Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

Effect of Phosphorylation on EGFR Dimer Stability Probed by Single-Molecule Dynamics and FRET/FLIM

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (k_off). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the k_off of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.     

Acceptability of Mini-Tablets in Young Children: Results from Three Prospective Cross-over Studies

To ensure optimal, reliable treatment, it is necessary to investigate the efficacy, safety and the optimal dose of drug substances and to develop suitable age-specific pharmaceutical formulations for the different paediatric age groups due to a lack of evidence-based therapeutic options for children. While WHO recommends the use of solid dosage forms in general, European Medicines Agency (EMA) requires evidence for the suitability of these dosage forms in the targeted age group. This review aims to summarize and discuss the data obtained in acceptability studies on the suitability of coated and uncoated mini-tablets in children of different ages in comparison to a sweet syrup considered as gold standard. The predefined outcome parameters ‘acceptability’ and ‘capability to swallow’ of the two different mini-tablet formulations (uncoated and film-coated) were statistically significantly higher than that of the syrup.     

Using antagonistic soil bacteria and their cell‐free filtrates to control the black rot pathogen Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacteria, and it is the causative agent of black rot in crucifers. Recent studies have shown that Bacillus species have strong biological control on Xanthomonas. One of the mechanisms of this control is secondary metabolites production. A collection of 257 bacteria isolated from a suppressive soil was evaluated for in vitro antagonistic activity against X. campestris, and 92 isolates (44.6%) were able to inhibit its growth. Among the 92 isolates evaluated in the double‐layer technique, 51 (55.43%) inhibited Xcc growth on the inhibition tests with cell‐free filtrates (CFF) in liquid medium. Thirteen of these isolates presented 50% or more growth inhibition, and five isolates presented 100% growth inhibition of Xcc. The CFF of the isolate TCDT‐08, which belongs to the Paenibacillus genus, was used for in vivo tests with kale crops. The artificial inoculation of kale with Xcc‐629IBSBF pretreated with CFF from the isolate TCDT‐08 demonstrated that the bacterium loses the ability of colonizing kale and of causing black rot. A Paenibacillus sp. isolate has strong inhibitory activity against X. campestris pv. campestris, and further studies can result in the use of this isolate to protect kale from Xcc infection.     

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

The burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post-treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi-irreversible inhibitor of cinnamate 4-hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4-coenzyme A ligase) to 60-day-old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin-removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.     

Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

Background

The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively.

Methodology/Principal Findings

In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-α, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production.

Conclusions/Significance

The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-α, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88^−/− mice.     

High Levels of Heterogeneity in the HIV Cascade of Care across Different Population Subgroups in British Columbia,Canada

Background

The HIV cascade of care (cascade) is a comprehensive tool which identifies attrition along the HIV care continuum. We executed analyses to explicate heterogeneity in the cascade across key strata, as well as identify predictors of attrition across stages of the cascade.

Methods

Using linked individual-level data for the population of HIV-positive individuals in BC, we considered the 2011 calendar year, including individuals diagnosed at least 6 months prior, and excluding individuals that died or were lost to follow-up before January 1^st, 2011. We defined five stages in the cascade framework: HIV ‘diagnosed’, ‘linked’ to care, ‘retained’ in care, ‘on HAART’ and virologically ‘suppressed’. We stratified the cascade by sex, age, risk category, and regional health authority. Finally, multiple logistic regression models were built to predict attrition across each stage of the cascade, adjusting for stratification variables.

Results

We identified 7621 HIV diagnosed individuals during the study period; 80% were male and 5% were <30, 17% 30–39, 37% 40–49 and 40% were ≥50 years. Of these, 32% were MSM, 28% IDU, 8% MSM/IDU, 12% heterosexual, and 20% other. Overall, 85% of individuals ‘on HAART’ were ‘suppressed’; however, this proportion ranged from 60%–93% in our various stratifications. Most individuals, in all subgroups, were lost between the stages: ‘linked’ to ‘retained’ and ‘on HAART’ to ‘suppressed’. Subgroups with the highest attrition between these stages included females and individuals <30 years (regardless of transmission risk group). IDUs experienced the greatest attrition of all subgroups. Logistic regression results found extensive statistically significant heterogeneity in attrition across the cascade between subgroups and regional health authorities.

Conclusions

We found that extensive heterogeneity in attrition existed across subgroups and regional health authorities along the HIV cascade of care in B.C., Canada. Our results provide critical information to optimize engagement in care and health service delivery.