made in understanding the biology. Recognition that excessive cytokine-induced apoptosis plays a significant role in the cytopenias of the majority of patients opened the doors to anti-cytokine therapy, with thalidomide being used with success in approximately 20% patients. Other therapies that have emerged include the thalidomide analog lenalidomide which is particularly beneficial for 5q- patients as well as a subset of non-5q- patients with low or intermediate-1 risk MDS. Other targeted therapies include vitamins, agents that are cytoprotective, differentiation inducers, anti-angiogenic, or immune modulatory. In addition, inhibitors of proteasome, methylation, histone deacetylation, farnesylation, receptor tyrosine kinases, topoisomerase, and matrix mettaloproteinases have yielded encouraging responses in subsets of patients. Specific therapies have also been developed for genetic abnormalities that lead to fusion genes (TEL-PDGFR-beta, or FIP1L1-PDGFR-alpha), or abnormal proteins due to mutations/functional inactivation (FLT3), dysregulated expression (EVI-1). In a short span of ten years, the field has evolved from having no effective therapy to offer the majority of MDS patients save chemotherapy, to having one FDA approved drug, several on the way to approval, and a number of novel agents producing exciting clinical results. This chapter summarizes the novel targets and targeted therapies in the rapidly evolving therapeutic landscape of MDS.     

A novel thermodynamic relationship based on Kramers Theory for studying enzyme kinetics under high viscosity

In most studies of enzyme kinetics it has been found sufficient to use the classical Transition State Theory (TST) of Eyring and others. This theory was based on the solvent being an ideal dilute substance treated as a heat bath. However, enzymes found in organisms adapted to very low (psychrophiles) and very high (thermophiles) temperatures are also subjected to variable solute concentrations and viscosities. Therefore, the TST may not always be applicable to enzyme reactions carried out in various solvents with viscosities ranging from moderate to very high. There have been numerous advances in the theory of chemical reactions in realistic non-ideal solvents such as Kramers Theory. In this paper we wish to propose a modified thermodynamic equation, which have contributions from kcat, Km and the viscosity of the medium in which the enzyme reaction is occurring. These could be very useful for determining the thermodynamics of enzymes catalyzing reactions at temperature extremes in the presence of substrate solutions of different compositions and viscosities.     

Quantitative Risk Stratification of Oral Leukoplakia with Exfoliative Cytology

Exfoliative cytology has been widely used for early diagnosis of oral squamous cell carcinoma (OSCC). Test outcome is reported as “negative”, “atypical” (defined as abnormal epithelial changes of uncertain diagnostic significance), and “positive” (defined as definitive cellular evidence of epithelial dysplasia or carcinoma). The major challenge is how to properly manage the “atypical” patients in order to diagnose OSCC early and prevent OSCC. In this study, we collected exfoliative cytology data, histopathology data, and clinical data of normal subjects (n=102), oral leukoplakia (OLK) patients (n=82), and OSCC patients (n=93), and developed a data analysis procedure for quantitative risk stratification of OLK patients. This procedure involving a step called expert-guided data transformation and reconstruction (EdTAR) which allows automatic data processing and reconstruction and reveals informative signals for subsequent risk stratification. Modern machine learning techniques were utilized to build statistical prediction models on the reconstructed data. Among the several models tested using resampling methods for parameter pruning and performance evaluation, Support Vector Machine (SVM) was found to be optimal with a high sensitivity (median>0.98) and specificity (median>0.99). With the SVM model, we constructed an oral cancer risk index (OCRI) which may potentially guide clinical follow-up of OLK patients. One OLK patient with an initial OCRI of 0.88 developed OSCC after 40 months of follow-up. In conclusion, we have developed a statistical method for qualitative risk stratification of OLK patients. This method may potentially improve cost-effectiveness of clinical follow-up of OLK patients, and help design clinical chemoprevention trial for high-risk populations.     

The Relationship between RTS,S Vaccine-Induced Antibodies,CD4+ T Cell Responses and Protection against Plasmodium falciparum Infection

Vaccination with the pre-erythrocytic malaria vaccine RTS,S induces high levels of antibodies and CD4⁺ T cells specific for the circumsporozoite protein (CSP). Using a biologically-motivated mathematical model of sporozoite infection fitted to data from malaria-naive adults vaccinated with RTS,S and subjected to experimental P. falciparum challenge, we characterised the relationship between antibodies, CD4⁺ T cell responses and protection from infection. Both anti-CSP antibody titres and CSP-specific CD4⁺ T cells were identified as immunological surrogates of protection, with RTS,S induced anti-CSP antibodies estimated to prevent 32% (95% confidence interval (CI) 24%–41%) of infections. The addition of RTS,S-induced CSP-specific CD4⁺ T cells was estimated to increase vaccine efficacy against infection to 40% (95% CI, 34%–48%). This protective efficacy is estimated to result from a 96.1% (95% CI, 93.4%–97.8%) reduction in the liver-to-blood parasite inoculum, indicating that in volunteers who developed P. falciparum infection, a small number of parasites (often the progeny of a single surviving sporozoite) are responsible for breakthrough blood-stage infections.     

Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium

Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.     

Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals

Commensal rodent species are key reservoirs for Toxoplasma gondii in the domestic environment. In rodents, different T. gondii strains show variable patterns of virulence according to host species. Toxoplasma gondii strains causing non-lethal chronic infections in local hosts will be more likely to persist in a given environment, but few studies have addressed the possible role of these interactions in shaping the T. gondii population structure. In addition, the absence of validated techniques for upstream detection of T. gondii chronic infection in wild rodents hinders exploration of this issue under natural conditions. In this study, we took advantage of an extensive survey of commensal small mammals in three coastal localities of Senegal, with a species assemblage constituted of both native African species and invasive species. We tested 828 individuals for T. gondii chronic infection using the modified agglutination test for antibody detection in serum samples and a quantitative PCR assay for detection of T. gondii DNA in brain samples. The infecting T. gondii strains were genotyped whenever possible by the analysis of 15 microsatellite markers. We found (i) a very poor concordance between molecular detection and serology in the invasive house mouse, (ii) significantly different levels of prevalence by species and (iii) the autochthonous T. gondii Africa 1 lineage strains, which are lethal for laboratory mice, only in the native African species of commensal small mammals. Overall, this study highlights the need to reconsider the use of MAT serology in natural populations of house mice and provides the first known data about T. gondii genetic diversity in invasive and native species of small mammals from Africa. In light of these results, we discuss the role of invasive and native species, with their variable adaptations to different T. gondii strains, in shaping the spatial structure of T. gondii genetic diversity in Africa.     

Antimicrobial and Cytotoxic Activities of Lepidium latifolium L. Hydrodistillate,Extract and Its Major Sulfur Volatile Allyl Isothiocyanate

The cultivated Lepidium latifolium L. was investigated to decipher its glucosinolate profile, antimicrobial, and cytotoxic activities. HPLC/ESI‐MS analyses of the intact glucosinolates and GC/MS analysis of their hydrolysis products showed the presence of sinigrin ( 1 ), glucocochlearin ( 2 ), glucotropaeolin ( 3 ), and 4‐methoxyglucobrassicin ( 4 ). Hydrodistillate, extract, and allyl isothiocyanate, the main volatile resulting from sinigrin degradation, showed antimicrobial activity against all eleven tested pathogenic and food spoilage bacteria and fungi, with highest effect observed against Candida albicans with MIC₅₀ 8 and 16 μg/mL. Hydrodistillate and extract showed the best cytotoxic activity on bladder cancer UM‐UC‐3 cell line during an incubation time of 24 h (IC₅₀ 192.9 and 133.8 μg/mL, respectively), while the best effect on glioblastoma LN229 cell line was observed after 48 h (IC₅₀ 110.8 and 30.9 μg/mL, respectively). Pure allyl isothiocyanate displayed a similar trend in cytotoxic effect on both cell lines (IC₅₀ 23.3 and 36.5 μg/mL after 24 h and 48 h, respectively).     

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.