Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events.

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

Relating individual cell division events to single-cell ERK and Akt activity time courses.

Biochemical correlates of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, and paired them to division events in the same single cells using the non-transformed MCF10A epithelial line. Following growth factor treatment, in cells that divide both ERK and Akt activities are significantly higher within the S-G2 time window (~ 8.5-40 h). Such differences were much smaller in the pre-S-phase, restriction point window which is traditionally associated with ERK and Akt activity dependence, suggesting unappreciated roles for ERK and Akt in S through G2. Simple metrics of central tendency in this time window are associated with subsequent cell division fates. ERK activity was more strongly associated with division fates than Akt activity, suggesting Akt activity dynamics may contribute less to the decision driving cell division in this context. We also find that ERK and Akt activities are less correlated with each other in cells that divide. Network reconstruction experiments demonstrated that this correlation behavior was likely not due to crosstalk, as ERK and Akt do not interact in this context, in contrast to other transformed cell types. Overall, our findings support roles for ERK and Akt activity throughout the cell cycle as opposed to just before the restriction point, and suggest ERK activity dynamics may be more important than Akt activity dynamics for driving cell division in this non-transformed context.

Functional Effects of Cardiomyocyte Injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

Transcriptomic profiling of human cardiac cells predicts protein kinase inhibitor-associated cardiotoxicity.

Kinase inhibitors (KIs) represent an important class of anti-cancer drugs. Although cardiotoxicity is a serious adverse event associated with several KIs, the reasons remain poorly understood, and its prediction remains challenging. We obtain transcriptional profiles of human heart-derived primary cardiomyocyte like cell lines treated with a panel of 26 FDA-approved KIs and classify their effects on subcellular pathways and processes. Individual cardiotoxicity patient reports for these KIs, obtained from the FDA Adverse Event Reporting System, are used to compute relative risk scores. These are then combined with the cell line-derived transcriptomic datasets through elastic net regression analysis to identify a gene signature that can predict risk of cardiotoxicity. We also identify relationships between cardiotoxicity risk and structural/binding profiles of individual KIs. We conclude that acute transcriptomic changes in cell-based assays combined with drug substructures are predictive of KI-induced cardiotoxicity risk, and that they can be informative for future drug discovery.

A Multi-center Study on the Reproducibility of Drug-Response Assays in Mammalian Cell Lines.

Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in practices that generate findable, accessible, interoperable, and reusable (FAIR) data. Multiple papers have identified specific examples of irreproducibility, but practical ways to make data more reproducible have not been widely studied. Here, five research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cellular networks, and patient stratification. While many experimental and computational factors impact intra- and inter-center reproducibility, the factors most difficult to identify and control are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways to identify such context-sensitive factors, thereby improving both the theory and practice of reproducible cell-based assays.

A mechanistic pan-cancer pathway model informed by multi-omics data interprets stochastic cell fate responses to drugs and mitogens.

Most cancer cells harbor multiple drivers whose epistasis and interactions with expression context clouds drug and drug combination sensitivity prediction. We constructed a mechanistic computational model that is context-tailored by omics data to capture regulation of stochastic proliferation and death by pan-cancer driver pathways. Simulations and experiments explore how the coordinated dynamics of RAF/MEK/ERK and PI-3K/AKT kinase activities in response to synergistic mitogen or drug combinations control cell fate in a specific cellular context. In this MCF10A cell context, simulations suggest that synergistic ERK and AKT inhibitor-induced death is likely mediated by BIM rather than BAD, which is supported by prior experimental studies. AKT dynamics explain S-phase entry synergy between EGF and insulin, but simulations suggest that stochastic ERK, and not AKT, dynamics seem to drive cell-to-cell proliferation variability, which in simulations is predictable from pre-stimulus fluctuations in C-Raf/B-Raf levels. Simulations suggest MEK alteration negligibly influences transformation, consistent with clinical data. Tailoring the model to an alternate cell expression and mutation context, a glioma cell line, allows prediction of increased sensitivity of cell death to AKT inhibition. Our model mechanistically interprets context-specific landscapes between driver pathways and cell fates, providing a framework for designing more rational cancer combination therapy.

Hazard identification of the potential for dieldrin carcinogenicity to humans.

Although dieldrin׳s use in the U.S. was partially banned in the 1970s and its use was completely eliminated in 1987, dieldrin continues to be a common contaminant at hazardous waste sites. The USEPA׳s current cancer potency estimate for dieldrin was derived in 1987 and is based on the production of mouse liver tumors. Because of its environmental persistence and its relatively high USEPA cancer potency estimate, dieldrin functions as a cleanup "driver" in many hazardous site remediations. Since 1987, new risk assessment perspectives and new data on dieldrin׳s carcinogenic potential have arisen. This review presents a reassessment of dielrin׳s human cancer potential in light of these new data and new perspectives. Based on this reassessment, dieldrin may be carcinogenic through multiple modes of action. These modes of action may operate within the same tissue, or may be specific to individual tissues. Of the several possible carcinogenic modes of action for dieldrin, one or more may be more relevant to human cancer risk than others, but the relative importance of each is unknown. In addition, neither the details of the possible modes of action, nor the shape of the tumor dose-response curves associated with each are sufficiently well known to permit quantitative cancer dose-response modeling. Thus, the mouse liver tumor data used by the USEPA in its 1987 assessment remain the only quantitative data available for cancer dose-response modeling.

Growth of flagellar filaments of Escherichia coli is independent of filament length.

Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel ∼2 nm in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, following an additional period of growth, with a red maleimide dye. The contour lengths of the green and red segments were measured. The average lengths of red segments (∼2.3 µm) were the same regardless of the lengths of the green segments from which they grew (ranging from less than 1 to more than 9 µm in length). Thus, flagellar filaments do not grow at a rate that decreases exponentially with length, as formerly supposed. If flagellar filaments were broken by viscous shear, the broken filaments continued to grow. Identical results were obtained whether flagellin was expressed from fliC on the chromosome under the control of its native promoter or on a plasmid under the control of the arabinose promoter.

Consumption patterns and risk assessment of crab consumers from the Newark Bay Complex, New Jersey, USA.

The Newark Bay Complex (NBC) is a significant historical repository of polychlorinated dibenzodioxins (PCDDs) and dioxin-like compounds. Detection of high levels of 2,3,7,8 tetrachloro-dibenzodioxins (TCDD) and its toxicological equivalents in blue crabs in the early 1990's led to a ban on the taking and distribution of crabs from the NBC. Despite this ban and ongoing communication outreach, surveys of crabbers in 1995, 2002 and 2005 by the New Jersey Department of Environmental Protection (NJDEP) showed that crabbing for recreational purposes and for significant dietary supplementation was continuing. At the time they were surveyed, the crabbers had been consuming these crabs for an average of 37% of their lives. Thus, exposure can be considered chronic. The surveys provided data on the duration, frequency and amount of NBC crab consumption. In 2004, the NJDEP sampled blue crabs in the NBC and analyzed the edible portions for 2,3,7,8 TCDD toxicity equivalent (TEQ) concentration. We have combined the survey-based exposure data and the 2,3,7,8 TCDD TEQ concentration data to produce an estimate of the lifetime cancer risk to NBC crabbers from dioxin-like compounds. We employed a point-estimate approach using discrete lower, central tendency and reasonable maximum exposure (RME) estimates of exposure factors and a probabilistic approach to exposure factors. Both approaches show central tendency lifetime cancer risk of greater than one-in-a-thousand (10(-3)) and an upper percentile/RME risk of approximately one-in-a-hundred (10(-2)). Little extrapolation is involved in applying the 2,3,7,8-TCDD TEQ concentration data in crabs to risk estimates in the population consuming those crabs. The ongoing and frequent nature of the crab collection minimizes the uncertainty often inherent in food recall surveys. These estimates point to the continued risk posed to NBC crab consumers and to the continuing importance of this resource which, with proper remediation, could provide ongoing benefit to the surrounding community.

A quantitative assessment of the carcinogenicity of hexavalent chromium by the oral route and its relevance to human exposure.

BACKGROUND: Hexavalent chromium (Cr(6+)) has long been recognized as an inhalation carcinogen. Useful data on its carcinogenicity by ingestion have been slower to develop. The 2008 NTP chronic bioassay of sodium dichromate dihydrate in drinking water found clear evidence of carcinogenicity in rodents and allows a generalizable estimate of the human ingestion cancer potency of Cr(6+). OBJECTIVES: To estimate the human ingestion cancer potency of Cr(6+) and evaluate its relevance for human exposure. METHODS: Tumors of the small intestine in male mice were selected as the critical endpoint, for the derivation of cancer potency. Following the 2005 USEPA Cancer Risk Assessment Guidelines, the point of departure in the dose-response data was defined using benchmark-dose modeling. Linear extrapolation was carried out from the point of departure and interspecies dose conversion was based on allometric scaling of body weight. RESULTS: Small intestine tumor incidence in male mice provided a robust and nearly identical fit with most available dose-response models using benchmark-dose modeling. A human equivalent cancer slope factor of 0.5 (mg/kg/day)(-1) was derived based on linear extrapolation from the point of departure. Statistical and kinetic analysis carried out on the NTP data as well as data reported in other studies support that the carcinogenicity of Cr(6+) did not result from an exceedance of the reduction capacity of the mouse gastrointestinal tract at the doses in the NTP study. CONCLUSIONS: Mouse gastric emptying time and human Cr(6+) dosing studies strongly suggest that even at doses considerably lower than those in the NTP study, Cr(6+) escapes reduction in both the mouse and human stomachs due to kinetic competition from Cr(6+) absorption and gastric emptying. The cancer potency derived from the NTP data is, therefore, deemed to be relevant and applicable to human exposure. Cr(6+) is, therefore, identified as "likely to be carcinogenic to humans" in accordance with the USEPA's cancer characterization rubric.

Hexavalent chromium in house dust--a comparison between an area with historic contamination from chromate production and background locations.

In contrast to Cr(+3), Cr(+6) is carcinogenic and allergenic. Although Cr(+6) can occur naturally, it is thought that most soil Cr(+6) is anthropogenic, however, the extent of Cr(+6) in the background environment is unknown. Cr(+6)-containing chromite ore processing residue (COPR) from chromate manufacture was deposited in numerous locations in Jersey City (JC), New Jersey. In the 1990's, significantly elevated concentrations of total Cr (Cr(+6)+Cr(+3)) were found in house dust near COPR sites. We undertook a follow-up study to determine ongoing COPR exposure. We compared Cr(+6) in house dust in JC to selected background communities with no known sources of Cr(+6). Samples were collected from living areas, basements and window wells. Cr(+6) was detected in dust from all JC and background houses. In the JC homes, the mean (+ or - SD) Cr(+6) concentration for all samples was 3.9 + or - 7.0 microg/g (range: non-detect-90.4 microg/g), and the mean Cr(+6) loading was 5.8 + or - 15.7 microg/m(2) (range: non-detect-196.4 microg/m(2)). In background homes, the mean Cr(+6) concentrations of all samples was 4.6 + or - 7.8 microg/g, (range, 0.05-56.6 microg/g). The mean loading was 10.0 + or - 27.9 microg/m(2) (range, 0.22-169.3 microg/m(2)). There was no significant difference between Cr(+6) dust concentrations in Jersey City and background locations. Stratification by sample location within houses and sampling method gave similar results. Samples exceeding 20 microg/g were obtained only from single wood surfaces in different homes. Lower concentrations in window well samples suggests transport from outside is not the major source of indoor Cr(+6). Landscaping and groundcover may influence indoor Cr(+6). There appears to be a widespread low level background of Cr(+6) that is not elevated in Jersey City homes despite its historic COPR contamination. It is possible that house dust, in general, is a source of Cr(+6) exposure with potential implications for persistence of chromium allergic contact dermatitis.

Comments on article "Toxicity and carcinogenicity of chromium compounds in humans" by Costa and Klein.

The recent article by Costa and Klein (2006) contains several inaccuracies about the federal drinking water standard for chromium. The federal drinking water standard for chromium is 100 ppb, not 50 ppb as stated by Costa and Klein, and it is based on non-carcinogenic effects, not a one in one million cancer risk level. The question of whether or not hexavalent chromium is carcinogenic via the oral route is the focus of much current interest and has major implications for regulation of chromium in drinking water and soil. The chronic drinking water study of hexavalent chromium currently being conducted by the National Toxicology Program will provide further information that will be useful in addressing this question.

Premenopausal sexual dimorphism in lipopolysaccharide-stimulated production and secretion of tumor necrosis factor.

OBJECTIVE: To establish whether sexual dimorphism in tumor necrosis factor (TNF) concentration in lipopolysaccharide (LPS)-stimulated whole blood culture is related to menopausal status or hormone concentrations. METHODS: Healthy volunteers (72 premenopausal female, 159 male, and 62 postmenopausal female) completed questionnaires and gave peripheral blood specimens for whole blood LPS-stimulated TNF assay and for selected hormone levels. TNFab microsatellite markers were genotyped. RESULTS: Mean LPS-stimulated TNF level in the premenopausal female group was 18% lower than the postmenopausal female mean (1579 +/- 913 pg/ml compared with 2257 +/- 881 in the men and 1965 +/- 950 in the postmenopausal women; p < 0.0003 and p 0.058, respectively). Analyzing a subset for which blood counts were obtained, mean stimulated TNF per monocyte was lower in the premenopausal female group than in the postmenopausal female group and appeared lower than in the male group (2.67 +/- 1.96 pg/ml per 10(3) monocytes vs 4.44 +/- 2.16 and 3.60 +/- 1.40; p = 0.018 and p = 0.12, respectively). Total plasma cortisol was higher in premenopausal women than men, and, in turn, higher in men than postmenopausal women (mean +/- SD 16.1 +/- 5.7, 12.2 +/- 3.6, and 10.4 +/- 4.3 microg/dl, respectively; p < 0.05 for each comparison). Using multiple linear regression to correct for covariates and TNF allelic effects, premenopausal status predicted TNF level independently from potential confounders or TNF genetic markers (covariate-adjusted decrement of 408 pg/ml; p = 0.0241). In the male group, total cortisol predicted lower TNF level (coefficient -67.5 pg/ml for each microg/dl cortisol; p = 0.0006 after stepwise selection), but total testosterone had no effect. In premenopausal women, LPS-stimulated TNF was not related to total estradiol, testosterone, or cortisol level. CONCLUSION: Premenopausal women had a lower mean whole blood LPS-stimulated TNF level than postmenopausal women, but there was no significant relation to total estradiol, testosterone, or cortisol levels in premenopausal women.

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain.

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.