Time Series Experiments Research Articles

Metabolic Response to XD14 Treatment in Human Breast Cancer Cell Line MCF-7.

XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.

Magma mixing induced by particle settling.

A time series of experiments at high temperature have been performed to investigate the influence of particle settling on magma mixing. A natural rhyolite glass was held above a natural basalt glass in a platinum crucible. After melting of the glasses at superliquidus temperatures, a platinum sphere was placed on the upper surface of the rhyolitic melt and sank into the experimental column (rhyolitic melt above basaltic melt). Upon falling through the rhyolitic–basaltic melt interface, the Pt sphere entrained a filament of rhyolitic melt in its further fall. The quenched products of the experiments were imaged using X-ray microCT methods. The images of our time series of experiments document the formation of a rhyolite filament as it is entrained into the underlying basalt by the falling platinum sphere. When the Pt particle reached the bottom of the crucible, the entrained rhyolitic filament started to ascend buoyantly up to the initial rhyolitic–basaltic interface. This generated a significant thickness increase of a comingled “melange” layer at the interface due to “liquid rope coiling” and piling up of the filament. As a consequence, the basalt/rhyolite interface was greatly enlarged and diffusive hybridisation greatly accelerated. Further, bubbles, originating at the interface, are observed to have risen into the overlying rhyolite dragging basalt filaments with them. Upon crossing the basalt/rhyolite interface, the bubbles have non-spherical shapes as they adapt to the differing surface tensions of basaltic and rhyolitic melts. Major element profiles, measured across the rhyolite filaments, exhibit asymmetrical shapes from the rhyolite into the basalt. Na and Ti reveal uphill diffusion from the rhyolite towards the interface in the filament cross sections. These results reveal the potential qualitative complexity of the mingling process between rhyolitic and basaltic magmas in the presence of sinking crystals. They imply that crystal-rich magma mingling may be expected to be accelerated with respect to crystal-poor systems. We urge the further fluid dynamic analysis of these phenomena, obtainable for the first time using detailed tomographic imaging.Electronic supplementary materialThe online version of this article (doi:10.1007/s00410-016-1305-1) contains supplementary material, which is available to authorized users.

The CaCO3–Fe interaction: Kinetic approach for carbonate subduction to the deep Earth’s mantle

The CaCO3–Fe0 system, as a model for redox reactions between carbonates and reduced lithologies at the slab-mantle interface during subduction or at core-mantle boundary, was investigated systematically at temperatures from 650 to 1400°C and pressures from 4 to 16GPa using multianvil apparatus. CaCO3 reduction via reaction: 3 CaCO3 (aragonite)+13 Fe0 (metal)=Fe7C3 (carbide)+3 CaFe2O3 (Ca-wüstite) was observed. The thickness of the reaction-product layer (Δx) increases linearly with the square root of time in the time-series experiments (t), indicating diffusion-controlled process. The reaction rate constant (k=Δx2/2t) is log-linear relative to 1/T. Its temperature dependences was determined to be k [m2/s]=2.1×10−7exp(−162[kJ/mol]/RT) at 4–6GPa and k [m2/s]=2.6×10−11exp(−65[kJ/mol]/RT) at 16GPa. The sluggish kinetics of established CaCO3–Fe0 interaction suggests that significant amount of carbonates could survive during subduction from metal saturation boundary near 250km depth down to the transition zone and presumably to the lower mantle if melting of carbonates is not involved.

Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes.

Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (-29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep.

Abstract SY12-03: MYC-mediated metabolic vulnerabilities and the circadian clock

Abstract SY12-03: MYC-mediated metabolic vulnerabilities and the circadian clock

Time is Money: Designing Cost-Effective Time Series Experiments

A new theoretical model helps to evaluate the tradeoffs between running technical replicates in high-throughput experiments and sampling at more time points.

Tradeoffs between Dense and Replicate Sampling Strategies for High-Throughput Time Series Experiments

An important experimental design question for high-throughput time series studies is the number of replicates required for accurate reconstruction of the profiles. Due to budget and sample availability constraints, more replicates imply fewer time points and vice versa. We analyze the performance of dense and replicate sampling by developing a theoretical framework that focuses on a restricted yet expressive set of possible curves over a wide range of noise levels and by analyzing real expression data. For both the theoretical analysis and experimental data, we observe that, under reasonable noise levels, autocorrelations in the time series data allow dense sampling to better determine the correct levels of non-sampled points when compared to replicate sampling. A Java implementation of our framework can be used to determine the best replicate strategy given the expected noise. These results provide theoretical support to the large number of high-throughput time series experiments that do not use replicates.

Statistical modeling of isoform splicing dynamics from RNA-seq time series data.

Isoform quantification is an important goal of RNA-seq experiments, yet it remains problematic for genes with low expression or several isoforms. These difficulties may in principle be ameliorated by exploiting correlated experimental designs, such as time series or dosage response experiments. Time series RNA-seq experiments, in particular, are becoming increasingly popular, yet there are no methods that explicitly leverage the experimental design to improve isoform quantification. Here, we present DICEseq, the first isoform quantification method tailored to correlated RNA-seq experiments. DICEseq explicitly models the correlations between different RNA-seq experiments to aid the quantification of isoforms across experiments. Numerical experiments on simulated datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. On real datasets, our results show that DICEseq provides substantially more reproducible and robust quantifications, increasing the correlation of estimates from replicate datasets by up to 10% on genes with low or moderate expression levels (bottom third of all genes). Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Our results have strong implications for the design of RNA-seq experiments, and offer a novel tool for improved analysis of such datasets. Python code is freely available at http://diceseq.sf.net G.Sanguinetti@ed.ac.uk Supplementary data are available at Bioinformatics online.

Measuring the steady state of pedestrian flow in bottleneck experiments

Experiments with pedestrians could depend strongly on initial conditions. Comparisons of the results of such experiments require to distinguish carefully between transient state and steady state. In this work, a feasible algorithm - Cumulative Sum Control Chart - is proposed and improved to automatically detect steady states from density and speed time series of bottleneck experiments. The threshold of the detection parameter in the algorithm is calibrated using an autoregressive model. Comparing the detected steady states with previous manually selected ones, the modified algorithm gives more reproducible results. For the applications, three groups of bottleneck experiments are analysed and the steady states are detected. The study about pedestrian flow shows that the difference between the flows in all states and in steady state mainly depends on the ratio of pedestrian number to bottleneck width. When the ratio is higher than a critical value (approximately 115 persons/m), the flow in all states is almost identical with the flow in steady state. Thus we have more possibilities to compare the flows from different experiments, especially when the detection of steady states is difficult.

Uptake and fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in coastal marine biota determined using a stable isotopic tracer, 15N – [RDX

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is globally one of the most commonly used military explosives and environmental contaminant. 15N labeled RDX was added into a mesocosm containing 9 different coastal marine species in a time series experiment to quantify the uptake of RDX and assess the RDX derived 15N retention into biota tissue. The 15N attributed to munitions compounds reached steady state concentrations ranging from 0.04 to 0.67 μg 15N g dw−1, the bulk 15N tissue concentration for all species was 1–2 orders of magnitude higher suggesting a common mechanism or pathway of RDX biotransformation and retention of 15N. A toxicokinetic model was created that described the 15N uptake, elimination, and transformation rates. While modeled uptake rates were within previous published values, elimination rates were several orders of magnitude smaller than previous studies ranging from 0.05 to 0.7 days−1. These small elimination rates were offset by high rates of retention of 15N previously not measured. Bioconcentration factors and related aqueous:organism ratios of compounds and tracer calculated using different tracer and non-tracer methods yielded a broad range of values (0.35–101.6 mL g−1) that were largely method dependent. Despite the method-derived variability, all values were generally low and consistent with little bioaccumulation potential. The use of 15N labeled RDX in this study indicates four possible explanations for the observed distribution of compounds and tracer; each with unique potential implications for possible toxicological impacts in the coastal marine environment.

Kinetics of anorthite dissolution in basaltic melt

We report convection-free anorthite dissolution experiments in a basaltic melt at 1280–1500°C and 0.5GPa on two different crystallographic surfaces, (121¯) and (3¯02) to investigate dissolution kinetics. The anisotropy of the anorthite dissolution rate along these two surfaces is negligible. Time series experiments at ∼1280°C show that anorthite dissolution is mainly controlled by diffusion in the melt within experimental uncertainty. Analytical solutions were used to model the dissolution and diffusion processes, and to obtain the diffusivities and the saturation concentrations of the equilibrium-determining component (Al2O3) for anorthite dissolution into the basaltic melt. For the first time, we are able to show the physical and chemical characteristics of quench growth effect on the near-interface melt using high spatial resolution (0.3μm) EDS analyses. For anorthite (An#⩾90) saturation in a melt with 39–53wt% SiO2 and ⩽0.4wt% H2O, the concentration of Al2O3 in wt% depends on temperature as follows:lnCs,Al2O3=8.032(±0.192)-7882(±287)T,where T is temperature in K, and errors are given at 1σ level. Al2O3 diffusivity in basaltic melt during plagioclase dissolution can be expressed as:lnDAl2O3EBD,plag diss=-13.69(±1.57)-19,313(±2485)T,where D is in m2/s, and the activation energy is 161±21kJ/mol. These results are applied to model the convective dissolution of anorthite in basaltic melts. The model indicates that though anorthite crystals can survive for a longer time compared to olivine crystals of the same size in the same melt, the former would rise for a much smaller distance compared to the olivine sinking distance, due to the smaller density contrast between anorthite and melt.

Which agricultural management interventions are most influential on soil organic carbon (using time series data)?

Loss of soil organic carbon (SOC) from agricultural land is identified as one of the major threats to soils, as it influences both fertility and the production of ecosystem services from agriculture. Losses of SOC across regions are often determined by monitoring in different land use systems. Results from agricultural field experiments can reveal increasing SOC stocks after implementation of specific management practices compared to a control, though in time series experiments the relative rate of change is often negative and implying an overall loss. Long-term agricultural field experiments are indispensable for quantifying absolute changes in SOC stocks under different management regimes. Since SOC responses are seldom linear over time, time series data from these experiments are particularly valuable. This systematic review is based on studies reporting time series data collated in a recently completed systematic map on the topic restricted to the warm temperate climate zone and the snow climate zone. These 53 studies were identified and selected systematically according to CEE guidelines. An update of the original search for studies will be repeated using Web of Science and Google Scholar to include newly published academic and grey literature in the time since the original search was performed in September 2013. Studies will be subject to critical appraisal of the internal and external validity, followed by full data extraction (meta-data describing study settings and quantitative study results). Where possible, studies will be included in a quantitative synthesis using time series meta-analytical approaches. The implications of the meta-analytical findings will be discussed in terms of policy, practice and research along with a discussion of the nature of the evidence base.

Microbial Response to Experimentally Controlled Redox Transitions at the Sediment Water Interface.

The sediment–water interface of freshwater lakes is characterized by sharp chemical gradients, shaped by the interplay between physical, chemical and microbial processes. As dissolved oxygen is depleted in the uppermost sediment, the availability of alternative electron acceptors, e.g. nitrate and sulfate, becomes the limiting factor. We performed a time series experiment in a mesocosm to simulate the transition from aerobic to anaerobic conditions at the sediment–water interface. Our goal was to identify changes in the microbial activity due to redox transitions induced by successive depletion of available electron acceptors. Monitoring critical hydrochemical parameters in the overlying water in conjunction with a new sampling strategy for sediment bacteria enabled us to correlate redox changes in the water to shifts in the active microbial community and the expression of functional genes representing specific redox-dependent microbial processes. Our results show that during several transitions from oxic-heterotrophic condition to sulfate-reducing condition, nitrate-availability and the on-set of sulfate reduction strongly affected the corresponding functional gene expression. There was evidence of anaerobic methane oxidation with NOx. DGGE analysis revealed redox-related changes in microbial activity and expression of functional genes involved in sulfate and nitrite reduction, whereas methanogenesis and methanotrophy showed only minor changes during redox transitions. The combination of high-frequency chemical measurements and molecular methods provide new insights into the temporal dynamics of the interplay between microbial activity and specific redox transitions at the sediment–water interface.

Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts, Revealed by Comparative Modeling of Pathway Dynamics.

The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., “overshoot”) was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr308 (Aktp308). The model was fit to experimental measurements of Aktp308, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp308 (at 30–60 minutes). From this we conclude that the peak and decline behavior of Aktp308 is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp308 is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient experiments.

Interactions between allelochemicals and the microbial community affect weed suppression following cover crop residue incorporation into soil

The objective of this study is to understand how soil microorganisms interact with cover crop-derived allelochemicals to suppress weed germination and growth following cover crop residue incorporation. We conducted a time series experiment crossing sterilized and non-sterilized soil with four different residue treatments. We measured weed seed germination rates, radicle elongation, and disease incidence in seed germination bioassays. We also monitored cover crop-derived, isoflavone allelochemicals in these bioassays. We partitioned the total weed suppression into three sources: microbe-only inhibition, residue-only inhibition, and the microbe-residue interaction. Microbial activity suppressed weed germination and growth for 30 days, while cover crop-derived allelochemicals provided suppression for a limited time. There was an antagonistic interaction between microbes and allelochemicals. This interaction was strongest for water-soluble allelochemicals, while residue fractions containing intact plant tissues retained greater suppressiveness even in the presence of a live microbial community. Microbial activity can directly suppress weed germination and growth, but microorganisms also indirectly help weeds by degrading cover crop-derived allelochemicals. As a result of these interactions, cover crop-derived weed suppression in agricultural soils shifts from an early allelochemical-dominated phase to a later phase where microbial suppression is more important.

A method for measurement of free cadmium species in waters using diffusive gradients in thin films technique with an ion-imprinted sorbent

A diffusive gradients in thin films (DGT) device for the analysis of free Cd(II) species, based on Cd(II) ion-imprinted sorbent (IIS) as the binding agents and commercial polyethersulfone membrane (PES) as diffusion layer, was developed (PES/IIS-DGT). DGT time-series experiments showed that the mass of free Cd(II) species accumulated by PES/IIS-DGT was linear vs. time (R2 = 0.9953) and the concentration of free Cd(II) species by PES/IIS-DGT was in good agreement with the total dissolved concentrations of free Cd(II) species in simple synthetic solutions where free ionic species dominated. PES/IIS-DGT performance was independent in the range of pH 4.5–7.5 and ionic strength range from 1.0 × 10−3 to 0.7 mol L−1. The measurement of free Cd(II) species in synthetic solution containing different concentrations of ligands by PES/IIS-DGT showed an excellent agreement with the value measured by Cd(II) ion selective electrodes (Cd-ISE), indicating that PES/IIS-DGT method is more suitable than Cd-ISE for the measurement of low concentration of free Cd(II) species due to the enrichment of IIS for the analytes.

Barberry Does Not Function as an Alternate Host for Puccinia striiformis f. sp. tritici in the U. S. Pacific Northwest Due to Teliospore Degradation and Barberry Phenology.

Sexual reproduction of the stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), on barberry (Berberis vulgaris) has been shown to provide initial inoculum for the development of the disease on wheat and barley and also generate diverse races of the pathogen. However, in our previous study, the stripe rust pathogen, P. striiformis f. sp. tritici (Pst), was not found on barberry in the U. S. Pacific Northwest. To determine why Pgt is able to infect the alternate host, while Pst cannot under the natural conditions, the viabilities of teliospores of both Pgt and Pst were investigated from 2011 to 2014 by determining the germination rates using telial samples collected periodically from wheat fields. Teliospores of Pst usually produced in July were physically degraded during winter, and their germination rate decreased from 50 to 90% in August to less than 1% in the following March and no germination after May. In contrast, Pgt teliospores usually produced in July and August remained physically intact and physiologically dormant, and could not germinate until February. Germination of Pgt teliospores gradually increased to 90% in May, at which time young leaves of barberry were susceptible to infection. In addition, a time-series experiment was conducted for inoculation of barberry plants with Pst teliospores. The results showed that Pst teliospores need a minimum of 32 h continual dew-forming conditions to infect barberry, and infection reaches a peak after incubation of inoculated plants for 88 h. The lack of a prolonged period of leaf wetness conditions during the season of telial maturity effectively negates Pst infection of barberry plants in the Pacific Northwest.

On Accounting for Sequence-Specific Bias in Genome-Wide Chromatin Accessibility Experiments: Recent Advances and Contradictions.

Uncovering the protein–DNA interactions involved in cell fate, development, and disease in a timeand cell-specific manner is a fundamental goal of molecular biology. The advent of the sequencing technologies has opened a new genomic era, uncovering the information encoded in genomes, epigenomes, and transcriptomes (McPherson, 2014). For example, the popular ChIPbased techniques ChIP-seq (Johnson et al., 2007; Robertson et al., 2007) and ChIP-exo (Rhee and Pugh, 2011) are widely used to detect transcription factor (TF)-binding sites using an antibody against a single protein of interest (Mahony and Pugh, 2015). Alternative protocols assaying the chromatin landscape, such as those based ondigestion byDNase I enzyme (DNase-seq),micrococcal nuclease (MNase-seq), and Tn5 transposase attack (ATAC-seq), enable the identification of DNAbinding protein footprints of many TFs in a single experiment (Tsompana and Buck, 2014). Timeseries experiments might be required for the identification of those TFs cataloged as pioneer factors, allowing their effects on chromatin to be investigated (Zaret and Carroll, 2011; Pajoro et al., 2014; Sherwood et al., 2014). Despite the initial promise of detecting the majority of TFs in one assay, DNA sequencespecific biases, together with TF-dependent binding kinetics, have been recently pinpointed as major confounding factors in DNase-seq experiments (Koohy et al., 2013; He et al., 2014; Raj and McVicker, 2014; Rusk, 2014; Sung et al., 2014). These influencing factors were not considered by any of the previous computational approaches for the analysis of next-generation sequencing chromatin accessibility data (Madrigal and Krajewski, 2012); neither those strategies based on TFgeneric DNase signature nor those based on TF-specific DNase signature (Luo and Hartemink, 2013).

Concentration variance decay during magma mixing: a volcanic chronometer.

The mixing of magmas is a common phenomenon in explosive eruptions. Concentration variance is a useful metric of this process and its decay (CVD) with time is an inevitable consequence during the progress of magma mixing. In order to calibrate this petrological/volcanological clock we have performed a time-series of high temperature experiments of magma mixing. The results of these experiments demonstrate that compositional variance decays exponentially with time. With this calibration the CVD rate (CVD-R) becomes a new geochronometer for the time lapse from initiation of mixing to eruption. The resultant novel technique is fully independent of the typically unknown advective history of mixing – a notorious uncertainty which plagues the application of many diffusional analyses of magmatic history. Using the calibrated CVD-R technique we have obtained mingling-to-eruption times for three explosive volcanic eruptions from Campi Flegrei (Italy) in the range of tens of minutes. These in turn imply ascent velocities of 5-8 meters per second. We anticipate the routine application of the CVD-R geochronometer to the eruptive products of active volcanoes in future in order to constrain typical “mixing to eruption” time lapses such that monitoring activities can be targeted at relevant timescales and signals during volcanic unrest.

Characteristics, Energetics, and Origins of Agulhas Current Meanders and Their Limited Influence on Ring Shedding

AbstractThe Agulhas Current intermittently undergoes dramatic offshore excursions from its mean path because of the downstream passage of mesoscale solitary meanders or Natal pulses. New observations and analyses are presented of the variability of the current and its meanders using mooring observations from the Agulhas Current Time-Series Experiment (ACT) near 34°S. Using a new rotary EOF method, mesoscale meanders and smaller-scale meanders are differentiated and each captured in a single mode of variance. During mesoscale meanders, an onshore cyclonic circulation and an offshore anticyclonic circulation act together to displace the jet offshore, leading to sudden and strong positive conversion of kinetic energy from the mean flow to the meander via nonlinear interactions. Smaller meanders are principally represented by a single cyclonic circulation spanning the entire jet that acts to displace the jet without extracting kinetic energy from the mean flow. Synthesizing in situ observations with altimeter data leads to an account of the number of mesoscale meanders at 34°S: 1.6 yr−1 on average, in agreement with a recent analysis by Rouault and Penven (2011) and significantly less than previously understood. The links between meanders and the arrival of Mozambique Channel eddies or Madagascar dipoles at the western boundary upstream are found to be robust in the 20-yr altimeter record. Yet, only a small fraction of anomalies arriving at the western boundary result in meanders, and of those, two-thirds can be related to ring shedding. Most Agulhas rings are shed independently of meanders.