Protein Dynamics in Individual Human Cells: Experiment and Theory

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells

Protein Dynamics in Individual Human Cells: Experiment and Theory

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells

Dynamic Proteomics: a database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells

Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/

The Risk of Immunosuppression: A Case of Salmonella Meningitis

Salmonella meningitis is a rare infection, particularly in adults. We report the case of a 75-year-old female with a history of rheumatoid arthritis on TNF-antagonist immunosuppressive therapy who initially presented to the hospital for management of back and leg pain and was ultimately diagnosed with bacterial meningitis secondary to Salmonella species infection. She was treated with ceftriaxone with slow improvement in neurological function. Though the source of infection was never clearly identified from multiple imaging studies, we suspect the severity of her presentation was due to her history of TNF-antagonist use

Generation of a fluorescently labeled endogenous protein library in living human cells

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100–200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3′ rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format

Exposure to environmental tobacco smoke in non - smoking adults in Israel: results of the second Israel biomonitoring survey

Abstract Background Exposure to environmental tobacco smoke (ETS) increases the risk of heart and respiratory disease, cancer, and premature mortality in non-smoking individuals. Results from the first Israel Biomonitoring Study in 2011 showed that over 60% of non-smoking adults are exposed to ETS. The purpose of the current study was to assess whether policies to restrict smoking in public places have been associated with reductions in exposure to ETS, and to examine predictors of exposure. Methods We analyzed urinary cotinine and creatinine concentrations in 194 adult participants in the National Health and Nutrition (RAV MABAT) Survey in 2015–2016. Study participants were interviewed in person on smoking status and exposure to ETS. We calculated creatinine-adjusted and unadjusted urinary cotinine geometric means and medians among smokers and non-smokers. We analyzed associations in univariable analyses, between socio-demographic variables and self – reported exposure, and urinary cotinine concentrations. Results There was no reduction in geometric mean urinary cotinine levels in non-smokers in the current study (1.7 μg/g) compared to that in 2011 (1.6 μg/g). Median cotinine levels among the non – smoking Arab participants were higher in comparison to the Jewish and other participants (2.97 versus 1.56 μg/l, p = 0.035). Participants who reported that they were exposed to ETS at home had significantly higher median levels of creatinine adjusted urinary cotinine than those reporting they were not exposed at home (4.19 μg/g versus 2.9 μg/g, p = 0.0039). Conclusions Despite additional restrictions on smoking in public places in 2012–2016, over 60% of non-smoking adults in Israel continue to be exposed to ETS. Urinary cotinine levels in non-smokers have not decreased compared to 2011. Results indicate higher exposure to ETS in Arab study participants and those reporting ETS exposure at home. There is an urgent need: (1) to increase enforcement on the ban on smoking in work and public places; (2) for public health educational programs and campaigns about the adverse health effects of ETS; and (3) to develop and disseminate effective interventions to promote smoke free homes. Periodic surveys using objective measures of ETS exposure (cotinine) are an important tool for monitoring progress, or lack thereof, of policies to reduce exposure to tobacco smoke in non-smokers

Protein Dynamics in Individual Human Cells: Experiment and Theory

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells

Dynamic Proteomics of Human Protein Level and Localization across the Cell Cycle

<div><p>Regulation of proteins across the cell cycle is a basic process in cell biology. It has been difficult to study this globally in human cells due to lack of methods to accurately follow protein levels and localizations over time. Estimates based on global mRNA measurements suggest that only a few percent of human genes have cell-cycle dependent mRNA levels. Here, we used dynamic proteomics to study the cell-cycle dependence of proteins. We used 495 clones of a human cell line, each with a different protein tagged fluorescently at its endogenous locus. Protein level and localization was quantified in individual cells over 24h of growth using time-lapse microscopy. Instead of standard chemical or mechanical methods for cell synchronization, we employed in-silico synchronization to place protein levels and localization on a time axis between two cell divisions. This non-perturbative synchronization approach, together with the high accuracy of the measurements, allowed a sensitive assay of cell-cycle dependence. We further developed a computational approach that uses texture features to evaluate changes in protein localizations. We find that 40% of the proteins showed cell cycle dependence, of which 11% showed changes in protein level and 35% in localization. This suggests that a broader range of cell-cycle dependent proteins exists in human cells than was previously appreciated. Most of the cell-cycle dependent proteins exhibit changes in cellular localization. Such changes can be a useful tool in the regulation of the cell-cycle being fast and efficient.</p> </div

Moderating Role of Depression on the Association of Tic Severity With Functional Impairment in Children

BACKGROUND: Chronic tic disorders (CTDs) commonly co-occur with other psychiatric disorders. CTDs have been linked to functional impairment and reduction in quality of life. Insufficient research is available on depressive symptoms in patients with CTD, especially children and adolescents, yielding conflicting findings. To investigate the presence of depressive symptoms in a cohort of children and young adolescents with CTD and to test whether they moderate the link between tic severity and functional impairment.METHODS: The sample consisted of 85 children and adolescents (six to 18 years) with a CTD who were treated in a large referral center. Participants were evaluated using gold-standard self- and clinician-reporting instruments to measure tic symptom severity and tic-related functional impairment (Yale Global Tic Severity Scale), depression (Child Depression Inventory), and obsessive-compulsive symptoms (Children Yale Brown Obsessive Compulsive Scale).RESULTS: Depressive symptoms (mild to severe) were exhibited by 21% of our sample. Study participants with CTD and comorbid obsessive-compulsive disorder (OCD) and/or attention-deficit/hyperactivity disorder had higher rates of depressive symptoms compared with those without comorbidities. Significant correlations were found within and among all tic-related and OCD-related measures, yet depressive symptoms only correlated to tic-related functional impairment. Depression significantly and positively moderated the correlation between tic severity and tic-related functional impairment.CONCLUSIONS: Findings suggest that depression plays an important part as a moderator in the link between tic severity and functional impairment in children and adolescents. Our study highlights the importance of screening for and treating depression in patients with CTD.</p