Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.     

The cellular prion protein and its role in Alzheimer disease

The cellular prion protein (PrP^C) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrP^Sc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrP^C is needed for the establishment and further evolution of prions.The present work compares the expression and localization of PrP^C between healthy human brains and those suffering from Alzheimer disease (AD).In both situations we have observed a rostrocaudal decrease in the amount of PrP^C within the CNS, both by immunoblotting and immunohistochemistry techniques. PrP^C is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrP^C in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands.With regards to amyloid plaques, those present in AD cases were positively labeled for PrP^C, a result which is further supported by the presence of PrP^C in the amyloid plaques of a transgenic line of mice mimicking AD.The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.Key words: cellular prion protein, Alzheimer disease, transgenic mice     

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.     

Mesenchymal stem cells transmigrate over the endothelial barrier

Mesenchymal stem cells (MSCs) seem to be a useful tool for cellular therapy in injured tissues, e.g. myocardial infarction or cardiomyopathies resulting in heart failure. For therapeutic approaches it is crucial that MSCs cross the endothelial barrier especially in intravascular or rather intracoronary application. Until today little is known about MSCs transmigrating across the endothelium. We performed co-culture experiments of MSCs on an endothelial monolayer to analyse direct interactions. An increasing flattened morphology of the MSCs was followed by a total integration into the monolayer after 2h. We repeated these experiments in isolated heart perfusions with gold-labelled MSCs. Using electron microscopy we detected MSCs exhibited direct cell-cell contacts. Tight junctions between the endothelial cells became abolished resulting in a distinct split between the cells. MSCs developed tight cell-cell contacts and became integrated into the endothelial wall of the capillary vessel. Finally, using confocal laser scanning microscopy, we assessed the ability of the MSCs to fully pass the endothelial barrier. Within the first 30 min, 30+/-8% of MSCs transmigrated, increasing to about half at 60 min (50+/-8%), whereas after 120 min the rate remained nearly unchanged (53+/-10%). This work demonstrates the capability of MSCs for transendothelial migration. Moreover we showed that the vast majority of MSCs migrated within 30 min, an important finding for the exposure times in clinical settings.     

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.