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.     

Conditional Survival in de novo Metastatic Urothelial Carcinoma

Background

Second-line therapy is frequently utilized for metastatic urothelial carcinoma, but there are limited data to guide this approach. While an assessment of overall survival based on registry data may not capture the impact of second- and third-line therapies on clinical outcome, this may be reflected in relative conditional survival (RCS).

Methods

Patients with stage IV urothelial carcinoma diagnosed from 1990–2010 were identified from the Surveillance, Epidemiology and End Results (SEER) dataset. The association of clinicopathologic variables with disease specific survival (DSS) was explored through univariate and multivariate analyses. DSS in subgroups divided by time period (1990–2000 v 2001–2010) was compared using the Kaplan-Meier method and log-rank test. One-year RCS at annual landmarks up to 5 years was compared in subgroups divided by time period.

Results

Of 261,987 patients diagnosed with urothelial carcinoma from 1990–2010, 3,110 patients met criteria for the current analysis. Characteristics of patients diagnosed between 1990 and 2000 (n = 810) and 2001 to 2010 (n = 2,300) were similar and there was no significant difference in DSS between the two groups. On multivariate analysis, older age (age ≥ 80) was associated with shorter DSS (HR 1.79, 95%CI 1.48–2.15), but no association was found between time period of diagnosis and outcome. One-year RCS improved substantially through successive annual landmarks up to 5 years, but no differences were seen in subgroups divided by time of diagnosis.

Conclusions

No difference in RCS was observed amongst patients with stage IV urothelial carcinoma diagnosed from 1990–2000 and 2001–2010. A lack of difference in RCS (more so than cumulative DSS) may reflect a lack of progress in salvage therapies for the disease.     

Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence

In this study, the role of Listeria monocytogenes ferritin was investigated. The fri gene encoding the ferritin was deleted and the phenotype of the mutant was analyzed demonstrating that ferritin is necessary for optimal growth in minimal medium in both presence and absence of iron, as well as after cold- and heat-shock. We also showed that ferritin provides protection against reactive oxygen species and is essential for full virulence of L. monocytogenes. A comparative proteomic analysis revealed an effect of the fri deletion on the levels of listeriolysin O and several stress proteins. Together, our study demonstrates that fri has multiple roles that contribute to Listeria virulence.     

Mapping of mglB, the structural gene of the galactose-binding protein of Escherichia coli

Summary The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%–95%, 85%, 36%, 20%–40%, 12%–15%, and 0.5% contransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his. gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk.Neither zee-700::Tn10 nor zef-703::Tn10 showed any (0/300) contransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-702::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at-20°C, colonies could be isolated that carried only mutant or wild-type protein.     

Perceiving a Stranger's Voice as Being One's Own: A ‘Rubber Voice’ Illusion?

We describe an illusion in which a stranger's voice, when presented as the auditory concomitant of a participant's own speech, is perceived as a modified version of their own voice. When the congruence between utterance and feedback breaks down, the illusion is also broken. Compared to a baseline condition in which participants heard their own voice as feedback, hearing a stranger's voice induced robust changes in the fundamental frequency (F0) of their production. Moreover, the shift in F0 appears to be feedback dependent, since shift patterns depended reliably on the relationship between the participant's own F0 and the stranger-voice F0. The shift in F0 was evident both when the illusion was present and after it was broken, suggesting that auditory feedback from production may be used separately for self-recognition and for vocal motor control. Our findings indicate that self-recognition of voices, like other body attributes, is malleable and context dependent.     

Characterisation of source-separated household waste intended for composting

Large-scale composting of source-separated household waste has expanded in recent years in the Nordic countries. One problem can be low pH at the start of the process. Incoming biowaste at four composting plants was characterised chemically, physically and microbiologically. The pH of food waste ranged from 4.7 to 6.1 and organic acid concentration from 24 to 81 mmol kg(-1). The bacterial diversity in the waste samples was high, with all samples dominated by Gammaproteobacteria, particularly Pseudomonas and Enterobacteria (Escherichia coli, Klebsiella, Enterobacter). Lactic acid bacteria were also numerically important and are known to negatively affect the composting process because the lactic acid they produce lowers the pH, inhibiting other bacteria. The bacterial groups needed for efficient composting, i.e. Bacillales and Actinobacteria, were present in appreciable amounts. The results indicated that start-up problems in the composting process can be prevented by recycling bulk material and compost.     

Developmentally Arrested Precursors of Pontine Neurons Establish an Embryonic Blueprint of the Drosophila Central Complex

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Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.