Unexpected role of sterol synthesis in RNA stability and translation in Leishmania

Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike animals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14-demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target. In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis in Leishmania plays an unexpected role in global gene regulation

Exploiting Molecular Symmetry Reduction to Enrich Liquid Crystal Phase Diversity

The strategic tuning of liquid crystalline phase behaviour by adjusting molecular symmetry was investigated. A family of sixteen symmetrical and unsymmetrical 2,6-di(4’-n-alkoxybenzoyloxy) naphthalene derivatives were prepared and their liquid crystal properties examined by differential scanning calorimetry, polarised optical microscopy, and x-ray diffraction. All mesogens formed nematic phases, with longer-chain analogues also exhibiting smectic C phases at lower temperatures. Melting temperatures of the compounds strongly depend on molecular symmetry, whereas clearing transitions are relatively insensitive to this effect. A detailed analysis indicates that the clearing point can be predicted based on the nature of the terminal alkyl chains, with only a secondary effect from molecular symmetry. Moreover, low symmetry molecules showed a greater tendency to form smectic C phases, which was ascribed to the selective depression of the melting point versus the SmC-N transition. This demonstrates that molecular symmetry-breaking is a valuable tool both for tuning liquid crystalline phase range and for increasing a material’s polymorphism

Значение предсуществующей лекарственной устойчивости, обусловленной гиперэкспрессией Р-гликопротеина, для формирования резистентности к бортезомибу

Objective of the study. In our work we investigated the effect of pre-existing drug resistance by the mechanism of activation of ABC transporters – P-glycoprotein (Pgp) overexpression – on the development of resistance to the proteasome inhibitor bortezomib.Materials and methods. Cultures RPMI8226 and K562 / i-S9 (with Pgp overexpression) and their bortezomib-resistant sublines RPMI8226 / btz-6 and K562 / i-S9vlc were used as models. The methods used were MTT test, flow cytometry, Western blot and real-time polymerase chain reaction using the Human Signal Transduction Pathway Finder system.Results. The expression of the main PI3K-AKT and NF-κB signaling pathways did not change in RPMI8226 / btz-6 subline cells. However, AKT kinase expression was significantly increased and PTEN protein expression was reduced in K562 / i-S9vlc cells with Pgp-overexpression. Significant changes in gene expression (42 %) were found in RPMI8226 / btz-6 cells related to a number of main signaling pathways in the tumor cell, namely: activation of 3–4 genes in signaling pathways related to hypoxia, oxidative stress, PPAR and p53. The highest activation in these cells was found in the TGFβ signaling pathway. In resistant K562 / i-S9vlc cells, expression of only 5 genes (10 %) increased: Fas, HMOX1, CPT2, ICAM, and SOCS3. Three genes were also identified that changed in both resistant sublines: Fas, HMOX1 and CPT2. Further, we showed that in the RPMI8226 / btz-6 subline, along with changes in the expression of signal transduction genes, there is a large pool of CD138-negative cells, and in the K562 / i-S9vlc subline, the number of cells expressing CD34 increases and the number of CD13 decreases.Conclusion. We found that different signaling pathways are involved in the formation of resistance to bortezomib in the absence of Pgp expression and its overexpression. In addition, a cell line without activated resistance pathways requires more extensive rearrangements in the signal system to acquire resistance to bortezomib. However, in both cases, bortezomib leads to a change in the immunophenotype of the cells – to the appearance of dedifferentiated subpopulations.Цель исследования – изучить влияние предсуществующей лекарственной устойчивости по механизму активации АВС-транспортеров – гиперэкспрессии Р-гликопротеина (Pgp) – на пути развития резистентности к протеасомному ингибитору бортезомибу.Материалы и методы. В качестве моделей использовали культуры RPMI8226 и K562 / i-S9 (с гиперэкспрессией Pgp) и их резистентные к бортезомибу сублинии RPMI8226 / btz-6 и K562 / i-S9vlc. Применяли методы МТТ-теста, проточной цитометрии, вестерн-блоттинга и полимеразной цепной реакции в реальном времени с использованием системы Human Signal Transduction Pathway Finder.Результаты. В клетках сублинии RPMI8226 / btz-6 не изменялась экспрессия основных белков PI3K-AKT и NF-κB-сигнальных путей. Однако в клетках сублинии K562 / i-S9vlc с гиперэкспрессией Pgp значительно повышалась экспрессия AKT-киназы и снижалась экспрессия белка PTEN. В клетках сублинии RPMI8226 / btz-6 были найдены значительные изменения в экспрессии генов (42 %), относящихся к ряду основных сигнальных путей в опухолевой клетке, а именно активация 3–4 генов в сигнальных путях, отно сящихся к гипоксии, оксидативному стрессу, PPAR и p53. Наибольшая активация в этих клетках обнаружена в TGFβ-сигнальном пути. В устойчивых клетках K562 / i-S9vlc усилилась экспрессия только 5 генов (10 %): Fas, HMOX1, CPT2, ICAM и SOCS3. Также были выделены 3 гена, экспрессия которых изменилась в обеих устойчивых сублиниях: Fas, HMOX1 и CPT2. Далее мы показали, что в сублинии RPMI8226 / btz-6, наряду с изменениями экспрессии генов сигнальной трансдукции, присутствует большой пул CD138-негативных клеток, а в сублинии K562 / i-S9vlc повышается количество клеток, экспрессирующих СD34, и снижается количество CD13.Заключение. Мы выявили, что в формировании устойчивости к бортезомибу в клетках с отсутствием экспрессии Pgp и в клетках с гиперэкспрессией этого белка задействованы разные сигнальные пути. Кроме этого, клеточной линии без активированных путей устойчивости необходимы более масштабные перестройки в сигнальной системе для приобретения резистентности к бортезомибу. Однако и в том и в другом случае бортезомиб приводит к изменению иммунофенотипа клеток – к появлению дедифференцированных субпопуляций

Germline-restricted chromosome (GRC) is widespread among songbirds

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages

Genes flanking Xist in mouse and human are separated on the X chromosome in American marsupials

X inactivation, the transcriptional silencing of one of the two X chromosomes in female mammals, achieves dosage compensation of X-linked genes relative to XY males. In eutherian mammals X inactivation is regulated by the X-inactive specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing of the chromosome from which it is transcribed. Marsupial mammals also undergo X inactivation but the mechanism is relatively poorly understood. We set out to analyse the X chromosome in Monodelphis domestica and Didelphis virginiana, focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes that in eutherians flank the Xist locus. The synteny of this region is retained on chicken chromosome 4 where other loci belonging to the evolutionarily ancient stratum of the human X chromosome, the so-called X conserved region (XCR), are also located. We show that in both M. domestica and D. virginiana an evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3 gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the evolutionary precursor of Tsx, a gene located immediately downstream of Xist in eutherians. We discuss these findings in relation to the evolution of Xist and X inactivation in mammals

Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers

Pattern recognition underpins innate immunity; the accurate identification of danger, including infection, injury, or tumor, is key to an appropriately targeted immune response. Pathogen detection is increasingly well defined mechanistically, but the discrimination of endogenous inflammatory triggers remains unclear. Tenascin-C, a matrix protein induced upon tissue damage and expressed by tumors, activates toll-like receptor 4 (TLR4)-mediated sterile inflammation. Here we map three sites within tenascin-C that directly and cooperatively interact with TLR4. We also identify a conserved inflammatory epitope in related proteins from diverse families, and demonstrate that its presence targets molecules for TLR detection, while its absence enables escape of innate immune surveillance. These data reveal a unique molecular code that defines endogenous proteins as inflammatory stimuli by marking them for recognition by TLRs

Low-Dosage Inhibition of DII4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing