The nuclear factor kappaB (NF-kappaB) transcription regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations abundance. To define function of these oscillations, we treated cells with repeated short pulses tumor necrosis factor-alpha at various intervals mimic pulsatile inflammatory signals. At all pulse that were analyzed, observed synchronous cycles translocation. Lower frequency stimulations gave full-amplitude translocations,...

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate origins this behavior understand its functional relevance, we quantitatively analyzed variability using new mathematical tools that allowed us reconstruct dynamic transcription rates different reporter controlled by identical promoters same living cell. Quantitative microscopic analysis two genes, firefly luciferase...

Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient progesterone (0-3 microM) simulate approach oocyte induced slowly developing increase [Ca(2+)](i) upon which, many cells, slow oscillations were superimposed. often started very low (<10 nm), and their frequency did not change during subsequent rise concentration. Oscillations also occurred, but much smaller proportion...

Heterogeneity between individual cells is a common feature of dynamic cellular processes, including signaling, transcription, and cell fate; yet the overall tissue level physiological phenotype needs to be carefully controlled avoid fluctuations. Here we show that in NF-κB signaling system, precise timing dual-delayed negative feedback motif [involving stochastic transcription inhibitor κB (IκB)-α -ε] optimized induce heterogeneous oscillations cells. We suggest this enables generate robust...

Fluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires development effective statistical methods calibrate such against available data. Given prevalence stochasticity noise biochemical systems inference for stochastic is special interest. In this paper we present a simple computationally efficient...

Nuclear factor kappa B (NF-kappaB) signalling is activated by cellular stress and inflammation regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses different doses of tumour necrosis alpha (TNFalpha). Lower fewer cells those responding showed an increasingly variable delay in the initial NF-kappaB nuclear translocation associated IkappaBalpha degradation. Robust 100 minute nuclear:cytoplasmic oscillations were observed over a wide range TNFalpha...

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability NF-κB activation. However, fewer cells shorter pulse intervals (<100 min) suggesting heterogeneous refractory state. This state is established in the signal transduction network downstream TNFR and upstream IKK, depends on level system negative feedback protein A20. If second within phase IL-1β instead TNFα, all respond. suggests...

Significance Inflammation is often accompanied by temperature change, but little known about the role of in inflammatory response. We show that physiologically relevant changes significantly perturb NF-κB dynamics following TNFα stimulation single cells. Using experimentation informed mathematical modeling, we found these were mediated, at least part, through key feedback gene TNFAIP3/A20. Curtailing A20 expression removed sensitivity across fever range (37 °C to 40 °C). Gene was generally...

Dynamic cellular systems reprogram gene expression to ensure appropriate fate responses specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and cell cycle are prioritised differently depending on timing an inflammatory signal. Using iterative experimental computational analyses, show physical functional interactions between NF-κB E2 1 (E2F-1) 4 (E2F-4) regulators. These modulate response. In S-phase, response was delayed or...

The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 μM) and cyclopiazonic acid (10 μM), failed to affect resting [Ca2+] in human spermatozoa. Slow progesterone-induced [Ca2+ i]i oscillations spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive these inhibitors. Non-selective doses (5-30 μM, 50-300 times the saturating dose for SERCA inhibition), caused elevation [Ca2+]i partial, dose-dependent...

Abstract Motivation: Promoter-driven reporter genes, notably luciferase and green fluorescent protein, provide a tool for the generation of vast array time-course data sets from living cells organisms. The aim this study is to introduce modeling framework based on stochastic differential equations (SDEs) ordinary (ODEs) that addresses problem reconstructing transcription profiles associated degradation rates. dynamical model embedded into Bayesian inference performed using Markov chain Monte...

Abstract The COVID-19 pandemic caused a rapid seismic shift to online delivery in otherwise face-to-face higher education settings worldwide. This quantitative research study sought investigate the effect of different styles and assessment types on student outcomes. Specifically, grades achieved by first year undergraduate Biological Science students at UK Higher Education institution were compared from seven modules across two academic years, namely 2018–2019 2020–2021. was delivered...

Mechanisms controlling the process and patterning of blood vessel development in placenta remain largely unknown. The close physical proximity early vessels observed cytotrophoblast, as well reported production vasculogenic growth factors by latter, suggests that signalling between these two niches may be important. Here, we have developed an vitro model to address hypothesis secretion soluble factors, drives differentiation resident sub-trophoblastic mesenchymal stem cells (MSCs) along a...

Estrogens have been implicated in the regulation of prolactin gene expression man, although previous studies not defined molecular mechanism whereby estradiol activates human promoter (hPrl). We found that induced a reproducible 1.8-fold activation hPrl promoter, using pituitary GH3 cells stably transfected with 5000-bp fragment linked to luciferase reporter gene. This was blocked by treatment estrogen receptor (ER) antagonists 4-hydroxytamoxifen and ICI-182,780. Promoter deletion...

An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) - exocytosis of a single large vesicle (the acrosome) that surrounds nucleus at apical head. The acrosomal and plasma membranes fuse, resulting both release factors might facilitate penetration zona pellucida (which invests egg) externalisation membrane components required for gamete fusion. Exocytosis somatic cells rapid process typically complete within milliseconds yet enzymes are throughout period minutes....

Gene expression in living cells is highly dynamic, but temporal patterns of gene intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used quantify reporter dispersed lactotrophic or tissue from bacterial artificial chromosome (BAC) transgenic rats which a large prolactin genomic fragment directed luciferase...

A central challenge in computational modeling of dynamic biological systems is parameter inference from experimental time course measurements. However, one would not only like to infer kinetic parameters but also study their variability cell cell. Here we focus on the case where single-cell fluorescent protein imaging series data are available for a population cells. Based van Kampen's linear noise approximation, derive state space model molecular populations which then extended hierarchical...

Progesterone induces a biphasic Ca(2+) influx and consequent acrosome reaction in human spermatozoa. We have used two procedures to vary the stimulus (dosage prior receptor desensitization) investigate encoding of strength by intracellular [Ca(2+)] ([Ca(2+)](i)). Acrosome amplitude (but not kinetics) transient [Ca(2+)](i) response (population measurement) showed sigmoidal dose sensitivity over range 0.3 nM-3 microM, saturating at approximately 300 nM (ED(50) 30 nM). The sustained saturated 3...

Progesterone is present at micromolar concentrations in the vicinity of oocyte. Human spermatozoa generate a biphasic rise intracellular calcium concentration ([Ca2+]i) and undergo acrosome reaction upon progesterone stimulation, suggesting that hormone acts as secondary inducer or ‘primer’ association with zona pellucida. However, sensitivity human to such many cells may prematurely, compromising their ability fertilize. We have shown exposing gradient, simulating stimulus encountered sperm...

Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-α activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control 5 kb human promoter. Similar regulation endogenous rat was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns promoter activity individual cells. In treated with TNF-α,...

Progesterone at 3 microM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca(2+)](i)) human sperm, which is believed to be prerequisite for progesterone-induced acrosome reaction (AR). As very little known about how AR occurrence, latency, completion relate the characteristics of [Ca(2+)](i) signal, we examined these events using fluorescence microscopy individual living sperm. Direct assessment acrosomal status after imaging showed no differences kinetics or...

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) locus as physiologically important model system.The approach combines advantages bacterial artificial chromosome recombineering to report appropriate regulation expression by distant elements, with double reporter activity study highly dynamic promoter and ex vivo.We show first that this allows quantitative pituitary gland, allowing...