Gene expression profiles are an increasingly common data source that can yield insights into the functions of cells at a system-wide level. The present work considers the limitations in information ...content of gene expression data for reverse engineering regulatory networks. An in silico genetic regulatory network was constructed for this purpose. Using the in silico network, a formal identifiability analysis was performed that considered the accuracy with which the parameters in the network could be estimated using gene expression data and prior structural knowledge (which transcription factors regulate which genes) as a function of the input perturbation and stochastic gene expression. The analysis yielded experimentally relevant results. It was observed that, in addition to prior structural knowledge, prior knowledge of kinetic parameters, particularly mRNA degradation rate constants, was necessary for the network to be identifiable. Also, with the exception of cases where the noise due to stochastic gene expression was high, complex perturbations were more favorable for identifying the network than simple ones. Although the results may be specific to the network considered, the present study provides a framework for posing similar questions in other systems.
The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In ...pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK), causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR), while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
1 The Cardeza Foundation for Hematologic Research and Division of Hematology, Department of Medicine, Jefferson Medical College
2 Daniel Baugh Institute for Functional Genomics and Computational ...Biology, Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Understanding regulation of fetal and embryonic hemoglobin expression is critical, since their expression decreases clinical severity in sickle cell disease and ß-thalassemia. K562 cells, a human erythroleukemia cell line, can differentiate along erythroid or megakaryocytic lineages and serve as a model for regulation of fetal/embryonic globin expression. We used microarray expression profiling to characterize transcriptomes from K562 cells treated for various times with hemin, an inducer of erythroid commitment. Approximately 5,000 genes were expressed irrespective of treatment. Comparative expression analysis (CEA) identified 899 genes as differentially expressed; analysis by the self-organizing map (SOM) algorithm clustered 425 genes into 8 distinct expression patterns, 322 of which were shared by both analyses. Differential expression of a subset of genes was validated by real-time RT-PCR. Analysis of 5'-flanking regions from differentially expressed genes by PAINT v3.0 software showed enrichment in specific transcription regulatory elements (TREs), some localizing to different expression clusters. This finding suggests coordinate regulation of cluster members by specific TREs. Finally, our findings provide new insights into rate-limiting steps in the appearance of heme-containing hemoglobin tetramers in these cells.
gene expression profiling; hemin; cell differentiation; transcription regulatory elements
The broad conceptual postulate that systems engineering techniques developed for complex chemical processes may be applicable to complex cell biological processes is very compelling. However, a ...naïve, “direct” application of systems engineering techniques to biological problems of practical significance may be rendered virtually ineffective by fundamental differences between cell biology and chemical processes. These differences and the problems they pose are illustrated in this paper with an example problem: modeling a gene regulatory network involved in the yeast cell cycle. We demonstrate how the biological essence complicates a straightforward “process modeling/identification” problem and subsequently recommend an alternative approach. The approach—a middle ground between a direct, “off the shelf” application of systems engineering tools and a “one-at-a-time” ad-hoc development—incorporates fundamental knowledge of the mechanisms and constraints intrinsic to biological systems. The principles and implementation details of the approach are illustrated with the case study.
Multisystem inflammatory syndrome in children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory illness characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ...antigenemia, cytokine storm, and immune dysregulation. In severe COVID-19, neutrophil activation is central to hyperinflammatory complications, yet the role of neutrophils in MIS-C is undefined. Here, we collect blood from 152 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 78 pediatric controls. We find that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism that is distinct from the neutrophil interferon-stimulated gene (ISG) response we observe in pediatric COVID-19. Moreover, we observe extensive spontaneous neutrophil extracellular trap (NET) formation in MIS-C, and we identify neutrophil activation and degranulation signatures. Mechanistically, we determine that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Our findings suggest that hyperinflammatory presentation during MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia, driven by uncontrolled neutrophil activation and NET release in the vasculature.
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•Neutrophils in pediatric COVID-19 express an interferon-stimulated gene response•MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell signature•Neutrophil activation and extensive spontaneous NETosis are seen in MIS-C•SARS-CoV-2 spike immune complexes, especially IgA immune complexes, trigger NETs
Boribong, LaSalle, et al. extensively profile gene expression, protein production, and functionality of neutrophils from children with acute COVID-19 or multisystem inflammatory syndrome in children (MIS-C) to define neutrophil responses driving distinct SARS-CoV-2 disease states. They propose a model whereby neutrophil activation elicits endothelial dysfunction and cardiovascular complications within MIS-C.
Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the ...mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.
Significance
Regulatory T cells (Tregs) are responsible for restraining excessive inflammation, a hallmark of COVID-19. We identified a striking phenotype in Tregs from patients with severe disease, ...as well as an interesting role for interleukin (IL)-6 and IL-18. An increased suppressive profile, including increased Treg proportions, combined with the expression of proinflammatory mediators, distinguished severe patients and persisted in some of those recovered. This phenotype is in notable similarity to that found in tumor-infiltrating Tregs, which are generally associated with poor prognosis, and suggests both a detrimental role for these cells in COVID-19 as well as a potential explanation for some of the still largely unexplored complications associated with recovery.
The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3
+
T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19–linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.
A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. ...The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca
2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at Ca
2+ well below those required to generate tension. Cross-bridges increased the Ca
2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca
2+ sensitivity and was required for full change in probe position. At submaximal Ca
2+, the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca
2+-bound cTnC significantly affected by Ca
2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release Ca
2+ by laser photolysis of NP-EGTA was Ca
2+ sensitive and biphasic: a rapid component ∼10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca
2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.
Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 ...symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.
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16% of COVID-19 patients display an atypical low-inflammatory plasma proteomeSevere COVID-19 is associated with heterogeneous plasma proteomic responsesDeath of virus-infected lung epithelial cells is a key feature of severe diseaseLung monocyte/macrophages drive T cell activation, together promoting epithelial damage
Filbin et al. use plasma proteomics in 306 coronavirus disease 2019 (COVID-19) patients and 78 symptomatic controls over time to better understand the role of circulating immune cells and tissue cells in inflammation, disease severity, and survival. They propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage.
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