Single-Cell Alternative Splicing Analysis with Expedition Reveals Splicing Dynamics during Neuron Differentiation.

Alternative splicing (AS) generates isoform diversity for cellular identity and homeostasis in multicellular life. Although AS variation has been observed among single cells, little is known about the biological or evolutionary significance of such variation. We developed Expedition, a computational framework consisting of outrigger, a de novo splice graph transversal algorithm to detect AS; anchor, a Bayesian approach to assign modalities; and bonvoyage, a visualization tool using non-negative matrix factorization to display modality changes. Applying Expedition to single pluripotent stem cells undergoing neuronal differentiation, we discover that up to 20% of AS exons exhibit bimodality. Bimodal exons are flanked by more conserved intronic sequences harboring distinct cis-regulatory motifs, constitute much of cell-type-specific splicing, are highly dynamic during cellular transitions, preserve reading frame, and reveal intricacy of cell states invisible to conventional gene expression analysis. Systematic AS characterization in single cells redefines our understanding of AS complexity in cell biology.

Stem Growth and Dehydration Responses of Mediterranean Tree Species to Atmospheric and Soil Drought.

Stem growth responses to soil and atmospheric drought are critical to forecasting the tree carbon sink strength. Yet, responses of drought-prone forests remain uncertain despite global aridification trends. Stem diameter variations at an hourly resolution were monitored in five Mediterranean tree species from a mesic and a xeric site for 6 and 12 years. Stem growth and dehydration responses to soil (REW) and atmospheric (VPD) drought were explored at different timescales. Annually, growth was determined by the number of growing days and hours. Seasonally, growth was bimodal (autumn growth ≈ 8%-18% of annual growth), varying among species and sites across the hydrometeorological space, while dehydration consistently responded to REW. Sub-daily, substantial growth occurred during daytime, with nighttime-to-daytime ratios ranging between 1.2 and 3.5 (Arbutus unedo ≈ Quercus faginea < Quercus ilex < Pinus halepensis in the mesic site, and Juniperus thurifera < P. halepensis in the xeric site). Overall, time windows favourable for growth were limited by soil (rather than atmospheric) drought, modulating annual and seasonal growth in Mediterranean species, and stems maintained non-negligible growth during daytime. These patterns contrast with observations from wetter or cooler biomes, demonstrating the growth plasticity of drought-prone species to more arid climate conditions.

A randomized controlled trial to study the effect of supplemental premilking stimulation on milking performance, teat tissue condition, udder health, and well-being in dairy cows.

The objectives of this study were to evaluate the effect of supplemental premilking stimulation, provided after manual stimulation, by means of high-frequency pulsation without reduction of the vacuum in the pulsation chamber on milking performance, teat tissue condition, udder health, and well-being in dairy cows. In a randomized controlled trial, Holstein cows (n = 491) from 1 commercial dairy farm with a thrice-per-day milking schedule were assigned to treatment and control groups over a 64-d period. Treatments consisted of a maximum of 20 s of pulsation stimulation at a pulsation rate of 100 (SPS100) or 300 (SPS300) cycles per minute and a pulsation ratio of 25:75, which were applied after completion of manual premilking stimulation upon milking unit attachment. Cows in the control group (CON) received only traditional premilking stimulation by manual forestripping for 6 s, and wiping. Milking characteristics were measured with on-farm milk flow meters. Milking machine-induced short-term (swelling at teat base, firmness at teat end, and teat discoloration) and long-term (teat-end callosity) changes to the teat tissue were assessed manually and visually. Composite milk samples were analyzed for somatic cell counts. Cow hind-leg activity was assessed with 3-dimensional accelerometers. Fecal 11,17-dioxoandrostanes (11,17-DOAs; a group of cortisol metabolites) were determined on wk 4 and 8 to assess the well-being of the cows. Generalized linear mixed models were used to study the effect of treatments on the outcome variables milk yield/milking and milking unit-on time. We observed no meaningful differences among groups for milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the SPS100, SPS300, and CON groups were 13.9 (13.3-14.5), 14.0 (13.4-14.6), and 13.9 (13.3-14.6) kg for milk yield and 218 (212-224), 218 (211-224), and 218 (212-224) s for milking unit-on time, respectively. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.30 (0.95-1.78) for the SPS100 group and 1.50 (1.10-2.05) for the SPS300 group. The odds of long-term changes were 0.94 (0.67-1.34) for cows in the SPS100 group and 0.71 (0.49-1.04) for cows in the SPS300 group. We observed no differences in SCC. In reference to the CON group, the hazard ratio (95% CI) in SPS100 and SPS300, respectively, were 0.35 (0.13-0.98) and 1.22 (0.57-2.64) for clinical mastitis, and 0.34 (0.12-0.95) and 1.28 (0.60-2.73) for culling. The least squares means (95% CI) of hind-leg activity during milking were 8.3 (6.5-10.5), 10.6 (8.1-13.7), and 9.1 (7.2-11.6) movements per milking for the SPS100, SPS300, and CON groups, respectively. The least squares means (95% CI) of fecal 11,17-DOAs concentration (ng/g) at the first and second test days, respectively, were 31.1 (28.1-34.2) and 22.3 (19.2-25.4) for the SPS100 group, 26.4 (23.4-29.4) and 25.2 (22.0-28.4) for the SPS300 group, and 24.8 (21.8-27.9) and 25.0 (21.7-28.3) for the CON group. We conclude that applying supplemental stimulation after manual stimulation through the high-frequency pulsation system tested here did not impart additional benefits to the milk harvesting process, teat tissue condition, somatic cell count, or the well-being of the animals.

The association between teat shape and bimodal milk ejection in Holstein dairy cows.

Our objectives were to investigate the association of teat shape with (1) bimodality, (2) incremental milk flow rates (average flow rates during the first 15 s, 15-30 s, 30-60 s, and 60-120 s of milking), and (3) peak milk flow rate in Holstein dairy cows milked 3 times/d. In this prospective cohort study, we analyzed 220,928 milking observations that were collected from 2,520 cows from a single dairy herd over a period of 31 d. Teat shape was visually assessed and classified into 1 of 4 categories as follows: (1) triangular barrel and pointed teat end (TP), (2) square barrel and round teat end (SR), (3) square barrel, round teat end, and flat in the area of the teat orifice (SRF), and (4) square barrel and flat teat end (SF). Individual cow-level milk flow rates were obtained using electronic on-farm milk meters. We considered bimodality to be present if any of the incremental milk flow rates (flow rates during 15-30 s, 30-60 s, or 60-120 s) were lower than any of the previous rates (flow rates during the first 15 s, 15-30 s, or 30-60 s). The frequency distribution of cows with different teat shapes was as follows: TP, 96 (3.8%); SR, 1,751 (69.5%); SRF, 617 (24.5%); and SF, 56 (2.2%). A generalized linear mixed model revealed differences in the odds of bimodality among cows with different teat shapes. Compared with cows in category SR, the odds (95% CI) of bimodality of cows in other categories were as follows: TP, 0.68 (0.48-0.97); SF, 1.96 (1.21-3.19); and SRF, 1.46 (1.23-1.72). General linear mixed models indicated an association between teat shape and all incremental milk flow rates, with higher milk flow rates being present for cows with teat shapes in the SRF and SF categories. The general linear mixed model for the dependent variable peak milk flow rate indicated an association between teat shape and peak milk flow rate. Least squares means and 95% CI were 5.1 (4.9-5.3) kg/min for TP, 5.8 (5.5-6.1) kg/min for SF, 5.6 (5.5-5.7) for SRF, and 5.3 (5.3-5.4) for SR, respectively. We conclude that in this study cohort, bimodality is more likely to occur in cows with flat teat ends compared with those with a round teat ends. We attribute this relationship to the difference in milking speed across cows with different teat shapes. Teat shape may serve as a useful phenotype to identify cows that are more likely to exhibit bimodality. Because our study population was unique, future studies considering different circumstances such as breed, milking system, and milking routine are needed before results can be extrapolated.

A randomized controlled trial to study the effects of an automated premilking stimulation system on milking performance, teat tissue condition, and udder health in Holstein dairy cows.

The objectives were to examine the effect of an automated premilking stimulation (APS) by means of a high pulsation frequency (300 cycles/min) without a reduction of the vacuum in the pulsation chamber or claw piece on (1) milking performance, (2) teat tissue condition, and (3) udder health in dairy cows. In a randomized controlled field study, Holstein cows (n = 427) from 1 commercial dairy farm with a milking schedule of 3 times per day were assigned to treatment and control groups over a 90-d period. Treatments consisted of a maximum of 80 s (APS80) or 99 s (APS99) of mechanical stimulation at a pulsation rate of 300 pulses per minute and a ratio of 25:75 (no reduction of the pulsation chamber or milking vacuum). Cows in the control group (CON) received traditional premilking stimulation by means of manual forestripping for 8 s. Milking characteristics were documented with on-farm milk meters. Short- and long-term changes in teat tissue condition induced by machine milking were assessed visually on a weekly basis. Composite milk samples were analyzed once per month to determine somatic cell count. Generalized linear mixed models were used to study the effect of the treatment on the outcome variables. We observed no meaningful differences in milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the APS80, APS99, and CON groups were 13.5 (13.1-14.0), 13.2 (12.8-13.7), and 13.2 (12.8-13.7) kg for milk yield and 222 (213-231), 219 (210-228), and 223 (214-232) s for milking unit-on time, respectively. The effect of treatment on bimodality was modified by milk yield such that the odds of bimodality increased in the treatment groups with increasing milk yield. Compared with cows in the CON group, the odds ratios (95% CI) of bimodality were 1.08 (0.62-1.89) in the APS80 group and 0.89 (0.55-1.42) in the APS99 group at a milk yield of 11 kg and 2.0 (1.24-3.22) in the APS80 group and 2.08 (1.29-3.35) in the APS99 group at a milk yield of 16 kg. We observed differences in short- and long-term changes in teat tissue condition between the treatment and control groups. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.87 (1.35-2.58) for the APS80 group and 1.49 (1.08-2.07) for the APS99 group, and the odds of long-term changes were 1.52 (1.24-1.85) for cows in the APS80 group and 1.59 (1.31-1.94) for cows in the APS99 group. The least squares means (95% CI) for somatic cell counts (log10-transformed) were 4.74 (4.68-4.81) for the APS80 group, 4.77 (4.71-4.83) for the APS99 group, and 4.79 (4.73-4.86) for the CON group. We conclude that the APS system tested here had no negative effects on milk yield or milking unit-on time. However, differences in bimodality and teat tissue condition suggest that the APS system did not provide sufficient stimulation to facilitate a gentle milk harvest and adversely affects teat tissue condition.

The interplay of continuous milk ejection and milking system with and without prestimulation at different vacuum settings.

Efficient machine milking requires an optimal interaction of alveolar milk ejection in the udder and milk removal by the milking machine. The aim of the present study was to test whether the equilibrium between continuous milk ejection and milk removal can also be maintained at very fast milking through a particularly high vacuum. Eight Holstein dairy cows were milked at 42, 52, or 60 kPa, with (PS) or without (nPS) prestimulation. Each of the 6 treatments was conducted at 2 afternoon milkings in each animal. The prestimulation lasted 40 s and consisted of forestripping and teat cleaning. The cluster attachment followed after a 20-s latency period. Throughout each milking, B-mode ultrasound videos of the gland cistern of 1 front quarter as well as milk flow and claw vacuum curves were recorded. Total milk yield was neither affected by nPS or PS nor by the vacuum level. Milk removed within the first minute and the first 2 min of milking and average milk flow were higher, and the duration of incline and time until peak milk flow were shorter at PS than at nPS milkings at all vacuum levels. Machine-on time was shorter at PS than at nPS milkings, although only at 42 and 52 kPa vacuum, obviously caused by the high percentage of bimodalities occurring in nPS milkings (17% bimodalities in PS vs. 92% bimodalities in nPS milkings). The frequency of bimodalities was higher at high than at low vacuum both in PS and nPS milkings. Peak flow rate and average milk flow were both higher at higher vacuum levels. The duration of milk flow plateau was shorter at 60 kPa than at 42 kPa milkings. At the highest vacuum (60 kPa), the shorter plateau phase indicated a declining milk ejection rate toward the end of the plateau phase, and milk ejection could no longer keep up with the fast milk removal; hence, a higher milking efficiency at a higher vacuum level could only be achieved as long as the gland cistern remained sufficiently filled by the continuous milk ejection. The ultrasound imaging confirmed this finding as the duration of cisternal area plateau in the recorded front quarter was shorter at high than at low vacuum. Thus, the highest vacuum of 60 kPa did not cause a shorter machine-on time than 52 kPa. In conclusion, milking at a very high vacuum can increase milking efficiency compared with a low vacuum. However, a vacuum reduction at the start and toward the end of milking is required to prevent overmilking if milking is performed at a very high vacuum.

Bridging Extremes: The Invertible Bimodal Gumbel Distribution.

This paper introduces a novel three-parameter invertible bimodal Gumbel distribution, addressing the need for a versatile statistical tool capable of simultaneously modeling maximum and minimum extremes in various fields such as hydrology, meteorology, finance, and insurance. Unlike previous bimodal Gumbel distributions available in the literature, our proposed model features a simple closed-form cumulative distribution function, enhancing its computational attractiveness and applicability. This paper elucidates the behavior and advantages of the invertible bimodal Gumbel distribution through detailed mathematical formulations, graphical illustrations, and exploration of distributional characteristics. We illustrate using financial data to estimate Value at Risk (VaR) from our suggested model, considering maximum and minimum blocks simultaneously.

Tipping the Balance: A Criticality Perspective.

Cell populations are often characterised by phenotypic heterogeneity in the form of two distinct subpopulations. We consider a model of tumour cells consisting of two subpopulations: non-cancer promoting (NCP) and cancer-promoting (CP). Under steady state conditions, the model has similarities with a well-known model of population genetics which exhibits a purely noise-induced transition from unimodality to bimodality at a critical value of the noise intensity σ2. The noise is associated with the parameter λ representing the system-environment coupling. In the case of the tumour model, λ has a natural interpretation in terms of the tissue microenvironment which has considerable influence on the phenotypic composition of the tumour. Oncogenic transformations give rise to considerable fluctuations in the parameter. We compute the λ-σ2 phase diagram in a stochastic setting, drawing analogies between bifurcations and phase transitions. In the region of bimodality, a transition from a state of balance to a state of dominance, in terms of the competing subpopulations, occurs at λ = 0. Away from this point, the NCP (CP) subpopulation becomes dominant as λ changes towards positive (negative) values. The variance of the steady state probability density function as well as two entropic measures provide characteristic signatures at the transition point.

Modeling the intracellular dynamics of the dengue viral infection and the innate immune response.

The interplay between the dengue virus and the innate immune response is not fully understood. Here, we use deterministic and stochastic approaches to investigate the dynamics of the interaction between the interferon-mediated innate immune response and the dengue virus. We aim to develop a quantitative representation of these complex interactions and predict their system-level dynamics. Our simulation results predict bimodal and bistable dynamics that represent viral clearance and virus-producing states. Under normal conditions, we determined that the viral infection outcome is modulated by the innate immune response and the positive-strand viral RNA concentration. Additionally, we tested system perturbations by external stimulation, such as the direct induction of the innate immune response by interferon, and a therapeutic intervention consisting of the direct application of mRNA encoding for several interferon-stimulated genes. Our simulation results suggest optimal regimes for the studied intervention approaches.

Bimodal expression of potential drug target CLL-1 (CLEC12A) on CD34+ blasts of AML patients.

OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.

Bivalent genes that undergo transcriptional switching identify networks of key regulators of embryonic stem cell differentiation.

BACKGROUND: Bivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to monovalency upon differntiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely. RESULTS: We develop an approach for capturing genes undergoing transcriptional switching by detecting 'bimodal' gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state, and identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudotime approximation and use a hidden Markov model to infer gene activity switching pseudotimes, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets. CONCLUSIONS: Genes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state. Since chromatin bivalency is a hallmark of gene promoters poised for activity, this approach provides an alternative that complements conventional scRNA-seq analysis while focusing on genes near the ON/OFF boundary of activity. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data.

Bimodal age distribution at diagnosis in breast cancer persists across molecular and genomic classifications.

PURPOSE: Female breast cancer demonstrates bimodal age frequency distribution patterns at diagnosis, interpretable as two main etiologic subtypes or groupings of tumors with shared risk factors. While RNA-based methods including PAM50 have identified well-established clinical subtypes, age distribution patterns at diagnosis as a proxy for etiologic subtype are not established for molecular and genomic tumor classifications. METHODS: We evaluated smoothed age frequency distributions at diagnosis for Carolina Breast Cancer Study cases within immunohistochemistry-based and RNA-based expression categories. Akaike information criterion (AIC) values compared the fit of single density versus two-component mixture models. Two-component mixture models estimated the proportion of early-onset and late-onset categories by immunohistochemistry-based ER (n = 2860), and by RNA-based ESR1 and PAM50 subtype (n = 1965). PAM50 findings were validated using pooled publicly available data (n = 8103). RESULTS: Breast cancers were best characterized by bimodal age distribution at diagnosis with incidence peaks near 45 and 65 years, regardless of molecular characteristics. However, proportional composition of early-onset and late-onset age distributions varied by molecular and genomic characteristics. Higher ER-protein and ESR1-RNA categories showed a greater proportion of late age-at-onset. Similarly, PAM50 subtypes showed a shifting age-at-onset distribution, with most pronounced early-onset and late-onset peaks found in Basal-like and Luminal A, respectively. CONCLUSIONS: Bimodal age distribution at diagnosis was detected in the Carolina Breast Cancer Study, similar to national cancer registry data. Our data support two fundamental age-defined etiologic breast cancer subtypes that persist across molecular and genomic characteristics. Better criteria to distinguish etiologic subtypes could improve understanding of breast cancer etiology and contribute to prevention efforts.

The emergence of the two cell fates and their associated switching for a negative auto-regulating gene.

BACKGROUND: Decisions in the cell that lead to its ultimate fate are important for fundamental cellular functions such as proliferation, growth, differentiation, development, and death. These cell fate decisions can be influenced by both the gene regulatory network and also environmental factors and can be modeled using simple gene feedback circuits. Negative auto-regulation is a common feedback motif in the gene circuits. It can act to reduce gene expression noise or induce oscillatory expression and is thought to lead to only one cell fate. Here, we present experimental and modeling data to suggest that a self-repressor circuit can lead to two cell fates under specific conditions. RESULTS: We show that the introduction of inducers capable of binding and unbinding to a self-repressing gene product (protein), thus regulating the associated gene, can lead to the emergence of two cell states. We suggest that the inducers can alter the effective regulatory binding and unbinding speed of the self-repressor regulatory protein to its destination DNA without changing the gene itself. The corresponding simulation results are consistent with the experimental findings. We propose physical and quantitative explanations for the origin of the two phenotypic cell fates. CONCLUSIONS: Our results suggest a mechanism for the emergence of multiple cell fates. This may explain the heterogeneity often observed among cell states, while illustrating that altering gene regulation strength can influence cell fates and their decision-making processes without genetic changes.

Computational methods to identify bimodal gene expression and facilitate personalized treatment in cancer patients.

Standard methods for detecting cancer-associated genes rely on comparison of sample means between cancer patients and healthy controls. While such methods have successfully identified several oncogenes and tumor suppressor genes, they neglect to account for heterogeneity within the cancer population. Genetic mutations, translocations, and amplifications are often inconsistent across tumors, and instead they often affect smaller subsets of patients. This concept gives rise to the idea of bimodally expressed genes, or genes that display two modes of expression within one population. Analysis of bimodal gene expression has been explored via a variety of techniques including test statistics and clustering. In this review, we summarize the methodologies used to quantify bimodal gene expression and address the utility of these genes in patient stratification and specialized therapeutics in breast and lung cancer. Finally we discuss the limitations and future directions for bimodal genes in the era of high-throughput sequencing and personalized medicine.

Asymmetric Bimodal Exponential Power Distribution on the Real Line.

The asymmetric bimodal exponential power (ABEP) distribution is an extension of the generalized gamma distribution to the real line via adding two parameters that fit the shape of peakedness in bimodality on the real line. The special values of peakedness parameters of the distribution are a combination of half Laplace and half normal distributions on the real line. The distribution has two parameters fitting the height of bimodality, so capacity of bimodality is enhanced by using these parameters. Adding a skewness parameter is considered to model asymmetry in data. The location-scale form of this distribution is proposed. The Fisher information matrix of these parameters in ABEP is obtained explicitly. Properties of ABEP are examined. Real data examples are given to illustrate the modelling capacity of ABEP. The replicated artificial data from maximum likelihood estimates of parameters of ABEP and other distributions having an algorithm for artificial data generation procedure are provided to test the similarity with real data. A brief simulation study is presented.

Bimodal expression of yeast GAL genes is controlled by a long non-coding RNA and a bifunctional galactokinase.

Bimodality in gene expression can generate phenotypic heterogeneity facilitating fitness and growth of isogenic cell populations in suboptimal environments. We investigated the mechanism by which, in conditions of limiting galactose, yeast cell populations activate GAL genes in a bimodal fashion with a cell fraction expressing GAL genes (ON), while the rest subpopulation is kept at the non-expressing (OFF) state. We show that a long non-coding RNA (GAL10-ncRNA) crossing the bidirectional GAL1-10 promoter, decreases the rate by which single cells commit transition to the ON state without affecting the rate of GAL transcription per se in ON cells. This is accomplished by repressing stochastic expression of the bifunctional Gal1p galactokinase, which besides its enzymatic activity acts as an essential inducer of the system under those conditions. We show that once single cells switch to the ON state, the GAL10-ncRNA effect is overridden by accumulating Gal1p levels sufficient to feedback positively on Gal4p, and not by the active transcription of GAL10 that occurs in opposite direction relative to that of GAL10-ncRNA. Conversely, GAL10-ncRNA does not influence transition of ON cells, where Gal4p is active, back to the OFF state. Our model suggests that the functional interplay between GAL10-ncRNA transcription, stochastic Gal1p expression and Gal1p positive feedback on Gal4p constitutes a novel molecular switch mechanism dictating the commitment of individual cells for either metabolic state.

Further evidence to support bimodality of oestrogen receptor expression in breast cancer.

AIMS: Although oestrogen receptor (ER)-negative breast cancers (BCs) do not respond to hormone therapy, the response of ER-positive BCs is reported to be variable, which may suggest a dose-dependent effect. The aim of this study was to assess the pattern of ER expression in BCs at the protein (immunohistochemistry) and transcriptome (microarray-based gene expression) levels. METHODS AND RESULTS: ER immunohistochemical (IHC) expression was assessed in a large series of BCs, including 3649 core biopsies and 1892 cases prepared as tissue microarrays (TMAs) stained with specific antibodies. ESR1 mRNA expression was assessed in the METABRIC study (1980 cases), by the use of the Linear Models for Microarray Data (limma) software, and the results were compared with protein levels. IHC data confirmed the bimodality of ER expression, with 92.2% and 89.2% of the cases showing completely negative (<1%) or highly positive (≥70%) expression on the cores and TMAs, respectively. Weakly positive cases (1-10%) and intermediately positive (11-69%) cases were infrequent (2.7% and 5.1%, and 1.6% and 9.2%, in cores and TMAs, respectively), and did not show survival difference from ER-negative tumours. When full-face sections of the corresponding excision specimens were immunostained, 47% of the ER-low/intermediate group were deemed to be ER-negative. Transcriptomic data not only showed a significant correlation between ESR1 mRNA and protein expression levels, but also confirmed the bimodality of ER expression at the mRNA level. CONCLUSIONS: Our study provides further evidence that ER expression is bimodal, and that it is observed at both the mRNA and protein levels. The reported poor survival of BC patients with low ER expression in the early clinical trials may be related to the inclusion of ER-negative cases.

Asymmetric competition causes multimodal size distributions in spatially structured populations.

Plant sizes within populations often exhibit multimodal distributions, even when all individuals are the same age and have experienced identical conditions. To establish the causes of this, we created an individual-based model simulating the growth of trees in a spatially explicit framework, which was parametrized using data from a long-term study of forest stands in New Zealand. First, we demonstrate that asymmetric resource competition is a necessary condition for the formation of multimodal size distributions within cohorts. By contrast, the legacy of small-scale clustering during recruitment is transient and quickly overwhelmed by density-dependent mortality. Complex multi-layered size distributions are generated when established individuals are restricted in the spatial domain within which they can capture resources. The number of modes reveals the effective number of direct competitors, while the separation and spread of modes are influenced by distances among established individuals. Asymmetric competition within local neighbourhoods can therefore generate a range of complex size distributions within even-aged cohorts.

Pollination, mating and reproductive fitness in a plant population with bimodal floral-tube length.

Mating patterns and natural selection play important roles in determining whether genetic polymorphisms are maintained or lost. Here, we document an atypical population of Lapeirousia anceps (Iridaceae) with a bimodal distribution of floral-tube length and investigate the reproductive mechanisms associated with this pattern of variation. Flowers were visited exclusively by the long-proboscid fly Moegistorhynchus longirostris (Nemestrinidae), which exhibited a unimodal distribution of proboscis length and displayed a preference for long-tubed phenotypes. Despite being visited by a single pollinator species, allozyme markers revealed significant genetic differentiation between open-pollinated progeny of long- and short-tubed phenotypes suggesting mating barriers between them. We obtained direct evidence for mating barriers between the floral-tube phenotypes through observations of pollinator foraging, controlled hand pollinations and measurements of pollen competition and seed set. Intermediate tube-length phenotypes produced fewer seeds in the field than either long- or short-tubed phenotypes. Although floral-tube length bimodality may not be a stable state over long timescales, reproductive barriers to mating and low 'hybrid' fitness have the potential to contribute to the maintenance of this state in the short term.

Strong selection on male plumage in a hybrid zone between a hybrid bird species and one of its parents.

Homoploid hybrid speciation (HHS) requires reproductive barriers between hybrid and parent species, despite incomplete reproductive isolation (RI) between the parents. Novel secondary sexual trait values in hybrids may cause prezygotic isolation from both parents, whereas signals inherited by the hybrid from one parent species may cause prezygotic isolation with the other. Here we investigate whether differences in male plumage function as a premating barrier between the hybrid Italian sparrow and one of its parent species, the house sparrow, in a narrow Alpine hybrid zone. Italian sparrow male plumage is a composite mosaic of the parental traits, with its head plumage most similar to its other parent, the Spanish sparrow. We use geographical cline analysis to examine selection on three plumage traits, 75 nuclear single nucleotide polymorphisms (SNPs) and hybrid indices based on these SNPs. Several SNPs showed evidence of restricted introgression in the Alps, supporting earlier findings. Crown colour exhibited the narrowest plumage cline, representing a 37% (range 4-65%) drop in fitness. The cline was too narrow to be due to neutral introgression. Only crown colour was significantly bimodal in the hybrid zone. Bimodality may be due to RI or a major QTL, although fitness estimates suggest that selection contributes to the pattern. We discuss the implications with respect to HHS and the species status of the Italian sparrow.