A sex-chromosome mutation in Silene latifolia

Silene latifolia is dioecious, yet rare hermaphrodites have been found, and such natural mutants can provide valuable insight into genetic mechanisms. Here, we describe a hermaphrodite-inducing mutation that is almost certainly localized to the gynoecium-suppression region of the Y chromosome in S. latifolia. The mutant Y chromosome was passed through the megaspore, and the presence of two X chromosomes was not necessary for seed development in the parent. This result supports a lack of degeneration of the Y chromosome in S. latifolia, consistent with the relatively recent formation of the sex chromosomes in this species. When crossed to wild-type plants, hermaphrodites performed poorly as females, producing low seed numbers. When hermaphrodites were pollen donors, the sex ratio of offspring they produced through crosses was biased towards females. This suggests that hermaphroditic S. latifolia would fail to thrive and potentially explains the rarity of hermaphrodites in natural populations of S. latifolia. These results indicate that the Y chromosome in Silene latifolia remains very similar to the X, perhaps mostly differing in the primary sex determination regions

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

Background: The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls

Three new chondrosarcoma cell lines: one grade III conventional central chondrosarcoma and two dedifferentiated chondrosarcomas of bone

BackgroundChondrosarcoma is the second most common primary sarcoma of bone. High-grade conventional chondrosarcoma and dedifferentiated chondrosarcoma have a poor outcome. In pre-clinical research aiming at the identification of novel treatment targets, the need for representative cell lines and model systems is high, but availability is scarce.MethodsWe developed and characterized three cell lines, derived from conventional grade III chondrosarcoma (L835), and dedifferentiated chondrosarcoma (L2975 and L3252) of bone. Proliferation and migration were studied and we used COBRA-FISH and array-CGH for karyotyping and genotyping. Immunohistochemistry for p16 and p53 was performed as well as TP53 and IDH mutation analysis. Cells were injected into nude mice to establish their tumorigenic potential.ResultsWe show that the three cell lines have distinct migrative properties, L2975 had the highest migration rate and showed tumorigenic potential in mice. All cell lines showed chromosomal rearrangements with complex karyotypes and genotypic aberrations were conserved throughout late passaging of the cell lines. All cell lines showed loss of CDKN2A, while TP53 was wild type for exons 5–8. L835 has an IDH1 R132C mutation, L2975 an IDH2 R172W mutation and L3252 is IDH wild type.ConclusionsBased on the stable culturing properties of these cell lines and their genotypic profile resembling the original tumors, these cell lines should provide useful functional models to further characterize chondrosarcoma and to evaluate new treatment strategies

Demographic responses of Daphnia magna fed transgenic Bt-maize

The food/feed quality of a variety of genetically modified (GM) maize expressing Cry1Ab Bt-toxin was tested over the life-cycle of Daphnia magna, an arthropod commonly used as model organism in ecotoxicological studies. Demographic responses were compared between animals fed GM or unmodified (UM) near isogenic maize, with and without the addition of predator smell. Age-specific data on survival and birth rates were integrated and analysed using life tables and Leslie matrices. Survival, fecundity and population growth rate (PGR) data generally disfavoured transgenic Bt-maize as feed for D. magna compared to animals fed the unmodified (UM) near isogenic line of maize. Decomposition of age-specific effects revealed that the most important contributions to a reduced PGR in the GM-fed group came from both fecundity and survival differences early in life. We conclude that juvenile and young adult stages are the most sensitive experimental units and should be prioritized in future research. These stages are often omitted in toxicological/ecotoxicological studies and in feeding trials

Effects of cell seeding density on real-time monitoring of anti-proliferative effects of transient gene silencing

WOS: 000390112500001PubMed ID: 27981039Background: Real-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the realtime changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well(-1), respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h. Results: TRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well(-1) seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well(-1) density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well(-1) for 72 h. Conclusions: Data suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells.Scientific and Technological Research Council of Turkey (TUBITAK Research Project)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108S072]; Novartis (Turkey)Novartis; Research Infrastructure Project, The State Planning Organization of Turkey (DPT) [2009K120640]This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK Research Project, 108S072) and Novartis (Turkey) to MT. The xCELLigence system was purchased within the Research Infrastructure Project, The State Planning Organization of Turkey (DPT, 2009K120640)

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization

RAPD-PCR as a rapid approach for the evaluation of genotoxin-induced damage to bacterial DNA

RAPD PCR is a sensitive and reliable approach useful for the detection of DNA lesions due to environmental contaminants. In addition, this method is cost-effective, and can be performed in any laboratory having a DNA thermocycler and gel electrophoresis system. Here, we describe its application to identify genotoxin-induced DNA damage in foodborne bacteria. DNA alterations are detected through the analysis of electrophoresis profiles with the appearance or disappearance of new bands as compared to the non-mutated control. The described RAPD PCR procedure takes 6 h for completion. It uses small amounts of DNA and can reveal even low mutation rates