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Visualization of mRNA molecules in single cells has revealed their core mechanisms as well as sources of cell-to-cell and spatiotemporal heterogeneity. Here, we describe a protocol to label, visualize, and quantify mRNA molecules by time-lapse imaging with the capability of resolving mRNA molecules over durations of hours to days. We provide links to mRNA-labeling plasmids as well as free software for a semi-automated image analysis pipeline. For complete details on the use and execution of this protocol, please refer to Guo and Lee (2022) and Kowalczyk et al. (2021).

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A myriad of inflammatory cytokines regulate signaling pathways to maintain cellular homeostasis. The IκB kinase (IKK) complex is an integration hub for cytokines that govern nuclear factor κB (NF-κB) signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here, we track dynamics of IKK complexes and nuclear NF-κB to identify upstream signaling features that determine same-cell responses. Experiments and modeling of single complexes reveal their size, number, and timing relays cytokine-specific control over shared signaling mechanisms with feedback regulation that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables cytokine-specific regulation and parsimonious information transfer. We propose that emergent properties of stochastic pooling are general principles of receptor signaling that have evolved for constructive information transmission in noisy molecular environments.

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MOTIVATION: Many biological processes are regulated by single molecules and molecular assemblies within cells that are visible by microscopy as punctate features, often diffraction limited. Here, we present detecting-NEMO (dNEMO), a computational tool optimized for accurate and rapid measurement of fluorescent puncta in fixed-cell and time-lapse images. RESULTS: The spot detection algorithm uses the à trous wavelet transform, a computationally inexpensive method that is robust to imaging noise. By combining automated with manual spot curation in the user interface, fluorescent puncta can be carefully selected and measured against their local background to extract high-quality single-cell data. Integrated into the workflow are segmentation and spot-inspection tools that enable almost real-time interaction with images without time consuming pre-processing steps. Although the software is agnostic to the type of puncta imaged, we demonstrate dNEMO using smFISH to measure transcript numbers in single cells in addition to the transient formation of IKK/NEMO puncta from time-lapse images of cells exposed to inflammatory stimuli. We conclude that dNEMO is an ideal user interface for rapid and accurate measurement of fluorescent molecular assemblies in biological imaging data. AVAILABILITY AND IMPLEMENTATION: The data and software are freely available online at https://github.com/recleelab. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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BACKGROUND: Inflammatory responses mediated by adipocytokines may affect both atherosclerosis development and progression, as well as the risk of in-stent restenosis. The aim of this study was to determine the relationships between blood leptin, adiponectin and tumor necrosis factor-α (TNF-α) concentrations and the 1-year outcome of superficial femoral artery (SFA) stenting. METHODS: Blood concentrations of leptin, adiponectin and TNF-α were determined in 70 patients undergoing SFA stenting due to intermittent claudication and in 40 patients undergoing carotid artery stenting (CAS). All subjects were followed up for at least 1 year in relation to the occurrence of clinically driven target lesion revascularization (TLR) or a major adverse cardiovascular event (MACE). RESULTS: Patients undergoing SFA stenting and CAS had similar blood adipocytokine concentrations. Patients with diabetes mellitus presented a higher leptin concentration, lower adiponectin-to-leptin ratio, and lower blood adiponectin concentration indexed to fat mass (FM) and to visceral adiposity score (VAS). In Kaplan-Meier analysis, blood concentration of TNF-α indexed to FM and to VAS was higher in patients who underwent TLR and MACE. However, in multifactorial analysis, the severity of atherosclerosis lesions in the femoropopliteal vascular region, estimated in relation to TASC-II classification, was the only predictor of TLR. CONCLUSIONS: Circulating adipocytokines did not distinguish patients with different clinical manifestations of atherosclerosis. Higher ratios of TNF-α -to-FM and to VAS before SFA stenting were related to TLR and MACE occurrence. Dysregulation in adipocytokine secretion may be a potential mediator of a proatherogenic action of diabetes mellitus in patients with peripheral artery disease.

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BACKGROUND: The pathogenesis of in-stent restenosis is still not clear. The aim of this study was to determine the nutritional status of patients with lower limb ischemia and the risk of target lesion revascularization (TLR) after superficial femoral artery (SFA) stenting. METHODS: Numerous parameters of nutritional status assessment were compared between 70 patients undergoing SFA endovascular intervention with a self-expandable plane stent due to life-limiting intermittent claudication and 40 patients undergoing carotid artery stenting (CAS). All subjects were followed up for at least 1 year in relation to outcomes such as clinically driven TLR occurrence. RESULTS: Patients undergoing SFA stenting had a lower prevalence of overweight and obesity than those who underwent CAS (51.43% vs. 72.50%; P=0.031). An increase in Ankle-Brachial Index of >0.15 after SFA stenting (early end-point) was positively associated with greater handgrip strength (HGS), fat-free mass, skeletal muscle mass and waist-to-hip ratio. Freedom from TLR (late end-point) was significantly related to a higher waist-to-height ratio (WHtR), HGS and Geriatric Nutritional Risk Index (GNRI) Score. The 1-year risk of TLR for patients with a WHtR of ≥61.39 amounted to odds ratio; 95% confidence interval: 0.21; 0.05-0.25; P=0.021. CONCLUSIONS: Parameters of nutritional status assessment were associated with early and late outcomes of SFA stenting in patients with intermittent claudication. Abdominal fat distribution and higher HGS and GNRI scores lowered the 1-year risk of TLR. Further study is needed to determine the pathomechanism of the obesity paradox, sarcopenia and undernutrition in relation to outcomes of endovascular interventions.

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PURPOSE: The aim of this study was to determine the relationships between total lymphocyte count (TLC) and prognosis among inpatients. PATIENTS AND METHODS: We retrospectively analyzed data from electronic medical documentation of 54 976 inpatients hospitalized in an urban university hospital during 3 consecutive years (2014-2017). RESULTS: TLC was available for 12 651 (23.01%) of the inpatients. Patients with TLC <0.8 G/L constituted about 15% of the inpatients studied and had the highest risk of death, hospital readmission within 14 days, hospital readmission within 30 days and hospital readmission within 1 year, the lowest values for biochemical parameters of nutritional status assessment, and the highest C-reactive protein levels. An increase in TLC was associated with reduced risk of in-hospital death (odds ratio [OR]; 95% confidence interval [CI]): 0.31; 0.27-0.36 and 14-day readmission: 0.78; 0.72-0.86. The risk of in-hospital death associated with the Nutritional Risk Screening 2002 (NRS-2002) score, blood albumin concentration, and the score for the combined values of hemoglobin, TLC, albumin and neutrophils (HLAN) was (OR; 95% CI): 2.44; 2.35-2.53; 0.32; 0.28-0.36; and 0.96; 0.94-0.97; respectively. CONCLUSIONS: TLC < 0.8 G/L is associated with the highest risk of in-hospital death, 14-day and 30-day readmission, and longer in-hospital stay. An increase in TLC predicted in-hospital survival and freedom from early readmission with a power similar to or greater than a number of prognostic formulas based on questionnaires (e.g. NRS-2002), biochemical parameters (e.g. albumin) and formulas composed of multiple parameters (e.g. HLAN).

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Aim: The aim of this study is to determine the prognostic value of blood albumin (BA) in an unselected population of inpatients. Materials & methods: We performed prospective analysis of the medical documentation of 7279 patients hospitalized between July 2014 and September 2017. Results: Individuals with BA ≥3.35 mg/dl had significantly lower risk of in-hospital death (odds ratio [OR]: 0.22; 95% CI: 0.19-0.27; p < 0.001) and 14-day readmission (OR: 0.64; 95% CI: 0.55-0.77; p < 0.0001). BA concentration was the strongest favorable factor predicting inpatient survival in a Cox hazard regression model (OR: 0.43; 95% CI: 0.36-0.50; p < 0.001), did not correlate with body mass index and actual-to-ideal bodyweight ratio and was strongly affected by numerous non-nutrient factors. Conclusion: BA concentration showed similar or better predictive and diagnostic power in relation to all-cause in-hospital mortality and 14-day readmission among inpatients than selected multifactorial scores.

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Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

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