Dynamical insight into <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> eIF4E recognition specificity for mono-and trimethylated structures of mRNA 5′ cap

Specific recognition and binding of the ribonucleic acid 5′ termini (mRNA 5′ cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting step in translation initiation. Contrary to mammalian and yeast eIF4Es that discriminate in favor of 7-methylguanosine cap, three out of five eIF4E isoforms from the nematode Caenorhabditis elegans as well as eIF4Es from the parasites Schistosome mansoni and Ascaris suum, exhibit dual binding specificity for both 7-methylguanosine-and N²,N²,7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N²,N²,7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N²-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs from a typical, static one in that the differences in the protein-ligand binding specificity cannot be ascribed to formation and/or disruption of well defined stabilizing contacts.     

Enhancement and optimization of plasmid expression in femtosecond optical transfection

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non‐invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3‐fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO‐K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ～70% without compromising the transfection efficiency. Also, we report for the first time the successful photo‐transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Increased intrathecal high-avidity anti-tau antibodies in patients with multiple sclerosis

Background

Antibodies against tau protein indicate an interaction between the immune system and the neurocytoskeleton and therefore may reflect axonal injury in multiple sclerosis (MS).

Methodology/Principal Findings

The levels and avidities of anti-tau IgG antibodies were measured using ELISA in paired cerebrospinal fluid (CSF) and serum samples obtained from 49 MS patients and 47 controls. Anti-tau antibodies were significantly elevated intrathecally (p<0.0001) in the MS group. The CSF anti-tau antibody levels were lower in MS patients receiving therapy than those without treatment (p<0.05). The avidities of anti-tau antibodies were higher in the CSF than in the serum (MS group p<0.0001; controls p<0.005). Anti-tau avidities in the CSF were elevated in MS patients in comparison with controls (p<0.05), but not in serum.

Conclusions

MS patients have higher levels of intrathecal anti-tau antibodies. Anti-tau antibodies have different avidities in different compartments with the highest values in the CSF of MS patients.     

Enhanced transduction and replication of RGD-fiber modified adenovirus in primary T cells

Background

Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD).

Methodology/Principal Finding

A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replication-competent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45% of splenic T cells were transduced by Ad-RGD.

Conclusions

Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary T cells of both murine and human origin.     

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b(+) cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b(+) cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.     

Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission

Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.     

Differential changes in GPR55 during microglial cell activation

We examined how lipopolysaccharide (LPS) and interferon gamma (IFN-γ), known to differentially activate microglia, affect the expression of G protein-coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV-2 mouse microglial cell line, and that LPS down-regulates this message. Conversely, IFN-γ slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV-2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV-2 stimulated with IFN-γ, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli-induced changes in GPR55 expression are similar to those observed with CB₂-R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.     

<Emphasis Type="Italic">LeFRK2</Emphasis> is required for phloem and xylem differentiation and the transport of both sugar and water

It has been suggested that LeFRK2, the major fructose-phosphorylating enzyme in tomato plants, may be required for stem xylem development. Yet, we do not know if this enzyme affects the development of individual vessels, whether it affects water conductance, or whether it affects phloem development and sugar transport. Here, we show that suppression of LeFRK2 results in a significant reduction in the size of vascular cells and slows fiber maturation. The vessels in stems of LeFRK2-antisense plants are narrower than in WT plants and have thinner secondary cell walls. Although the cambium produces rounded secondary vessels, these vessels become deformed during the early stages of xylem maturation. Water conductance is then reduced in stems, roots, and leaves, suggesting that LeFRK2 influences xylem development throughout the entire vascular system. Interestingly, the build-up of positive xylem pressure under static (no-flow) conditions was also decreased. Suppression of LeFRK2 reduced the length and width of the sieve elements, as well as callose deposition. To examine the effect of LeFRK2 suppression on phloem transport, we created triple-grafted plants in which a portion of the wild-type stem was replaced with an antisense interstcok, and compared the contents of the transported sugar, sucrose, in the different portions of these stems. Sucrose contents above and within the LeFRK2-antisense interstock were significantly higher than those below the graft. These results show that the antisense interstock restricted the downward movement of sucrose, suggesting that LeFRK2 is required for both phloem and xylem development. Contribution No. 114/2009 from the Volcani Center ARO.     

The mechanism of acidic hydrolysis of esters explains the HDV ribozyme activity

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme that catalyzes the site-specific trans-esterification reaction. Using high hydrostatic pressure (HHP) technique we showed that HDV ribozyme catalyzes the reaction of RNA cleavage in the absence of magnesium ions according to mechanism of acidic hydrolysis of esters. HHP induces changes of water structure, lowering pH and effect ribozyme catalytic site structure formation without magnesium. HHP, similarly to magnesium ion at ambient pressure stabilizes the higher order RNA structure of HDV, but Mg²⁺ is not involved in the catalysis. Our results clearly support the new mechanism of HDV hydrolysis and show advantages of using HHP in analysis of macromolecules interaction.