Towards an In Vitro Model of Plasmodium Hypnozoites Suitable for Drug Discovery

Contains fulltext : 96475.pdf (publisher's version ) (Open Access)BACKGROUND: Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites. METHODOLOGY: P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured. RESULTS: The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine. CONCLUSION: Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs

Methods designed for the identification and characterization of in vitro and in vivo chromatin assembly mutants in Saccharomyces cerevisiae

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and in vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks

Andreev reflection spectroscopy of the pure and Cd-doped heavy-fermion superconductor CeCoIn5: Detecting order parameter symmetry and competing phases

Andreev reflection conductance spectra are obtained on nanoscale ballistic junctions between Au tips and single crystals of the pure and Cd-doped heavy-fermion superconductor CeCoIn5. Background conductance asymmetry starting at the heavy-fermion coherence temperature T* (∼45 K) and increasing with decreasing temperature down to Tc (2.3 K) signifies the emerging heavy-fermion liquid in CeCoIn5. Below Tc, enhancement of the sub-gap conductance arises from Andreev reflection, but the Blonder-Tinkham-Klapwijk theory dictates that the Fermi velocity mismatch should yield no Andreev reflection. The signal we do observe is several times weaker than that observed in conventional superconductors, but consistent with other heavy-fermion superconductor data reported. Data taken in the (0 0 1), (1 1 0), and (1 0 0) orientations provide consistent and reliable spectroscopic evidence for dx2 - y2 symmetry of the superconducting order parameter. Conductance spectra on the (1 0 0) surface of 10% Cd-doped CeCoIn5 show intriguing behaviors following antiferromagnetic and subsequent superconducting transitions

Point-contact spectroscopy of competing/coexisting orders in Cd-doped CeCoIn5

Differential conductance spectra are taken from metallic point-contact junctions on Cd-doped CeCoIn5. For (100) junctions with nominal 10% doping, we observe signatures for subsequent antiferromagnetic and superconducting transitions in qualitative agreement with bulk measurements. In the superconducting state, two conductance channels compete, both with enhanced subgap conductance, one due to superconductivity and the other antiferromagnetism. The conductance data exhibit variances for different doping levels and crystallographic orientations and junction to junction. This issue will be addressed further in terms of surface inhomogeneity or intrinsic nonuniform phase formation. © 2009 IOP Publishing Ltd

Point-contact spectroscopy of competing/coexisting orders in Cd-doped CeCoIn5

Differential conductance spectra are taken from metallic point-contact junctions on Cd-doped CeCoIn5. For (100) junctions with nominal 10% doping, we observe signatures for subsequent antiferromagnetic and superconducting transitions in qualitative agreement with bulk measurements. In the superconducting state, two conductance channels compete, both with enhanced subgap conductance, one due to superconductivity and the other antiferromagnetism. The conductance data exhibit variances for different doping levels and crystallographic orientations and junction to junction. This issue will be addressed further in terms of surface inhomogeneity or intrinsic nonuniform phase formation. © 2009 IOP Publishing Ltd

Hybridization gap and Fano resonance in SmB6.

Hybridization between conduction electrons and the strongly interacting f-electrons in rare earth or actinide compounds may result in new states of matter. Depending on the exact location of the concomitant hybridization gap with respect to the Fermi energy, a heavy fermion or an insulating ground state ensues. To study this entanglement locally, we conducted scanning tunneling microscopy and spectroscopy (STS) measurements on the "Kondo insulator" SmB6. The vast majority of surface areas investigated were reconstructed, but infrequently, patches of varying sizes of nonreconstructed Sm- or B-terminated surfaces also were found. On the smallest patches, clear indications for the hybridization gap with logarithmic temperature dependence (as expected for a Kondo system) and for intermultiplet transitions were observed. On nonreconstructed surface areas large enough for coherent cotunneling, we were able to observe clear-cut Fano resonances. Our locally resolved STS indicated considerable finite conductance on all surfaces independent of their structure, not proving but leaving open the possibility of the existence of a topologically protected surface state