LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues

<p>Abstract</p> <p>Background</p> <p>Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium.</p> <p>Findings</p> <p>We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids.</p> <p>Conclusions</p> <p>The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.</p

LeishIF3d is a non-canonical cap-binding protein in Leishmania

Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the pre-initiation complex at the 5′ end of mRNAs driving translation initiation. The genome of Leishmania encodes a large repertoire of cap-binding complexes that fulfill a variety of functions possibly involved in survival along the life cycle. However, most of these complexes function in the promastigote life form that resides in the sand fly vector and decrease their activity in amastigotes, the mammalian life form. Here we examined the possibility that LeishIF3d drives translation in Leishmania using alternative pathways. We describe a non-canonical cap-binding activity of LeishIF3d and examine its potential role in driving translation. LeishIF3d is required for translation, as reducing its expression by a hemizygous deletion reduces the translation activity of the LeishIF3d(+/−) mutant cells. Proteomic analysis of the mutant cells highlights the reduced expression of flagellar and cytoskeletal proteins, as reflected in the morphological changes observed in the mutant cells. Targeted mutations in two predicted alpha helices diminish the cap-binding activity of LeishIF3d. Overall, LeishIF3d could serve as a driving force for alternative translation pathways, although it does not seem to offer an alternative pathway for translation in amastigotes

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E–eIF4G interactions

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m7GTP cap structure at the 5′-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m7GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X4)Lϕ], where X is any amino acid and Φ is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Φ) is dispensable. The LeishIF4E–LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E–eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery

In the Hunt for Therapeutic Targets: Mimicking the Growth, Metastasis, and Stromal Associations of Early-Stage Lung Cancer Using a Novel Orthotopic Animal Model

BackgroundThe existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor–stromal cell interactions under conditions that reflect early-stage lung cancer.MethodsUnlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment.ResultsWe successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment.ConclusionOur orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor–stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells

A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways

In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways

Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D(high) or NK30(high) donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities

“Not Unsympathetic”: Freud’s Overlooked 1920 Case on the Female Homosexuality of Margarethe Csonka (1900-1999)

Sigmund Freud’s case studies, dedicated to the analysis of the histories of individual patients, are among his most well-known and well-researched writings. The literature on Freud usually notes the cases of Dora (1905), Little Hans (1909), Rat Man (1909), Dr. Daniel Schreber (1911), and Wolf Man (1918). However, his sixth and last case, written in 1920 and dedicated to homosexuality in a young woman, has received much less attention among psychoanalytic scholars, and undoubtedly less among historians. This was despite the fact that this four-month-long treatment of Margarethe Csonka, a girl who fell in love with another woman ten years older, was the only explicit instance of female homosexuality that Freud analyzed. Historians of sexuality repeatedly focus on Freud’s writings on male homosexuality, largely omitting the study of his views on female homosexuality. In what follows, I highlight why we should pay more attention to this paper and see it as part of the history of sexuality; and why, with revealed new details on the young woman in modern Vienna and its assimilated Jewish life, we can gain entirely new insights about the context of the paper and this patient’s world beyond the scientific discourse. The first part of this article analyzes Freud’s text methodically and historically in order to examine what kind of Freud Margarethe Csonka met as a young female patient. I highlight the radicalism of the text, discussing female sexuality beyond reproduction in light of Freud’s other writings and in view of the work of other medical men during the interwar period. The second part of the article juxtaposes Freud’s narrative with that of Margarethe Csonka who rejected the language of scientia sexualis and experienced same-sex love outside the scientific-medical purview. I will suggest that beyond drawing on models of heterosexual romantic love, she effectively utilized other resources as she took advantage of new possibilities that opened up for women and assimilated Jews through emancipation and the modernization of urban life. Vienna and its dazzling, new, modern architecture and culture was an active agent in her affair. The city was very much part of the one-sided love story that was set against the background of Jewish life in the early twentieth century during a shift between increasing gender and ethnic equality and rising anti-Semitism. &nbsp