Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Pathway alterations reflected as changes in gene expression regulation and gene interaction can result from cellular exposure to toxicants. Such information is often used to elucidate toxicological modes of action. From a risk assessment perspective, alterations in biological pathways are a rich resource for setting toxicant thresholds, which may be more sensitive and mechanism-informed than traditional toxicity endpoints. Here we developed a novel differential networks (DNs) approach to connect pathway perturbation with toxicity threshold setting.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

RNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.

The goal of many human disease-oriented studies is to detect molecular mechanisms different between healthy controls and patients. Yet, commonly used gene expression measurements from blood samples suffer from variability of cell composition. This variability hinders the detection of differentially expressed genes and is often ignored. Combined with cell counts, heterogeneous gene expression may provide deeper insights into the gene expression differences on the cell type-specific level. Published computational methods use linear regression to estimate cell type-specific differential expression, and a global cutoff to judge significance, such as False Discovery Rate (FDR). Yet, they do not consider many artifacts hidden in high-dimensional gene expression data that may negatively affect linear regression. In this paper we quantify the parameter space affecting the performance of linear regression (sensitivity of cell type-specific differential expression detection) on a per-gene basis.

A challenge in gene expression studies is the reliable identification of differentially expressed genes. In many high-throughput studies, genes are accepted as differentially expressed only if they satisfy simultaneously a p value criterion and a fold change criterion. A statistical method, TREAT, has been developed for microarray data to assess formally if fold changes are significantly higher than a predefined threshold. We have recently applied the NanoString digital platform to study expression of mouse odorant receptor genes, which form with 1,200 members the largest gene family in the mouse genome. Our objectives are, on these data, to decrease false discoveries when formally assessing the genes relative to a fold change threshold, and to provide a guided selection in the choice of this threshold.

In adult mouse trigeminal ganglion (TG) neurones we identified three neuronal subpopulations, defined in terms of their firing response to protracted depolarizations, namely MF neurones, characterized by a multiple tonic firing; DMF neurones, characterized by a delay before the beginning of repetitive firing; and SS neurones, characterized by a strongly adapting response. The three subpopulations also differed in several other properties important for defining their functional role in vivo, namely soma size, action potential (AP) shape and capsaicin sensitivity. MF neurones had small soma, markedly long AP and mostly responded to capsaicin, properties typical of a subgroup of C-type nociceptors. SS neurones had large soma, short AP duration and were mostly capsaicin insensitive, suggesting that most of them have functions other than nociception. DMF neurones were all capsaicin insensitive, had a small soma size and intermediate AP duration, making them functionally distinct from both MF and SS neurones. We investigated the ionic basis underlying the delay to the generation of the first AP of DMF neurones, and the strong adaptation of SS neurones. We found that the expression of a fast-inactivating, 4-AP- and CP-339,818-sensitive K+ current (I(A)) in DMF neurones plays a critical role in the generation of the delay, whereas a DTX-sensitive K+ current (I(DTX)) selectively expressed in SS neurones appeared to be determinant for their strong firing adaptation. A minimal theoretical model of TG neuronal excitability confirmed that I(A) and I(DTX) have properties congruent with their suggested role.

Norovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT-PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29-22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT-PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease.

A mathematical model was proposed to predict the role played by apneic threshold in periodic breathing in preterm infants. Prior models have mainly applied linear control theory which predicted instability but could not explain sustained periodic breathing. Apneic threshold to CO2 which has been postulated to play a major role in infant periodic breathing is a nonlinear effect and cannot be described by linear theory. Another previously unexplored nonlinear factor affecting instability is brain vascular volume change with CO2 which affects time delay to chemoreceptors. The current model explored the influences of apneic threshold, central and peripheral chemoreceptor gains, cardiac output, lung volume, and circulatory time delay on periodic breathing. Apneic threshold was found to play a major role in ventilatory responses to spontaneous sighs. Sighs led to apneic pauses followed by periods of periodic breathing with peripheral chemoreceptor CO2 gain, cardiac output, and lung volume were at reported normal levels. Apneic threshold when exceeded was observed to cause an asymmetry in the periodic breathing cycling and an increased periodic breathing frequency. Sighs in infants occur frequently enough to lead to repeated stimulation within the epoch duration of periodic breathing for a single sigh. Multiple sighs may then play a major role in promoting continuous periodic breathing in infants. Peripheral chemoreceptor gain estimated using endogenous CO2 led to validated predicted periodic breathing cycle duration as a function of age. Brain vascular volume increase with CO2 contributes to periodic breathing in very young (1-2 day old) preterm infants.

We formulate a statistical model for the regulation of global gene expression by multiple regulatory programs and propose a thresholding singular value decomposition (T-SVD) regression method for learning such a model from data. Extensive simulations demonstrate that this method offers improved computational speed and higher sensitivity and specificity over competing approaches. The method is used to analyze microRNA (miRNA) and long noncoding RNA (lncRNA) data from The Cancer Genome Atlas (TCGA) consortium. The analysis yields previously unidentified insights into the combinatorial regulation of gene expression by noncoding RNAs, as well as findings that are supported by evidence from the literature.

In central neurons, the threshold for spike initiation can depend on the stimulus and varies between cells and between recording sites in a given cell, but it is unclear what mechanisms underlie this variability. Properties of ionic channels are likely to play a role in threshold modulation. We examined in models the influence of Na channel activation, inactivation, slow voltage-gated channels and synaptic conductances on spike threshold. We propose a threshold equation which quantifies the contribution of all these mechanisms. It provides an instantaneous time-varying value of the threshold, which applies to neurons with fluctuating inputs. We deduce a differential equation for the threshold, similar to the equations of gating variables in the Hodgkin-Huxley formalism, which describes how the spike threshold varies with the membrane potential, depending on channel properties. We find that spike threshold depends logarithmically on Na channel density, and that Na channel inactivation and K channels can dynamically modulate it in an adaptive way: the threshold increases with membrane potential and after every action potential. Our equation was validated with simulations of a previously published multicompartemental model of spike initiation. Finally, we observed that threshold variability in models depends crucially on the shape of the Na activation function near spike initiation (about -55 mV), while its parameters are adjusted near half-activation voltage (about -30 mV), which might explain why many models exhibit little threshold variability, contrary to experimental observations. We conclude that ionic channels can account for large variations in spike threshold.

We review and revisit the predictive processing inspired "Gestalts as predictions" hypothesis. The study of Gestalt phenomena at and below threshold can help clarify the role of higher-order object selective areas and feedback connections in mid-level vision. In two psychophysical experiments assessing manipulations of contrast and configurality we showed that: (1) Gestalt phenomena are robust against saliency manipulations across the psychometric function even below threshold (with the accuracy gains and higher saliency associated with Gestalts being present even around chance performance); and (2) peak differences between Gestalt and control conditions happened around the time where responses to Gestalts are starting to saturate (mimicking the differential contrast response profile of striate vs. extra-striate visual neurons). In addition, Gestalts are associated with steeper psychometric functions in all experiments. We propose that these results reflect the differential engagement of object-selective areas in Gestalt phenomena and of information- or percept-based processing, as opposed to energy- or stimulus-based processing, more generally. In addition, the presence of nonlinearities in the psychometric functions suggest differential top-down modulation of the early visual cortex. We treat this as a proof of principle study, illustrating that classic psychophysics can help assess possible involvement of hierarchical predictive processing in Gestalt phenomena.

Epigenome-wide association studies (EWAS) are designed to characterise population-level epigenetic differences across the genome and link them to disease. Most commonly, they assess DNA-methylation status at cytosine-guanine dinucleotide (CpG) sites, using platforms such as the Illumina 450k array that profile a subset of CpGs genome wide. An important challenge in the context of EWAS is determining a significance threshold for declaring a CpG site as differentially methylated, taking multiple testing into account. We used a permutation method to estimate a significance threshold specifically for the 450k array and a simulation extrapolation approach to estimate a genome-wide threshold. These methods were applied to five different EWAS datasets derived from a variety of populations and tissue types. We obtained an estimate of α=2.4×10-7 for the 450k array, and a genome-wide estimate of α=3.6×10-8. We further demonstrate the importance of these results by showing that previously recommended sample sizes for EWAS should be adjusted upwards, requiring samples between ∼10% and ∼20% larger in order to maintain type-1 errors at the desired level.

Previous studies disclosed the pivotal role of methyltransferase complex G9a/Glp in the pathogenesis of neuropathic hypersensitivity induced by peripheral nerve injury. We observed that higher dose of G9a inhibitor improved nociceptive behavior, but the lower dose worsened pain. The aim of this study is to extensively observe the differential effect of various dosages of G9a/Glp inhibitors on nerve injury-induced allodynia.

Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications.

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.

Gene co-expression networks are often constructed by computing some measure of similarity between expression levels of gene transcripts and subsequently applying a high-pass filter to remove all but the most likely biologically-significant relationships. The selection of this expression threshold necessarily has a significant effect on any conclusions derived from the resulting network. Many approaches have been taken to choose an appropriate threshold, among them computing levels of statistical significance, accepting only the top one percent of relationships, and selecting an arbitrary expression cutoff.

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Cluster-based analysis methods in neuroimaging provide control of whole-brain false positive rates without the need to conservatively correct for the number of voxels and the associated false negative results. The current method defines clusters based purely on shapes in the landscape of activation, instead of requiring the choice of a statistical threshold that may strongly affect results. Statistical significance is determined using permutation testing, combining both size and height of activation. A method is proposed for dealing with relatively small local peaks. Simulations confirm the method controls the false positive rate and correctly identifies regions of activation. The method is also illustrated using real data. •A landscape-based method to define clusters in neuroimaging data avoids the need to pre-specify a threshold to define clusters.•The implementation of the method works as expected, based on simulated and real data.•The recursive method used for defining clusters, the method used for combining clusters, and the definition of the "value" of a cluster may be of interest for future variations.

The mammalian somatosensory system is comprised of multiple neuronal populations that form specialized, highly organized sensory endings in the skin. The organization of somatosensory endings is essential to their functions, yet the mechanisms which regulate this organization remain unclear. Using a combination of genetic and molecular labeling approaches, we examined the development of mouse hair follicle-innervating low-threshold mechanoreceptors (LTMRs) and explored competition for innervation targets as a mechanism involved in the patterning of their receptive fields. We show that follicle innervating neurons are present in the skin at birth and that LTMR receptive fields gradually add follicle-innervating endings during the first two postnatal weeks. Using a constitutive Bax knockout to increase the number of neurons in adult animals, we show that two LTMR subtypes have differential responses to an increase in neuronal population size: Aδ-LTMR neurons shrink their receptive fields to accommodate the increased number of neurons innervating the skin, while C-LTMR neurons do not. Our findings suggest that competition for hair follicles to innervate plays a role in the patterning and organization of follicle-innervating LTMR neurons.