Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Dramatic mitochondrial gene rearrangements in the hermit crab Pagurus longicarpus (Crustacea, anomura)

The entire mitochondrial gene order of the crustacean Pagurus longicarpus was determined by sequencing all but approximately 300 bp of the mitochondrial genome. We report the first major gene rearrangements found in the clade including Crustacea and Insecta. At least eight mitochondrial gene rearrangements have dramatically altered the gene order of the hermit crab P. longicarpus relative to the putatively ancestral crustacean gene order. These include two rearrangements of protein-coding genes, the first reported for any nonchelicerate arthropod. Codon usage and amino acid sequences do not deviate substantially from those reported for other crustaceans. Investigating the phylogenetic distribution of these eight rearrangements will add additional characters to help resolve decapod phylogeny.     

Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

The aggregate site frequency spectrum for comparative population genomic inference

Understanding how assemblages of species responded to past climate change is a central goal of comparative phylogeography and comparative population genomics, an endeavour that has increasing potential to integrate with community ecology. New sequencing technology now provides the potential to perform complex demographic inference at unprecedented resolution across assemblages of nonmodel species. To this end, we introduce the aggregate site frequency spectrum (aSFS), an expansion of the site frequency spectrum to use single nucleotide polymorphism (SNP) data sets collected from multiple, co‐distributed species for assemblage‐level demographic inference. We describe how the aSFS is constructed over an arbitrary number of independent population samples and then demonstrate how the aSFS can differentiate various multispecies demographic histories under a wide range of sampling configurations while allowing effective population sizes and expansion magnitudes to vary independently. We subsequently couple the aSFS with a hierarchical approximate Bayesian computation (hABC) framework to estimate degree of temporal synchronicity in expansion times across taxa, including an empirical demonstration with a data set consisting of five populations of the threespine stickleback (Gasterosteus aculeatus). Corroborating what is generally understood about the recent postglacial origins of these populations, the joint aSFS/hABC analysis strongly suggests that the stickleback data are most consistent with synchronous expansion after the Last Glacial Maximum (posterior probability = 0.99). The aSFS will have general application for multilevel statistical frameworks to test models involving assemblages and/or communities, and as large‐scale SNP data from nonmodel species become routine, the aSFS expands the potential for powerful next‐generation comparative population genomic inference.     

Model misspecification confounds the estimation of rates and exaggerates their time dependency

While welcoming the comment of Ho et al. ( 2015 ), we find little that undermines the strength of our criticism, and it would appear they have misunderstood our central argument. Here we respond with the purpose of reiterating that we are (i) generally critical of much of the evidence presented in support of the time‐dependent molecular rate (TDMR) hypothesis and (ii) specifically critical of estimates of μ derived from tip‐dated sequences that exaggerate the importance of purifying selection as an explanation for TDMR over extended timescales. In response to assertions put forward by Ho et al. ( 2015 ), we use panmictic coalescent simulations of temporal data to explore a fundamental assumption for tip‐dated tree shape and associated mutation rate estimates, and the appropriateness and utility of the date randomization test. The results reveal problems for the joint estimation of tree topology, effective population size and μ with tip‐dated sequences using beast . Given the simulations, beast consistently obtains incorrect topological tree structures that are consistent with the substantial overestimation of μ and underestimation of effective population size. Data generated from lower effective population sizes were less likely to fail the date randomization test yet still resulted in substantially upwardly biased estimates of rates, bringing previous estimates of μ from temporally sampled DNA sequences into question. We find that our general criticisms of both the hypothesis of time‐dependent molecular evolution and Bayesian methods to estimate μ from temporally sampled DNA sequences are further reinforced.     

Lack of support for the time‐dependent molecular evolution hypothesis

There is increasing momentum surrounding the hypothesis that rates of molecular evolution between individuals within contemporary populations are high, and that these rates decrease as a function of time, perhaps over several millions of years, before reaching stationarity. The implications of this are powerful, potentially reshaping our view of how climate history impacts upon both species distribution patterns and the geographic structuring of genetic variation within species. However, our assessment of the hypothesis reveals a lack of theoretical support and empirical evidence for hypothesized magnitudes of time‐dependent rates of molecular evolution, with much of the apparent rate changes coming from artefacts and biases inherent in the methods of rate estimation. Our assessment also reveals a problem with how serial sampling is implemented for mutation rate estimation using ancient DNA samples, rendering published estimates unreliable.     

MTML-msBayes: Approximate Bayesian comparative phylogeographic inference from multiple taxa and multiple loci with rate heterogeneity

Background  

MTML-msBayes uses hierarchical approximate Bayesian computation (HABC) under a coalescent model to infer temporal patterns of divergence and gene flow across codistributed taxon-pairs. Under a model of multiple codistributed taxa that diverge into taxon-pairs with subsequent gene flow or isolation, one can estimate hyper-parameters that quantify the mean and variability in divergence times or test models of migration and isolation. The software uses multi-locus DNA sequence data collected from multiple taxon-pairs and allows variation across taxa in demographic parameters as well as heterogeneity in DNA mutation rates across loci. The method also allows a flexible sampling scheme: different numbers of loci of varying length can be sampled from different taxon-pairs.