The role of incoherent feedforward circuits in regulating precision of event timing

AbstractTriggering of cellular events often relies on the level of a key gene product crossing a critical threshold. Achieving precision in event timing in spite of noisy gene expression facilitates high-fidelity functioning of diverse processes from biomolecular clocks, apoptosis and cellular differentiation. Here we investigate the role of an incoherent feedforward circuit in regulating the time taken by a bacterial virus (bacteriophage lambda) to lyse an infected Escherichia coli cell. Lysis timing is the result of expression and accumulation of a single lambda protein (holin) in the E. coli cell membrane up to a critical threshold level, which triggers the formation of membrane lesions. This easily visualized process provides a simple model system for characterizing event-timing stochasticity in single cells. Intriguingly, lambda’s lytic pathway synthesizes two functionally opposite proteins: holin and antiholin from the same mRNA in a 2:1 ratio. Antiholin sequesters holin and inhibits the formation of lethal membrane lesions, thus creating an incoherent feedforward circuit. We develop and analyze a stochastic model for this feedforward circuit that considers correlated bursty expression of holin/antiholin, and their concentrations are diluted from cellular growth. Interestingly, our analysis shows the noise in timing is minimized when both proteins are expressed at an optimal ratio, hence revealing an important regulatory role for antiholin. These results are in agreement with single cell data, where removal of antiholin results in enhanced stochasticity in lysis timing.

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Distinguishing different modes of growth using single-cell data

eLife ◽

10.7554/elife.72565 ◽

2021 ◽

Vol 10 ◽

Author(s):

Prathitha Kar ◽

Sriram Tiruvadi-Krishnan ◽

Jaana Männik ◽

Jaan Männik ◽

Ariel Amir

Keyword(s):

Data Analysis ◽

Single Cell ◽

Statistical Methods ◽

Synthetic Data ◽

Cellular Growth ◽

Biological Mechanisms ◽

E Coli ◽

High Throughput Data ◽

Consistent Method ◽

Cell Data

Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

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Intracellular Complexes of Viral Spike and Cellular Receptor Accumulate during Cytopathic Murine Coronavirus Infections

Journal of Virology ◽

10.1128/jvi.72.4.3278-3288.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 3278-3288 ◽

Cited By ~ 19

Author(s):

Pasupuleti V. Rao ◽

Thomas M. Gallagher

Keyword(s):

Trypan Blue ◽

Hepatitis Virus ◽

Syncytium Formation ◽

Threshold Level ◽

Inducible Promoter ◽

Critical Threshold ◽

Murine Hepatitis Virus ◽

Trypan Blue Exclusion ◽

Hela Cell Lines

ABSTRACT Murine hepatitis virus (MHV) infections exhibit remarkable variability in cytopathology, ranging from acutely cytolytic to essentially asymptomatic levels. In this report, we assess the role of the MHV receptor (MHVR) in controlling this variable virus-induced cytopathology. We developed human (HeLa) cell lines in which the MHVR was produced in a regulated fashion by placing MHVR cDNA under the control of an inducible promoter. Depending on the extent of induction, MHVR levels ranged from less than ∼1,500 molecules per cell (designated Rlo) to ∼300,000 molecules per cell (designated Rhi). Throughout this range, the otherwise MHV-resistant HeLa cells were rendered susceptible to infection. However, infection in the Rlo cells occurred without any overt evidence of cytopathology, while the corresponding Rhi cells died within 14 h after infection. When the HeLa-MHVR cells were infected with vaccinia virus recombinants encoding MHV spike (S) proteins, the Rhi cells succumbed within 12 h postinfection; Rlo cells infected in parallel were intact, as judged by trypan blue exclusion. This acute cytopathology was not due solely to syncytium formation between the cells producing S and MHVR, because fusion-blocking antiviral antibodies did not prevent it. These findings raised the possibility of an intracellular interaction between S and MHVR in the acute cell death. Indeed, we identified intracellular complexes of S and MHVR via coimmunoprecipitation of endoglycosidase H-sensitive forms of the two proteins. We suggest that MHV infections can become acutely cytopathic once these intracellular complexes rise above a critical threshold level.

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Optimum Threshold Minimizes Noise in Timing of Intracellular Events

10.1101/2020.02.14.949891 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sherin Kannoly ◽

Tianhui Gao ◽

Supravat Dey ◽

Ing-Nang Wang ◽

Abhyudai Singh ◽

...

Keyword(s):

Fundamental Problem ◽

Single Cells ◽

Threshold Level ◽

Site Directed Mutagenesis ◽

Critical Threshold ◽

Cellular Processes ◽

Lysis Time ◽

Escherchia Coli ◽

Stochastic Expression ◽

Optimum Threshold

ABSTRACTHow the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage λ to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherchia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene was used to generate phage variants that vary in their timing of lysis from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding – the noise in lysis timing first decreases with increasing lysis time to reach a minimum, and then sharply increases at longer longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile, and identify holin accumulation thresholds that generate precision in lysis timing.

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E2f3 is critical for normal cellular proliferation

Genes & Development ◽

10.1101/gad.14.6.690 ◽

2000 ◽

Vol 14 (6) ◽

pp. 690-703 ◽

Cited By ~ 6

Author(s):

Patrick O. Humbert ◽

Raluca Verona ◽

Jeffrey M. Trimarchi ◽

Catherine Rogers ◽

Savita Dandapani ◽

...

Keyword(s):

Transcriptional Activation ◽

Cellular Proliferation ◽

Threshold Level ◽

Critical Threshold ◽

Cellular Immortalization ◽

Proliferation And Differentiation ◽

Rate Limiting ◽

Deficient Mice

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, we generated an E2f3 mutant mouse strain. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, we analyzed the properties of embryonic fibroblasts derived from E2f3 mutant mice. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. We have been able to identify a number of genes, including B-myb,cyclin A, cdc2, cdc6, and DHFR, whose expression is dependent on the presence of E2F3 but not E2F1. We further show that a critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. Finally, we show that E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. We conclude that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells.

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A Sensitivity Analysis on the Consequences Assessment in Cases of Toxic Substance Released by Pipelines

Journal of Konbin ◽

10.2478/v10040-008-0009-7 ◽

2008 ◽

Vol 4 (1) ◽

pp. 39-56

Author(s):

Gagliardi Valentina ◽

Citro Lucia

Keyword(s):

Sensitivity Analysis ◽

Toxic Substance ◽

Threshold Level ◽

Critical Threshold ◽

Toxic Substances ◽

Threshold Values ◽

Hazard Area ◽

The Impact ◽

Harmful Effects

A Sensitivity Analysis on the Consequences Assessment in Cases of Toxic Substance Released by Pipelines This paper presents preliminary results of a study aimed at evaluating the impact on man and environment of major accident hazards related to transmission pipelines carrying toxic substances. In particular, it describes the assessment of the consequences of potential toxic release from pipelines, expressed in terms of hazard area, that is the zone in which the concentration of a toxic substance exceed a critical threshold level and induce harmful effects on people and the environment. Moreover, a sensitivity analysis has been undertaken, emphasizing the role of threshold values on the results obtained.

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To bin or not to bin: analyzing single-cell growth data

10.1101/2021.07.27.453901 ◽

2021 ◽

Author(s):

Prathitha Kar ◽

Sriram Tiruvadi-Krishnan ◽

Jaana Männik ◽

Jaan Männik ◽

Ariel Amir

Keyword(s):

Data Analysis ◽

Single Cell ◽

Statistical Methods ◽

Synthetic Data ◽

Cellular Growth ◽

Growth Data ◽

Biological Mechanisms ◽

E Coli ◽

Consistent Method ◽

Cell Data

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The Local Sanarelli-Shwartzman Phenomenon in Rats Induced by Human Leukaemic Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647777 ◽

1973 ◽

Vol 29 (02) ◽

pp. 353-362

Author(s):

J Lisiewicz ◽

A Pituch ◽

J. A Litwin

Keyword(s):

Chronic Lymphocytic Leukaemia ◽

Intravenous Injection ◽

Peripheral Blood ◽

Lymphocytic Leukaemia ◽

Acute Myeloblastic Leukaemia ◽

Demarcation Line ◽

E Coli ◽

Leukaemic Cells ◽

Shwartzman Phenomenon

SummaryThe local Sanarelli-Shwartzman phenomenon (SSP-L) in the skin of 30 rats was induced by an intr a cutaneous sensitizing injection of leukaemic leucocytes isolated from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL), acute myeloblastic leukaemia (AL) and chronic granulocytic leukaemia (CGL) and challenged by an intravenous injection of 100(μ of E. coli endotoxin. SSP-L was observed in 7 rats after injection of CLL lymphocytes and in 6 and 2 rats after AL myeloblasts and the CGL granulocytes, respectively. The lesions in the skin after AL myeloblasts appeared in a shorter time and were of longer duration compared with those observed after CLL lymphocytes and CGL granulocytes. Histologically, the lesions consisted of areas of destruction in the superficial layers of the skin ; the demarcation line showed the presence of neutrophils, macrophages and erythrocytes. Haemorrhages and fibrin deposits near the demarcation line were larger after injection of CLL lymphocytes and AL myeloblasts than after CGL granulocytes. The possible role of leucocyte procoagulative substances in the differences observed have been discussed.

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Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

10.26434/chemrxiv.12046125 ◽

2020 ◽

Author(s):

Feifei Jia ◽

Jie Wang ◽

Yanyan Zhang ◽

Qun Luo ◽

Luyu Qi ◽

...

Keyword(s):

Mass Spectrometry ◽

Secondary Ion Mass Spectrometry ◽

Single Cells ◽

Time Of Flight ◽

E Coli ◽

Tof Sims ◽

Ion Mass Spectrometry ◽

Chemical Tags ◽

Secondary Ion

In situ visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.

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Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2016.9.2.8 ◽

1970 ◽

Vol 9 (2) ◽

pp. 3212-3214

Author(s):

Pramod Dhakal ◽

Ankit a Achary ◽

Vedamurthy Joshi

Keyword(s):

Antibacterial Activity ◽

Azadirachta Indica ◽

Pathogenic Bacteria ◽

Ethanol Extract ◽

Watery Diarrhea ◽

Diffusion Method ◽

E Coli ◽

Drug Molecules ◽

Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

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E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

Cells ◽

10.3390/cells8090982 ◽

2019 ◽

Vol 8 (9) ◽

pp. 982

Author(s):

Xiaoyan Peng ◽

Rongguang Zhang ◽

Chen Wang ◽

Feiyan Yu ◽

Mingyang Yu ◽

...

Keyword(s):

Cellular Immunity ◽

Protective Efficacy ◽

Gastric Injury ◽

Oral Vaccines ◽

Gastric Mucus ◽

E Coli ◽

Non Invasive ◽

Considerable Impact ◽

H Pylori

Current studies indicate that the anti-H. pylori protective efficacy of oral vaccines to a large extent depends on using mucosal adjuvants like E. coli heat-lable enterotoxin B unit (LtB). However, the mechanism by which Th17/Th1-driven cellular immunity kills H. pylori and the role of LtB remains unclear. Here, two L. lactis strains, expressing H. pylori NapA and LtB, respectively, were orally administrated to mice. As observed, the administration of LtB significantly enhanced the fecal SIgA level and decreased gastric H. pylori colonization, but also markedly aggravated gastric inflammatory injury. Both NapA group and NapA+LtB group had elevated splenocyte production of IL-8, IL-10, IL-12, IL-17, IL-23 and INF-γ. Notably, gastric leukocytes’ migration or leakage into the mucus was observed more frequently in NapA+LtB group than in NapA group. This report is the first that discusses how LtB enhances vaccine-induced anti-H. pylori efficacy by aggravating gastric injury and leukocytes’ movement into the mucus layer. Significantly, it brings up a novel explanation for the mechanism underlying mucosal cellular immunity destroying the non-invasive pathogens. More importantly, the findings suggest the necessity to further evaluate LtB’s potential hazards to humans before extending its applications. Thus, this report can provide considerable impact on the fields of mucosal immunology and vaccinology.

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