“Bouquet arrest”, monopolar chromosomes segregation, and correction of the abnormal spindle

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F₁). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality—monopolar chromosome orientation—is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.     

The Diversity of Karyotypes and Genomes within Section Syllinum of the Genus Linum (Linaceae) Revealed by Molecular Cytogenetic Markers and RAPD Analysis

The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.     

Inhibition of Pediatric Glioblastoma Tumor Growth by the Anti-Cancer Agent OKN-007 in Orthotopic Mouse Xenografts

Pediatric glioblastomas (pGBM), although rare, are one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. Here, we describe the use of conventional and advanced in vivo magnetic resonance imaging (MRI) techniques to assess a novel orthotopic xenograft pGBM mouse (IC-3752GBM patient-derived culture) model, and to monitor the effects of the anti-cancer agent OKN-007 as an inhibitor of pGBM tumor growth. Immunohistochemistry support data is also presented for cell proliferation and tumor growth signaling. OKN-007 was found to significantly decrease tumor volumes (p<0.05) and increase animal survival (p<0.05) in all OKN-007-treated mice compared to untreated animals. In a responsive cohort of treated animals, OKN-007 was able to significantly decrease tumor volumes (p<0.0001), increase survival (p<0.001), and increase diffusion (p<0.01) and perfusion rates (p<0.05). OKN-007 also significantly reduced lipid tumor metabolism in responsive animals [(Lip1.3 and Lip0.9)-to-creatine ratio (p<0.05)], as well as significantly decrease tumor cell proliferation (p<0.05) and microvessel density (p<0.05). Furthermore, in relationship to the PDGFRα pathway, OKN-007 was able to significantly decrease SULF2 (p<0.05) and PDGFR-α (platelet-derived growth factor receptor-α) (p<0.05) immunoexpression, and significantly increase decorin expression (p<0.05) in responsive mice. This study indicates that OKN-007 may be an effective anti-cancer agent for some patients with pGBMs by inhibiting cell proliferation and angiogenesis, possibly via the PDGFRα pathway, and could be considered as an additional therapy for pediatric brain tumor patients.     

IL-17 in spondyloarthritis: is the T-party over?

The past decade has witnessed significant progress in revealing an important role for IL-17 in the pathogenesis of several immune-mediated inflammatory diseases. Recent studies have provided new insights into the cellular source of IL-17, originally identified as the signature cytokine of a distinct CD4⁺ T-cell subset known as Th17. Accumulating evidence suggests that the majority of the IL-17 released in inflammatory arthritis is produced by innate immune cells rather than T cells. Understanding molecular mechanisms behind these early innate immune responses will be the key to designing rational therapies targeting these important inflammatory pathways.     

A prokaryotic S1P lyase degrades extracellular S1P in vitro and in vivo: implication for treating hyperproliferative disorders

Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling.     

Epoxyeicosatrienoic Acids (EETs) Regulate Epithelial Sodium Channel Activity by Extracellular Signal-regulated Kinase 1/2 (ERK1/2)-mediated Phosphorylation

The epithelial sodium channel (ENaC) participates in the regulation of plasma sodium and volume, and gain of function mutations in the human channel cause salt-sensitive hypertension. Roles for the arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), in ENaC activity have been identified; however, their mechanisms of action remain unknown. In polarized M1 cells, 14,15-EET inhibited amiloride-sensitive apical to basolateral sodium transport as effectively as epidermal growth factor (EGF). The EET effects were associated with increased threonine phosphorylation of the ENaC β and γ subunits and abolished by inhibitors of (a) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal regulated kinases 1 and 2 (MEK/ERK1/2) and (b) EGF receptor signaling. CYP2C44 epoxygenase knockdown blunted the sodium transport effects of EGF, and its 14,15-EET metabolite rescued the knockdown phenotype. The relevance of these findings is indicated by (a) the hypertension that results in mice administered cetuximab, an inhibitor of EGF receptor binding, and (b) immunological data showing an association between the pressure effects of cetuximab and reductions in ENaCγ phosphorylation. These studies (a) identify an ERK1/2-dependent mechanism for ENaC inhibition by 14,15-EET, (b) point to ENaC as a proximal target for EET-activated ERK1/2 mitogenic kinases, (c) characterize a mechanistic commonality between EGF and epoxygenase metabolites as ENaC inhibitors, and (d) suggest a CYP2C epoxygenase-mediated pathway for the regulation of distal sodium transport.     

Voluntary Running in Young Adult Mice Reduces Anxiety-Like Behavior and Increases the Accumulation of Bioactive Lipids in the Cerebral Cortex

Combinatorial therapies using voluntary exercise and diet supplementation with polyunsaturated fatty acids have synergistic effects benefiting brain function and behavior. Here, we assessed the effects of voluntary exercise on anxiety-like behavior and on total FA accumulation within three brain regions: cortex, hippocampus, and cerebellum of running versus sedentary young adult male C57/BL6J mice. The running group was subjected to one month of voluntary exercise in their home cages, while the sedentary group was kept in their home cages without access to a running wheel. Elevated plus maze (EPM), several behavioral postures and two risk assessment behaviors (RABs) were then measured in both animal groups followed immediately by blood samplings for assessment of corticosterone levels. Brains were then dissected for non-targeted lipidomic analysis of selected brain regions using gas chromatography coupled to mass spectrometry (GC/MS). Results showed that mice in the running group, when examined in the EPM, displayed significantly lower anxiety-like behavior, higher exploratory and risky behaviors, compared to sedentary mice. Notably, we found no differences in blood corticosterone levels between the two groups, suggesting that the different EPM and RAB behaviors were not related to reduced physiological stress in the running mice. Lipidomics analysis revealed a region-specific cortical decrease of the saturated FA: palmitate (C16:0) and a concomitant increase of polyunsaturated FA, arachidonic acid (AA, omega 6-C20: 4) and docosahexaenoic acid (DHA, omega 3-C22: 6), in running mice compared to sedentary controls. Finally, we found that running mice, as opposed to sedentary animals, showed significantly enhanced cortical expression of phospholipase A2 (PLA₂) protein, a signaling molecule required in the production of both AA and DHA. In summary, our data support the anxiolytic effects of exercise and provide insights into the molecular processes modulated by exercise that may lead to its beneficial effects on mood.     

Species distinctions among closely related strains of Eustigmatophyceae (Stramenopiles) emphasizing ITS2 sequence-structure data: Eustigmatos and Vischeria

ABSTRACT

There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.