Several scores and algorithms have been proposed for diagnosing chronic heart failure with preserved ejection fraction (HFpEF), however, so far, there is no definition of acute HFpEF.Acute HFpEF seems to be overdiagnosed in patients presenting with acute dyspnoea.Definition of acute HFpEF should comprise both (1) features of chronic HFpEF and (2) markers of increased left ventricular filling pressures and/or of pulmonary congestion.Heart failure with preserved ejection fraction (HFpEF) might soon become the most prevalent type of acute heart failure. Still, despite more than 30 years of research on HFpEF, not only do we lack specific treatment, but also a generally accepted definition of HFpEF. Since 2016, several definitions and algorithms have been proposed for diagnosing both diastolic dysfunction and overt HFpEF. However, all of them focus exclusively on chronic (and not acute) HFpEF. Recent studies showed that acute HFpEF may be overdiagnosed in patients presenting with acute dyspnoea. The aim of our article was to address two questions: (1) why there is a need for specific diagnostic criteria for acute HFpEF, and (2) what such definition of acute HFpEF should encompass. According to projected trajectories of the proportion of heart failure (HF) types among patients hospitalised for HF decompensation, HF with preserved ejection fraction (EF) – HFpEF – might soon become the most prevalent type of acute HF . HoweverIn 2019, the PARAGON-HF trial missed its primary endpoint of cardiovascular death and total HF hospitalisations (“hard endpoints”), and in 2020, the PARALLAX trial failed to demonstrate improvement over the control group in functional capacity, and quality of life improved at 4 but not 24 weeks (“soft endpoints”) with sacubitril-valsartan in HFpEF, despite achieving the anticipated reduction in concentrations of natriuretic peptides (NPs) . Some hoBoth the PARAGON-HF and the PARALLAX trial included not only patients with normal EF but also those with “borderline” EF  ,3. Earli2FPEF score includes 6 dichotomised, clinical and echocardiographic variables: H1 for “heavy” (body mass index >30 kg/m2), H2 for hypertension treated with 2 or more medicines, F for the presence of atrial fibrillation (AF), P for echocardiographic signs of elevated pulmonary artery pressure, E for elderly (age of >60 years), and F for elevated left ventricular (LV) filling pressures on echocardiography (E/e’ ratio >9). If positive, these variables are attributed 1–3 points with a total maximum of 9 points and a high probability of HFpEF at scores of 6–9 [2FPEF score has the advantage of validation against invasive measurements [Following the HFpEF definition from the 2016 ESC guidelines, several algorithms were proposed for diagnosing both diastolic dysfunction and overt HFpEF . The 201s of 6–9 . In conts of 6–9 . Compareurements . Still, urements .2FPEF score did not include NPs at all, as their addition to the score did not increase its diagnostic ability [Diagnosing acute HFpEF relies mostly on clinical judgement. For example, in an elderly patient with acute dyspnoea, classical HFpEF risk factors and comorbidities , rales at the basis of the lungs, elevated NPs concentrations, and preserved EF on echocardiography, it is tempting to diagnose HFpEF. However, such patients often have other cardiac and extracardiac comorbidities, which may, at least in part, be responsible for their clinical presentation. Basal rales on lung auscultation are not pathognomonic for pulmonary congestion and may result from inflammation, fibrosis, interstitial lung disease, or atelectasis (the latter often observed in obese and less mobile patients). Concentrations of NPs may rise above the adopted “HF cut-points” in patients with no HF, but with other cardiac  and extracardiac  diseases, especially if they coexist, which is common in the elderly. Older age and lower body mass index are also associated with higher levels of NPs ,18,19. T ability . In the Thus, there is a need for separate diagnostic criteria for acute HFpEF. Most parameters included in algorithms for the diagnosis of chronic HFpEF or diastolic dysfunction, such as NPs, E/e’ ratio, mitral inflow velocities, tricuspid regurgitation velocity, and to a lesser extent left atrial volume index and e’ velocities, change with volume and pressure overload, as well as heart rate, and thus cut-offs used for chronic HFpEF may not be applicable in patients with acute symptoms .de novo, i.e. in an individual with previously normal LV structure and function). In an acute HFpEF definition, cut-offs for NPs should be adopted for acute HFpEF if possible, and preferably adjusted for the presence or absence of AF. A proposed concept of an integrated approach to acute HFpEF definition is graphically presented in In a patient with preserved EF and acute symptoms suggestive of HF, two questions need to be addressed: (1) whether the patient has HFpEF, and (2) whether HFpEF is the reason for present symptom exacerbation. Regarding the first question, of patients hospitalised for acute HFpEF, as many as half might not meet even the relatively mild criteria for chronic HFpEF during the index hospitalisation, and three quarters might not meet chronic HFpEF criteria at stable follow-up ,21. EvenAt the same time, we acknowledge the limitations of such a complex approach in the acute setting. In the emergency department, treatment of pulmonary congestion must often be prioritised and based on clinical judgement before applying more sophisticated diagnostic tests. Such “empirical” treatment, usually consisting of intravenous diuretics and/or vasodilators will lead to a reduction in LAP and, consequently and desirably, an improvement in symptoms. If echocardiography is delayed, then, at the time of assessment, LAP might have already normalised with treatment, hindering the “post-hoc” confirmation of acute HFpEF. However, the use of point-of-care ultrasound is becoming more and more common in emergency departments and intensive care units, and NP concentrations can also be measured within the first few hours of admission. Finally, as emphasised above, features of chronic HFpEF , which can be assessed after achieving clinical stabilisation, are obligatory to secure the diagnosis of acute HFpEF decompensation. Typical features of chronic HFpEF are gathered under the functional and morphological domains of the HFA-PEFF score . If LAP Regardless of EF category, when validated externally, HF could be potentially misdiagnosed in 15–50% of hospitalised patients and half of the ambulatory, primary care patients ,23–29. TYet, given that there is no evidence-based treatment for acute HFpEF, does it matter if we correctly diagnose it? Diuretics may help resolve symptoms of increased LAP irrespective of its aetiology. However, in patients with normal LAP and no hypervolemia, diuretic use may be detrimental, especially in the elderly with reduced glomerular filtration. Furthermore, overdiagnosing HFpEF may lead to disregarding other potential cardiac or extracardiac causes of symptoms. In patients with preserved EF and a clinical diagnosis of acute HF, compared to patients with increased LAP (and thus “confirmed” acute HFpEF), those with normal LAP (and thus possibly misdiagnosed with HFpEF) were subsequently twice as often hospitalised for non-cardiovascular reasons and significantly more often hospitalised for cardiovascular non-HF reasons, despite a lower prevalence of cardiac and extracardiac comorbidities at baseline . This suA reliable definition of acute HFpEF is needed also because many patients are first diagnosed with HFpEF during hospitalisation for acute symptoms. Once “labeled” with HFpEF, their chronic symptoms such as dyspnoea, fatigue, exercise intolerance or peripheral oedema may later be automatically attributed to HFpEF, delaying the diagnosis of other relevant comorbidities, such as chronic obstructive pulmonary disease, interstitial lung diseases, pulmonary arterial hypertension, venous thromboembolism and chronic thromboembolic pulmonary hypertension, chronic coronary syndromes, chronotropic incompetence, constrictive pericarditis, thyroid dysfunction, kidney disease, liver disease, anaemia, myelodysplastic and myeloproliferative syndromes, systemic autoimmune diseases, neuromuscular disorders, or cancer disease. Given the natural history of HFpEF, the definition of acute HFpEF should comprise both (1) features of chronic HFpEF (such as LV hypertrophy and decreased e’ velocities) and, obligatory, (2) markers of increased LV filling pressures  and/or of pulmonary congestion (such as B-lines on lung ultrasound). Almost every chronic disease has a definition of its acute exacerbation, and HFpEF should be no exception.


This volume presents a special issue on selected papers from the 2019 & 2020 editions of the International Conference on Theory and Practice of Digital Libraries (TPDL). They cover different research areas within Digital Libraries, from Ontology and Linked Data to quality in Web Archives and Topic Detection. We first provide a brief overview of both TPDL editions, and we introduce the selected papers. The TPDL conference constitutes a leading scientific forum that brings together researchers, developers, content providers, and practitioners in the field of Digital Libraries. Digital libraries and repositories store, manage, represent, and disseminate rich and heterogeneous data that are often of enormous cultural, scientific, educational, artistic, and social value. Serving as digital ecosystems for empowering researchers and practitioners they provide unparalleled opportunities for novel knowledge extraction and discovery. New applications raise novel challenges that can only be addressed in an interdisciplinary community of researchers and practitioners from various disciplines including the Digital Humanities, Information Sciences, and others.connecting with communities to reflect the need for digital libraries to interact with researchers and practitioners for effective data utilization, management, and exploitation. The proceedings contain 17 long research papers, 11 short research papers, and 18 poster and demonstration papers [The 23Following the tradition of previous TPDL editions , 6, the Papers accepted in both editions of TPDL had already been thoroughly reviewed by three reviewers and one senior meta-reviewer. Additionally, all papers were discussed during a meeting of the Program and General chairs for producing the list of TPDL accepted papers. From the pool of papers accepted in TPDL 2019 & 2020, the General and the Program Committee Chairs nominated ten papers based on their reviewing scores: three from TPDL 2019 and seven from TPDL 2020. Authors of these papers were invited to extend their original paper by at least 30% in a four-months period. Nine new versions were submitted. As required by IJDL’s policy, these extended papers went through another round of reviews by at least three reviewers, supported by a senior reviewer. Finally, 6 papers were accepted for publication in this special issue. Below is a short description of these papers.An Extended Analysis of the Persistence of Persistent Identifiers of the Scholarly Web by Martin Klein and Lyudmila Balakireva investigates the notion of persistence of DOIs by analyzing their resolution on the web. Digital Object Identifiers (DOIs) are common standard to persistently identify resources. The DOI concept is based on the assumption that mappings between the resources and the DOIs are updated when the resource location is changing. The authors show that persistence is in reality not guaranteed. Instead content providers respond differently to varying request methods and network environments and even change their response to requests against the same DOI. The paper presents quantitative analysis results and aims informing the scholarly communication community about this disconcerting lack of consistency.The paper Correspondence as the Primary Measure of Information Quality for Web Archives: A Human-Centered Grounded Theory Study, a grounded theory of quality specifically for web archives. She analyzed support tickets submitted by clients of the Internet Archive’s Archive-It (AIT). Therefore, the theory is human-centered and grounded on the perception of users and creators on the quality of web archives. The resulting theory compromises the dimensions correspondence, relevance, and archivability. Web archivists and cultural heritage institutions will benefit from the clarified notion of quality in a web archive.Brenda Reyes Ayala presents Analysing the Requirements for an Open Research Knowledge Graph: Use Cases, Quality Requirements and Construction Strategies tackles the issue of exploring and comparing scientific research literature in a semantic way using an Open Research Knowledge Graph (ORKG). They first describe the typical tasks of a researcher (use cases) that can be supported by an ORKG, such as finding related work or obtaining a deep understanding of a paper. Minimal requirements (in terms of granularity or completeness for instance) are defined for each use case. Next, manual, semi-automatic, and automatic approaches for building an ORKG are surveyed, both for designing ontologies and for populating the graph. This article thus provides a broad overview of the different requirements for an ORKG, and it fosters future research on this emerging topic.The next paper by Arthur Brack, Anett Hoppe, Markus Stocker, Sören Auer, and Ralph Ewerth on VeTo+: improved expert set expansion in academia article by Serafeim Chatzopoulos, Thanasis Vergoulis, Theodore Dalamagas, and Christos Tryfonopoulos extends their work on identifying experts in a given field, for example, in order to identify new reviewers for a conference. Rather than relying on data containing explicit links between experts and topics, the authors identify potential experts by considering the venues the experts publish in and the topics attached to the publications. The extended algorithm presented here builds on this by enabling dynamic weighting of the two factors and filtering of the considered publication venues. The second is of particular interest to interdisciplinary venues, such as TPDL or JCDL, where except for the interdisciplinary venue, there is often little publishing overlap between experts.The Multi-Label Classification of Legislative Contents with Hierarchical Label Attention Networks by Danielle Caled, Mário J. Silva, Bruno Martins, and Miguel Won deals with the issue of annotating legislative documents using the EuroVoc hierarchical thesaurus. Contrary to existing annotation methods, authors propose a deep learning model to take into account the three levels of the thesaurus rather than predicting for a single level. In order to evaluate this approach, more than 200,000 legal documents in Portuguese, which have been classified according to EuroVoc, are integrated into the dataset EUR-Lex PT. This article finally highlights recurring challenges for automatic annotation of large collection of documents, such as the long tail label distribution which degrades accuracy.The following paper CSO Classifier 3.0: A Scalable Unsupervised Method for Classifying Documents in Terms of Research Topics extends their work presented at TPDL 2019 by introducing an improved design of the Computer Science Ontology (CSO) Classifier for automatic classification of research papers according to the CSO and presenting novel mechanisms for detecting outlier topics. To measure the performance of their solutions, the authors created a gold standard using 70 documents, manually annotated by domain experts. The paper also describes how the classifier has been adopted by other researchers since 2019 and how the classifier could be adapted for classifying with other knowledge organization systems as the code is freely available to the wider research community.Angelo Salatino, Francesco Osborne, and Enrico Motta’s paper To conclude, we believe that this volume of selected Digital Libraries papers reflects the ongoing work and topic trends in our field. We hope that our readers will find them insightful, and we invite them to take note about the next editions of TPDL (online in 2021 [


However, molecular mechanisms for the association remained elusive. In this study, we first found that both leptin (10 nM) and TNF-α (20 ng/ml) significantly inhibited glucose-stimulated insulin secretion (GSIS) of INS-1E cells. When in combination, the GSIS function of INS-1E cells was significantly increased compared with that of the leptin alone treatment, indicating that TNF-α attenuated the inhibiting effect of leptin on GSIS of INS-1E cells. Similarly, we found that TNF-α has the same inhibitory effect on leptin in regulating insulin synthesis and secretion, and the survival and apoptosis of insulin cells. Further studies showed that TNF-α blocks leptin pathway by reducing the expression of leptin receptor  and inhibiting the activation of STAT3, a key molecule involved in the leptin signaling pathway in INS-1E cells. Besides, the downregulated expression of phosphorylated FOXO1 was found to be involved in the possible mechanism of TNF-α. Overexpression of constitutively active FOXO1 markedly aggravated the LepRb reduction by TNF-α treatment of INS-1E cells, and the endogenous FOXO1 knockdown abolished the effect of TNF-α on INS-1E cells. Furthermore, we have proved that FOXO1 could directly bind to the promoter of LepRb as a negative transcription regulator. Taken together, the results of this study reveal that TNF-α-induced LepRb downregulated in pancreatic β cells and demonstrate that transcriptional reduction of FOXO1 might be the primary mechanism underlying TNF-α promoting INS-1E leptin resistance and β cell dysfunction. Conclusions. Our current studies based on INS-1E cells in vitro indicate that the inflammatory factor TNF-α plays an important role in the development of INS-1E leptin resistance and glucose metabolism disorders, probably through FOXO1-induced transcription reduction of LepRb promoter in pancreatic β cells, and FOXO1 may be a novel target for treating β cell dysfunction in obesity-induced hyperinsulinemia and T2DM.Proinflammatory cytokines play a causal role in the development of hyperinsulinemia and T2MD. FOXO1, a transcription factor which is known to enhance proinflammation, was recently shown to be involved in obesity-induced  The antigen-antibody complex was then detected by incubating the membranes for 1 hour in a buffer containing a 1 : 5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies .INS-1E cells treated with leptin (10 nM) or/and TNF-escribed . Brieflyα for 48 h. Then, the cells were incubated with 20 μl 3--2,5-diphenyl-tetrazolium bromide  at 37°C for 3 h. After removal of MTT, 150 μl DMSO was added to the cells and the absorbance was measured at 492 nm with a microplate reader. Apoptosis assay was performed with Annexin V-FITC apoptosis assay kit (Bender MedSystems) in accordance with the manufacturer's directions.Cell proliferation of INS-1E cells was determined by means of a colorimetric MTT-assay as previously reported . BrieflyHuman FOXO1-AAA plasmid, a constitutive active form construct with the three conserved Akt/PKB sites  within FOXO1 specifically mutated into alanine, was kindly provided by Dr. Terry Unterman (University of Illinois at Chicago). Both FOXO1-TSS plasmid and pcDNA3.1 were preserved in our laboratory. The DNA fragments for the three human LepRb promoter fusion reporter constructs shown in 5/well and then transfected with 100 μl of LepRb reporter gene plasmid or equal molar amounts of its truncated promoter reporter constructs. After transfection for 6 h, the medium was removed and replaced with complete growth medium containing 20 ng/ml TNF-α or PBS as control. After 36 hours of incubation, cells were washed twice and lysed with specific reporter lysis buffer. Then, the luciferase activities of the cell lysate were evaluated according to the manufacturer's instructions , and the total protein concentration in each well was measured as an internal control.Amplified DNA fragments were inserted into the pGL3-Basic vector. Transient transfections were conducted using Lipofectamine 2000 according to the protocol as previously reported . Briefly6 INS-1E cells were seeded in 6-well dishes and normally cultured until 80% confluence and then harvested in PBS after cross-linking with 1% formaldehyde at room temperature for 10 min. Cells were then collected and transferred to a centrifuge tube. After centrifugation, the cells were incubated with SDS lysis buffer supplemented with proteinase inhibitor for 1 h at 4°C. The mixture was sonicated on ice to obtain 200 to 500 base pair DNA fragments. The ChIP assay was performed according to the manufacturer's protocol (Millipore Corp). The input group accounted for 1% of the total DNA, while the IgG and FOXO1 groups were added with their ChIP grade antibodies. The immunoprecipitates were analyzed by PCR for detecting the protein binding sites of the DNA samples. The primers were designed as follows: LR1 promoter forward: GCACGCGTGAATGGATTTGATGCCCTGT, LR1 promoter reverse: GCCTCGAGTTTAACGCCCGCCATGTCT; LR2 promoter forward: GCACGCGTCAGGGCTTGTCTGATACGCAGG, LR2 promoter reverse: GCCTCGAGTTTAACGCCCGCCATGTCT; and LR3 promoter forward: GCACGCGTGTGCTTTTCTAGTACCGTGTAC, LR3 promoter reverse: GCCTCGAGTTTAACGCCCGCCATGTCT. The calculation of the ChIP signal is %input = 1% × 2(CTinput − CTsample).MAGnify™ System (Thermo Fisher Scientific) was used for ChIP assay . BrieflyFOXO1-specific binding sites in the human LepRb promoter region LR3 were mutated using MutanBEST Kit  according to the manufacturer's protocol. Briefly, the required primers were designed as follows: for LR3-mut1, forward: GCATCAGCAGTGAGTCAGGTA, reverse: TCTCCTCATAGACGGATGGCA; for LR3-mut2, forward: GACATTTCTTGAAACAGTAACATGC, reverse: GTTTCATGATCCTTGCTCAGTGG; for LR3-mut3, forward: CCCGCTAGTTTCAGTAAAGCG, reverse: CCCCTTAGGAAACACTCCTCA; and for LR3-mut4, forward: GCGGCCTCTGCGAGC, reverse: GGCGCCTGCTGCTCC.The DNA pull-down was performed with a DNA pull-down test kit (Thermo Fisher Scientific) according to the manual . Brieflyα (20 ng/ml) was added for an additional 24 h after 48 h of transfection with 20 nmol/ml of siRNA.Human FOXO1-specific siRNA (si-FOXO1): 5-AAGCCCTGGCTCTCACAGC-AA-3 and the scrambled control RNA (siNC): 5-UUCUCCGAACGUGUCACGUT-T-3 were designed to knock down the expression of endogenous FOXO1 genes according to our previous report . INS-1E P < 0.05 was considered significant.Statistical analysis was performed using software SPSS V18 . All measurement data were presented as the mean ± SEM. The significance of each group was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test. α and leptin under the state of obesity, we firstly investigated the effects of leptin and TNF-α on the GSIS function in INS-1E cells. Results showed that leptin (10 nM) and TNF-α (20 ng/ml) alone could significantly reduce the GSIS function of INS-1E cells  in combination, the GSIS function of INS-1E cells was significantly increased compared with the leptin alone treatment group  and TNF-α (20 ng/ml). RT-PCR results showed that the expression of proinsulin mRNA was reduced by leptin, and TNF-α significantly reversed the inhibitory of leptin on proinsulin mRNA  and (20 ng/ml) alone or in combination, and then, the percentage of apoptosis and cell viability were measured. Flow cytometry results showed that both leptin (10 nM) and TNF-α (20 ng/ml) significantly accelerated the apoptosis of INS-1E cells. However, when INS-1E cells were treated with leptin (10 nM) and TNF-α (20 ng/ml) in combination, the percentage of apoptosis was relatively decreased compared with those treated with leptin (10 nM) but not with TNF-α (20 ng/ml)  significantly inhibited the expression level of LepRb , which play a key role in the leptin signaling pathway , 32. To  Figures , despiteα stimulus on the signaling cascade of the leptin receptor, we studied the activation of STAT3 in INS-1E cells treated with leptin and TNF-α. Intriguingly, the level of p-STAT3 was not further increased by leptin (10 nM) compared with the PBS group (α (20 ng/ml) successfully inhibited the activation of STAT3  significantly decreased the expression level of p-FOXO1 (α reduced the leptin pathway probably by inhibiting the phosphorylation of FOXO1.The Janus kinase-2 (JAK2) and signal transducer and activator of transcription (STAT3) pathway were regarded as a major leptin signaling pathway. To investigate the effects of TNF-BS group . This isof STAT3 , indicat p-FOXO1 , suggestα inhibit the effect of leptin by directly mediating SOCS3, we next examined the expressing level changes of SOCS3 in INS-1E cells treated with TNF-α. However, no significant effect on the expression of SOCS3 was observed , which is an inhibitor of OBR signaling –37. To eobserved , indicatα. The results showed that FOXO1 overexpression significantly decreased LepRb mRNA levels  specifically targeting human FOXO1 (siFOXO1) was transfected into INS-1E cells to silence endogenous FOXO1 expression in INS-1E cells (α (20 ng/ml) for an additional 24 h; the result showed that FOXO1 silencing significantly attenuated the inhibitory effect of TNF-α on LepRb mRNA levels , LR2 (-2376/+105), and LR3 (-1824/+105) harboring different LepRb promoter regions were generated   , and witenhanced .α treatment, confirming the FOXO1 transcriptional activity on target gene expression. Intriguingly, opposite effects of FOXO1 on LepRb promoter activity were observed when HEK293 cells were treated with TNF-α using a LR3 (-1824/+105) DNA fragment  gene, a known target of FOXO1, was used as a positive control for the transcription activity of FOXO1 in this experiment. As expected, FOXO1 overexpression increased , and sil Figures . These rhttps://jaspar.genereg.net/) database and found that there are four potential FOXO1 binding sites in LR3, named region 1 (-1682 bp to -1662 bp), region 2 (-1520 bp to -1507 bp), region 3 (-960 bp to -945 bp), and region 4 (-124 bp to -105 bp)  . We next-105 bp) . Then, tth TNF-α , indicatTo verify the direct binding between FOXO1 and LepRb promoter, we performed ChIP-PCR assay. Results showed that all of the three LepRb promoter regions were detected by qRT-PCR from the DNA fragments eluted from FOXO1-bound beads, which indicated the direct binding between FOXO1 and LepRb promoter . Next, wα disturbed the regulation of leptin on β cell functions including GSIS, insulin synthesis and secretion, and β cell survival and apoptosis. Further studies confirmed that TNF-α could inhibit the expression of LepRb located on the surface of β cells. Besides, we explored the potential mechanism effect of FOXO1 on TNF-α-induced β cell dysfunction and proved that FOXO1 plays a crucial role in the regulation of TNF-α in the expression of LepRb by directly binding to the promoter of LepRb as a negative regulatory transcription factor. This study thus provides a novel mechanism that FOXO1 involves in proinflammatory cytokine-induced β cell dysfunction and would be a new target for the treatment of T2DM.The current study provides evidence that TNF-β cell dysfunction. Both obesity and T2DM have been considered as a chronic low-grade inflammatory [α is well characterized to induce insulin resistance in cultured adipocytes and in mouse [α may also have a role in impaired β cell function. In vitro studies have shown that TNF-α can reduce glucose-induced insulin release from pancreatic β cells by activating nuclear factor kappaB (NF-κB) and local production of nitrous oxide [α levels after transient insulin therapy correlate with improved β cell function in type 2 diabetics; however, the mechanism effect of TNF-α on β cell function remains to be established.T2DM is characterized by a progressive decrease of insulin sensitivity and pancreatic ammatory . It is win mouse . TNF-α mus oxide . Previouβ cell dysfunction in the course of T2DM. It has been reported that the transcription of proinsulin mRNA is inhibited by TNF-α and leptin [α (20 ng/ml) and leptin (10 nM) significantly reduced the GSIS function of β cells and inhibited the expression of insulin in gene level, which is consistent with previous reports. Intriguingly, when combined with TNF-α, the inhibitory effect of leptin on the transcription of proinsulin gene and the synthesis of insulin was significantly attenuated. Previous studies have reported that high concentration of leptin induces activation of NF-κB through the MAPK/JNKs pathway, and TNF-α cause cell-selective destruction by promoting NO production and inducing apoptosis [Numerous studies have reported that obesity, insulin resistance, glucocorticoids, estrogens, and chronic inflammation are closely related to the high levels of leptin , 40. In d leptin . In the poptosis , 44.β cell mass is essential for the compensatory response of the endocrine pancreas to situations of increased insulin demand such as obesity. Previous study reported that the mass of β cell increased during insulin resistance [β cell mass in subjects with obesity is assumed to increase, since plasma insulin levels in obese subjects increase to compensate for insulin resistance, a process known as hyperinsulinemia [α promote β cell mass, thus leading to overabundance of cells. These results are consistent with some previous reports. Therefore, we hypothesize that TNF-α is a potential inducer to deteriorate hyperinsulinemia and subsequent T2DM by promoting β cell mass and insulin secretion.Additionally, the regulation of sistance . Other rulinemia . Our curα in changes of LepRb, STAT3, and SOCS-3 protein level in this study. We found that TNF-α significantly inhibited LepRb expression and the activation of STAT3, but had no effect on the expression of SOCS3. These results are indicative of the JAK2/STAT3 pathway may be involved in the regulation of TNF-α in inhibiting the effects of leptin on β cell function.JAK/STAT is a vital pathway mediating the leptin signaling. JAK2, a member of the JAK family of protein tyrosine kinases, is the major subtype that recognizes and binds to the membrane proximal specific region of LepRb, which is considered the primary receptor for leptin . Studiesα, thereby regulating downstream target genes to promote gluconeogenesis [β cell dysfunction, because healthy β cells can compensate for insulin resistance by increasing in number and functional output. Despite the FoxO1 central role in the progression of insulin resistance, the effect of FoxO1 on β cell function has not been explored in detail and remains somewhat conjectural. Thus, we sought to test the hypothesis that FoxO1 involves in the regulation of TNF-α in β cell function and to identify the underlying mechanism.Transcription factor FOXO1 was reported to modulate the production and secretion of various cytokines and chemokines . Previouogenesis . It is rα reduced the phosphorylation level of FOXO1, thus leading to an increase of nucleus FOXO1 in the INS-1E cells. Accumulating evidence has proven that FOXO1 is involved in the insulin signaling pathway as a regulator and can directly bind to a series of target [α reduces LepRb expression, and this effect is significantly reinforced when FOXO1 is overexpressed and attenuated when endogenous FOXO1 is silenced. These data have confirmed that FOXO1 directly binds to the LepRb promoter as a negative regulator, thereby inhibiting the expression of LepRb, and ultimately reduced the leptin signaling pathway. Therefore, we pursued the hypothesis that preventing the transfer of dephosphorylated FOXO1 into the nucleus represents as a means of enhancing leptin receptor signaling to recover leptin pathway which was reduced by inflammatory factors, and a cell signaling mechanism diagram is shown in In the present study, we found that TNF-f target . Thus, wα aggravating obesity-related β cell dysfunction and described a novel function of FOXO1 involved in TNF-α-induced LepRb reduction of INS-1E cells. Thus, we conclude that TNF-α disrupts the regulation of leptin on the β cell GSIS function by inhibiting leptin receptor LepRb and thus producing excessive insulin secretion related to hyperinsulinemia, which is postulated to be a cause of insulin resistance. Our findings may provide a potential therapeutic means for the treatment of obesity-induced hyperinsulinemia and T2DM. This study has potential limitations. We have not elucidated the precise mechanism by interactions between TNF-α and the loss of insulin receptor and subsequent insulin resistance. In addition, we confirmed four binding sites of FOXO1 in LepRb promoter, but we have not explored the priority of FOXO1 for these binding sites, and more studies are required to solve these questions.In conclusion, our studies based on INS-1E cells in vitro have clarified the molecular mechanism of TNF-


With the help of computational fluid dynamics (CFD), hemodynamics of the pulmonary arteries (PA's) can be studied in detail and varying physiological circumstances and treatment options can be simulated. This offers the opportunity to improve the diagnostics and treatment of PA stenosis in biventricular congenital heart disease (CHD). The aim of this review was to evaluate the methods of computational studies for PA's in biventricular CHD and the level of validation of the numerical outcomes. A total of 34 original research papers were selected. The literature showed a great variety in the used methods for (re) construction of the geometry as well as definition of the boundary conditions and numerical setup. There were 10 different methods identified to define inlet boundary conditions and 17 for outlet boundary conditions. A total of nine papers verified their CFD outcomes by comparing results to clinical data or by an experimental mock loop. The diversity in used methods and the low level of validation of the outcomes result in uncertainties regarding the reliability of numerical studies. This limits the current clinical utility of CFD for the study of PA flow in CHD. Standardization and validation of the methods are therefore recommended. Stenosis of the pulmonary arteries (PA's) is commonly seen in patients with congenital heart disease (CHD). It can occur as a native substrate or after surgery . Diagnosst of January 2021 were included.A PubMed and Embase search was performed. The searched papers needed to include “pulmonary arteries” and “computational fluid dynamics” or a synonym in their title or abstract. The language for the search was restricted to English. All papers published before 1All papers on numerical analysis of the PA's in biventricular hearts were included. Pediatric and adult as well as animal studies were considered. Papers with the focus on pulmonary hypertension were excluded as well as papers from before 2001, reviews, and if no full text was available. All inclusion and exclusion criteria are shown in The search in the PubMed and Embase databases resulted in a total of, respectively, 266 and 286 papers. After undoubling and title abstract screening, 45 papers remained. The full-text screening of these papers revealed exclusion criteria for 11 articles. This resulted in a total of 34 eligible papers for this review. The flowchart for the selection process is shown in The 34 selected papers included four animal studies and 25 papers presenting human cases. In one paper, both human and animal cases were described . In six Over half of the included papers focused on the surgical treatment of PA stenosis. These 19 studies described the use of CFD in surgical planning, for comparison of different shunt configurations or to study (post)surgical complications , 18–31. Of the 34 studies, 13 were published in journals with a clinical orientation , 37, 43.In the following sections, the various used methods for numerical analysis of the PA's will be compared. The sections are subdivided into the major steps necessary for solving a CFD case. First is the anatomic reconstruction, then the meshing and setting the boundary conditions followed by the numerical setup and finishing with postprocessing and validation of the results. Most groups (re)constructed a geometry consisting of the main PA bifurcation and the right and left pulmonary artery , 38–40. An artificial geometry was used in ten studies , 28, 38.After geometry reconstruction, inlet and outlet extensions were added in four of the 34 studies , 30, 40.http://simvascular.org). For commercial software, this was MIMICS . Other software for segmentation included ITK-SNAP , OsiriX , and CardioViz3D .A variety of software was used for segmentation of the patient-specific anatomies. The most mentioned open-source software package was SimVascular  and ABAQUS . Four groups calculated their solution with special build-in-house software [The program most used for solving the numerical cases was ANSYS Fluent . This software package was utilized by 12 groups included in this review , 42, 44.software , 35, 36.software , 43, 45.3. Only three studies assigned a different density of 1050 kg/m3 and 1000 kg/m3, respectively [In 29 of the 34 included papers, blood was assumed to behave as a Newtonian fluid while in one paper, it was assumed to be a non-Newtonian fluid . Three pectively , 35, 40.ectively , 42, 45.ectively , 43, 44.ectively , 36, 40.ectively . Two autectively , 40. TheAs 10 studies were performed with a constant inlet flow, the number of simulated cardiac cycles here is irrelevant. In 11 of the other 24 papers, the number of simulated cycles was specified , 38–41. Information on time step size for the simulation was provided in 13 papers , 38–42. e-4 [The used convergence criteria were specified in 13 papers , 42, 44.e-4 , 38, 39.e-4 , 42.The computational time for the cases was described in six papers. The reported time per case varied between a couple of hours up to 1-2 months , 39, 41.The results presented in the included papers varied according to the research question proposed. In most papers, two or three numerical outcomes were presented, i.e., pressure and WSS or streamlines and flow rates. In the majority of the papers, figures showed the results of the peak systolic and one or two diastolic time steps. Velocity and WSS were the results most reported followed by, respectively, pressure, streamlines, and flow rates. Pressure outcomes were presented either by the peak systolic numbers and energy loss over a stenosis or as the time-pressure curve over the cardiac cycle.In nine papers, clinical data of the included patients was used to validate the CFD results  12, 19,, 40, 45.The use of advanced imaging modalities to describe the hemodynamic impact of PA stenosis is increasing. CFD is one of these techniques providing detailed visualization of patient-specific hemodynamics. The aim of this paper was to review the numerical methods and—clinical—validation of CFD for evaluation of PA's in biventricular CHD. All of the papers included in this review emphasize the importance of hemodynamic evaluation of the PA's in biventricular CHD. They show the use and feasibility of CFD for this purpose and the wide variety of applications of the technique, i.e., for surgical or interventional treatment planning, research on complications, and exercise simulation. However, this review also shows limitations of the current available literature.The literature reveals a large diversity in the setup for the numerical analysis of PA stenosis. This heterogeneity is important as variations in the numerical case setup significantly influence the outcomes. Results of patient-specific analysis are highly dependable on the source and quality of the anatomic reconstruction. In addition, small differences in the applied inlet or outlet boundary conditions can have a major impact. Outcomes of WSS and velocity can differ up to 30% with different boundary conditions –49. In tThe heterogeneity, assumptions, and generalizations in the computational setup result in uncertainties regarding the outcomes. The validation of methods and results is therefore of major importance. It provides direct feedback on the used methods and increases confidence in the reliability of the technique. This review shows that the level of validation of the CFD outcomes is very low. Studies with the main aim to validate CFD outcomes were completely missing, and only nine of the 31 papers compared their outcomes to clinical data. This lowers the translational value of the studies.Another important limitation for the clinical utility of CFD is the computational time. This was reported to be as long as several days to even months per case. However, more and more progress is made in speeding up the computational process. By use of “super computers,” improved algorithms, and cloud-based environments, the simulation time can be significantly reduced. Great examples of these efforts are shown by the two papers of Kong et al. included in this review. They show how the use of multiple cores and adjustment of algorithms can decrease computational time with several hours , 36. ThiThe aim of this review was to evaluate the available literature on numerical analysis of the PA's in biventricular CHD. The focus was on the used methods and the rate of—clinical—validation of the outcomes. To the best of our knowledge, this is the first review evaluating the different strategies for numerical studies of the PA's. The included literature shows the wide variety of applications of CFD and emphasizes the added value of numerical studies for hemodynamic assessment of the PA's. However, this review also shows the large heterogeneity in used methods in all parts of the numerical setup and little validation of the results. This limits the current clinical utility of CFD. To increase the translation towards clinical use, standardization of the methodologies is desirable. Future research should therefore be pointed towards validation of methods of numerical studies.


This review summarizes available data from current clinical practice and mechanistic studies in terms of epidemiology, etiology, clinical indicators, mechanisms, and treatments to discuss the potential association between HFmrEF and HFpEF patients.The new guidelines classify heart failure (HF) into three subgroups based on the ejection fraction (EF): HF with reduced EF (HFrEF), HF with mid-range EF (HFmrEF), and HF with preserved EF (HFpEF). The new guidelines regarding the declaration of HFmrEF as a unique phenotype have achieved the goal of stimulating research on the basic characteristics, pathophysiology, and treatment of HF patients with a left ventricular EF of 40–49%. Patients with HFmrEF have more often been described as an intermediate population between HFrEF and HFpEF patients; however, with regard to etiology and clinical indicators, they are more similar to the HFrEF population. Concerning clinical prognosis, they are closer to HFpEF because both populations have a good prognosis and quality of life. Meanwhile, growing evidence indicates that HFmrEF and HFpEF show heterogeneity in presentation and pathophysiology, and the emergence of this heterogeneity often plays a crucial role in the prognosis and treatment of the disease. To date, the exact mechanisms and effective treatment strategies of HFmrEF and HFpEF are still poorly understood, but some of the current evidence, from observational studies and  In 2016, heart failure (HF), a clinical syndrome with typical signs and symptoms triggered by a structural and/or functional cardiac abnormality resulting in reduced cardiac output (CO) and/or elevated intracardiac pressures at rest or during stress, was categorized into three subgroups based on left ventricular ejection fraction (LVEF): HF with reduced EF , HF with mid-range EF , and preserved EF (EF ≥ 50%)  mortality was numerically higher in patients with HFmrEF and HFpEF than in those with HFrEF. The incidences of 1-year death and hospitalization for HF among HFmrEF and HFpEF patients were less than those among HFrEF patients , 4. AddiTherefore, in this review, we briefly describe the existing knowledge on the epidemiology, etiology, and clinical indicators of HFpEF and HFmrEF and discuss the underlying mechanisms of these two subgroups. Finally, we present the evidence on current potential treatment options for these two groups of patients.In some registries and clinical trials, a significant proportion of patients with HF belong to the HFmrEF and HFpEF subgroups, with percentages ranging 8.1–24 and 11–43%, respectively , 8, 9—foAlthough patients with HFrEF and HFpEF share many similar risk factors , some coHigher laboratory parameters, including blood urea nitrogen, creatinine, N-terminal pro-B-type natriuretic peptide (NT-proBNP), potassium, uric acid, and ferritin levels, were shown in populations of HFmrEF compared with HFpEF. Compared with the HFpEF patients, the HFmrEF patients had larger LV end-diastolic and end-systolic dimensions, a higher left atrial volume index and LV mass index, and a lower LVEF. Patients in the HFmrEF and HFpEF groups did not differ significantly in valvular disease  or diastolic dysfunction parameters . A SwediExtensive data are lacking regarding the mechanism of HFmrEF. Therefore, we speculate on the underlying mechanism of HFmrEF, relying on current clinical registries and investigations that have reported the etiology and comorbidities of different EF values.However, numerous HFpEF mechanistic studies have been performed recently, including those on coronary microvascular dysfunction centered on inflammation and endothelial damage and energy production disturbance and myocardial metabolic abnormalities centered on mitochondrial injury as well as diastolic dysfunction related to myocardial fibrosis and calcium homeostasis disorder.2+ homeostasis, and mitochondrial dysfunction.Therefore, in the following section, we discuss the underlying mechanism of HFmrEF and HFpEF from inflammation, cardiomyocyte injury, endothelial dysfunction, cardiac fibrosis, CaSince the new HFpEF paradigm of coronary microvascular inflammation was proposed in 2013, more preclinical and clinical data have emerged . The newvia released intracellular cytokines, and/or (c) activation of pathways unrelated to microbial recognition receptors, such as cluster of differentiation 36 ; their structural and/or functional abnormalities will strongly affect cardiac function, especially in HFpEF . EndotheHowever, the role of endothelial dysfunction in the pathophysiology of HFmrEF has not yet been reported, and further study is needed. Another study demonstrated that the levels of endothelial dysfunction biomarkers (endothelin-1 and E-selectin) were interrelated with EF .In the pathophysiology of HFpEF, growing evidence shows that coronary microvascular dysfunction leads to cardiomyocyte injury, especially in women with cardiometabolic risk factors and LVH . In a reDiastolic dysfunction, as a hallmark of HFpEF, is triggered by abnormalities in excitation–contraction coupling  and ventMyocardial fibrosis, as an important pathophysiological mechanism, plays a key role in the emergence and progression of the disease, regardless of EF . In patiMoreover, fibrosis-related biomarkers, including C-terminal propeptide of procollagen type I (PICP) and N-terminal propeptide of procollagen type III (PIIINP), which are regarded as biomarkers of type I and III collagen formation, respectively , were siVentricular stiffness is not only caused by cardiac fibrosis (as described above) but also related to abnormal active relaxation and passive stiffness (F-passive) . Titin, 2+ is directly involved in the process of contraction and relaxation of cardiomyocytes. Ca2+ released from the sarcoplasmic reticulum (2+) levels  sarcolemmal Ca2+-ATPase, and (4) mitochondrial Ca2+ UniProt  and then use it to regulate energy metabolism, such as in ATP regeneration . As an indispensable regulator of ECG activity, Caeticulum  acts as eticulum . Cardiaceticulum , depends) levels , which c UniProt , 52. Myom levels  are not m levels . The mitneration . In rat P supply .Exercise intolerance is common in the HFpEF population  and may 2 diff), and peak exercise oxygen consumption (peak VO2), a widely validated measure of exercise capacity, were all significantly reduced  .In Dahl-sensitive rats fed a high-salt diet, changes in energy metabolism during the early stage of HFpEF mainly include an increase in glycolysis, but the increased glycolysis is not accompanied by an increase in glucose oxidation, suggesting that glycolysis and glucose oxidation are decoupled. The fatty acid oxidation rates were in a state of progressive decrease following the high-salt diet .In HFpEF patients, fatty acid oxidation is increased and glucose oxidation is decreased; moreover, there is a state of uncoupling between glucose uptake and glucose oxidation, leading to an increased rate of glycolysis . Choi etKetone bodies, consisting of β-hydroxybutyrate (β-OHB), acetoacetate, and acetone, are produced predominantly in the liver from FAO-derived acetyl-coenzyme A (CoA) and are transported to extrahepatic tissues for terminal oxidation . AlternaThe current ESC guidelines recommend therapies for HFmrEF based on the evidence for HFpEF rather than that for HFrEF. However, in actual clinical practice, the treatment of HFmrEF is closer to that of HFrEF .p = 0.040), regardless of ischemic or non-ischemic etiology. Despite the small number of events, its benefits were similar to those observed in HFrEF, with reductions in both HF-related death and sudden death , while there was no significant difference in HFpEF patients  and 26%  of the study patients, respectively , and alleviating myocardial fibrosis. Additionally, the only side effects of spironolactone that are of concern are hyperkalemia and gynecomastia . The greSacubitril/valsartan has shown better effects in reducing the risks of death and hospitalization in patients with HFrEF than enalapril , but wheStatins have been shown to reduce the morbidity and the mortality in CVD and other related diseases, such as MI, stroke, and revascularization –110, butpost-hoc analysis of DECLARE-TIMI 58, among HFrEF populations who had a pre-specified EF cutoff point of <45%, including the population that is now defined as HFmrEF with an ejection fraction of 40–49%, dapagliflozin showed a greater benefit in HF hospitalization and CV death than patients with an EF known to be ≥45% or those without EF history. Furthermore, a similar reduction in HF hospitalization was found in patients with HF regardless of EF, but a greater reduction with dapagliflozin in CV death was only found in HFrEF patients  inhibitors (canagliflozin and dapagliflozin) may be considered for patients with T2DM and those with CV disease or high CV risks to prevent or delay the onset of hospitalizations associated with HF . SGLT2 ipatients . Althougpatients , cardiacpatients , 120, thIn HFpEF, the intracellular NO-cGMP-PKG signal cascade is disturbed , and dowCardiac systolic modulation (CCM) is administered through an implantable pulse generator. It can enhance the contractility of the right ventricle by transmitting the CCM pulse to the right ventricle during the absolute refractory period . The 201P < 0.001), NYHAC (P < 0.001), and 6MWD (P = 0.02) scores. This study further confirmed that, in the affected population, cases with an EF between 35 and 45%, including some cases of HFmrEF (EFs 40–49%), gain greater clinical benefits than those with EF <35% , the National Natural Science Foundation of China , the Frontier Research Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory , and the Youth Science and Technology Innovation Talent of Guangdong TeZhi Plan .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Medical educators are constantly seeking methods to increase engagement in the era of coronavirus disease 2019 (COVID-19) where virtual and blended learning formats are increasingly common. Educational escape rooms have previously been used to motivate learners, enhance communication skills, and cultivate teamwork. However, it is not known whether escape rooms increase learner knowledge as compared to a lecture format.This quasi-experimental study included 30 emergency medicine residents at two programs who participated in both a virtual escape room and a lecture on infectious disease content. Learners completed a pre- and post-quiz and a tool to gauge resident motivation for each activity (the Intrinsic Motivation Inventory [IMI]). The primary objective was to determine a change in knowledge as a result of the activities, and a secondary objective was to determine resident motivation for each format.At both programs learners demonstrated a significant improvement in their pre- vs. post-quiz scores for the escape rooms , whereas the lectures did not impact a statistical improvement . Learners at UCI noted equivalent results on the IMI for both formats, while residents at Prisma noted they were more motivated by the escape room.Emergency medicine residents at two programs participating in a virtual escape room demonstrated a statistical increase in knowledge on infectious disease content as compared to a lecture format and reported positive motivation ratings for both formats, with one program preferring the escape room. Medical educators are constantly seeking methods to increase learner engagement, particularly in the era of coronavirus disease 2019 (COVID-19) where blended and virtual learning formats are increasingly common. One innovative modality of teaching used by educators is escape rooms. As described by Nicholson,Few experimental studies have shown an increase in knowledge in a pre- vs post-test fashion,19Escape rooms have a theoretical basis to motivate learners as described by self-determination theory. The theory states that motivation comprises three psychological needs: competence; autonomy; and social relatedness. As stated by Guckian et al, As learning environments transform, innovative modalities of teaching that motivate learners must be urgently explored and researched in an experimental fashion. Therefore, in this study we sought to understand whether a virtual escape room on infectious disease topics would increase learner knowledge as compared to a didactic lecture, as assessed by a pre- and post-quiz. A secondary objective was to assess learners’ self-rated interest and enjoyment with the activities as determined by the Intrinsic Motivation Inventory (IMI) held after the escape room.What do we already know about this issue?Educational escape rooms have previously been shown to motivate learners, however it is not known if they positively impact knowledge compared to a lecture format.What was the research question?Does a virtual escape room on infectious disease topics increase knowledge compared to a lecture format?What was the major finding of the study?Learners improved their pre vs. post-quiz scores on escape room content, but not the lecture content.How does this improve population health?A virtual educational escape room may be a unique method to engage learners in an online synchronous format without sacrificing knowledge acquisition.We surveyed 30 emergency medicine (EM) resident learners at two different postgraduate year (PGY) 1–3 EM programs, the University of California-Irvine (UCI) in Orange, California, and Prisma Health-Upstate in Greenville, South Carolina, in March 2021. Residents at both programs were selected by convenience sample as attendees at a weekly didactic conference during their infectious disease block in March–April 2021. Study participation was voluntary. This study was determined to be exempt after review by the Prisma Health-Upstate Institutional Review Board.Prior to study implementation, three faculty with fellowships in medical education developed six learning objectives related to infectious disease topics guided by the Model of the Clinical Practice of Emergency Medicine PracticeOn the study dates, participating residents completed a survey that included basic demographics, including their PGY year, identified gender, identified generation based on birth year, and previous experience with educational escape rooms. Then they completed a nine-item, multiple-choice pre-quiz relevant to objectives 1–3. After the pre-quiz, residents from UCI participated in the escape room format, whereas residents from Prisma received the same content in a lecture format. During the second half of the session, both programs completed another nine-item, multiple-choice pre-quiz, this time covering objectives 4–6. Residents from UCI this time received the lecture format, whereas residents from Prisma instead participated in an escape room activity . DirectlThe entire session lasted 90 minutes total, including 25 minutes for each activity followed by 5–10 minutes for debrief and questions. All content was delivered virtually via Zoom. The lecture format was delivered using Google Slides. To maintain the highest quality of lecture we used best practices in multimedia design based on Mayer’s principles of multimedia learning,The residents were randomly divided into teams of 4–5 participants that included a mix of PGY levels. They were split into breakout rooms and provided with a quick response code linking to a Google Form, which guided them through four escape room puzzles with a 25-minute time limit. They were allowed to use any source for information and up to two hints provided at the study authors’ discretion. The first team that completed all the puzzles correctly was recognized as the winner. See Survey data was stored in a secure Research Electronic Data Capture survey tool . We calculated mean aggregated scores on the pre- and post-quizzes for the escape room and the lectures using the two proportions z-test, as well as IMI results for each program using the paired t-test for normally distributed data and the Wilcoxon signed-rank test paired for non-normally distributed data . Data analysis was conducted using the software program R version 4.0.4 .A total of 30 EM residents participated in this quasi-experimental study, 14 from UCI and 16 from Prisma. The demographics were similar at both programs, with >90% of the residents self-identifying as being born in the millennial generation see . There wResidents at both programs improved their pre- vs post-quiz scores on the content related to the escape room; however, there was no significant improvement in the pre- vs post-quiz scores pertaining to the lecture activities at either program see .p = 0.1434), whereas Prisma reported a significant difference in the median score of 6 for the escape room format vs 3.5 for the lecture format (p = 0.0145). Learners at UCI responded to the question “The activity did not hold my attention at all” with a median score of 2 for both the escape and lecture formats (p = 0.4606), whereas learners at Prisma reported a significant difference of 0 for the escape room format vs 3 for the lecture format (p = 0.0259).The IMI interest/enjoyment subscale results are listed in This study demonstrated a statistically significant increase in knowledge as a result of participation in an escape room at two EM residency programs compared to a lecture format, where there was no statistical increase in knowledge. The IMI results demonstrate that residents enjoyed the escape room and found it interesting at both programs, although the learners at Prisma noted a statistical difference in their enjoyment vs the learners at UCI. While no qualitative data was collected to learn why this distinction existed, residents at UCI reported slightly more experience with educational escape rooms. Also, in our experience UCI includes more educational games as part of its didactics as compared to Prisma. A novelty effect, or a waning motivation level, has been described in previous gamification literature and could explain this difference.There are many potential limitations of this study. Firstly, it was a limited convenience sample of residents attending their weekly didactic conference at two programs. There may be baseline differences in the participants that were not identified, such as their enjoyment of gamification techniques, an inherent variation in resident baseline knowledge on infectious disease topics due to different curricula at different programs, and varying familiarity with escape rooms, and in particular virtual escape rooms. The pre- and post-quizzes were the same for a given activity; therefore, recall of the questions may have affected the results . Competition and the increased cognitive load of the game itself could have negatively affected some learners.It is not clear whether learners will retain the knowledge they gained through the escape room as opposed to a lecture format as we did not assess for this. While every attempt was made to ensure consistency across the content delivered, there is a possibility that the content was not presented in a similar manner, as it was delivered on two different days and the learning objectives were delivered in opposite formats to the program. The faculty did train together prior to the sessions to rehearse the teaching scripts and used similar templates for both the escape room and lecture content despite it covering different objectives. Regarding the IMI results, despite it being a validated tool this was a limited sample size and therefore may not have accurately reflected learners’ motivation.As a result of the COVID-19 pandemic, it has become apparent that educators must be able to adapt to virtual settings to reach their learners. Delivering a virtual escape room may be a feasible way to do this. This study helps establish the utility of using escape rooms to enhance learning as compared to a lecture format. While didactic lectures remain an efficient way for medical educators to disseminate information to learners, virtual escape rooms may be an equally if not a more effective way to provide knowledge to learners while creating a fun, motivating, and interactive environment for learning with minimal to no cost. Future research comparing traditional teaching methods to in-person escape rooms may be helpful, as well as testing long-term retention of knowledge as a result of the activities.


Multiple sclerosis (MS) is the most common disabling disease of the central nervous system (CNS) with a progressive neurodegenerative pattern. It is characterized by demyelination of white matter in CNS and apoptosis of oligodendrocytes. Tumor necrosis factor (TNF) alpha is a major cytokine in the pathogenesis of MS. However, the failure of TNF alpha inhibitors in preclinical and clinical trials disapproved of their use in MS patients. Nevertheless, failures and misses sometimes open avenues for new hits. In the later years, it was discovered that TNF signaling is mediated via two different receptors, TNFR1 and TNFR2, both of which have paradoxical effects. TNFR1 mediates demyelination and apoptosis, while TNFR2 promotes remyelination and neuroprotection. This explained the cause of the failure of non-selective TNF alpha-blockers in MS. It also enlightened researchers that repurposing the previously formulated non-selective TNF alpha-blockers using a receptor-selective approach could lead to discovering novel biologic agents with a broader spectrum of indications and better safety profiles. This review focuses on a novel premier TNFR1 blocker, atrosab, which has been tested in animal models of MS, experimental autoimmune encephalomyelitis (EAE), where it demonstrated a reduction in symptom severity. The early promise shown by atrosab in preclinical studies has given us hope to find another revolutionary drug for MS in the future. Clinical trials, which will finally decide whether this drug can be used as a better therapeutic agent for MS or not, are still going on, but currently, there is no approved evidence regarding efficacy of these agents in treating MS. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of white matter in the central nervous system . Young women are affected more commonly, and it is one of the frequent causes of disability in young adults . There aA definitive cure for this disease is still a challenge and focus of research for neuroscientists worldwide . TreatmeTumor necrosis factor-alpha  is an immunomodulatory cytokine involved in physiological and pathological processes in the CNS . HoweverTNF-alpha inhibitors are currently used as an efficient therapeutic choice in many autoimmune and inflammatory diseases . SeveralHowever, these failed attempts prompted researchers to enhance the scope of research in TNF signaling pathways and critically revise assumed immunopathological mechanisms . FurtherAtrosab is a humanized monovalent antibody against TNFR1 developed for the treatment of inflammatory disorders. In vitro, atrosab successfully inhibited TNF-mediated responses such as apoptosis and nuclear factor (NF) kappa B-dependent gene expression that is responsible for the production of interleukin 6 (IL-6) and interleukin 8 (IL-8) . These fWe searched the PubMed database using the Medical Subject Headings (MeSH) keywords: receptors, tumor necrosis factor, type 1. We conducted additional data search on PubMed using regular keywords: TNF alpha selective inhibitors in multiple sclerosis and atrosab. In this review, we included all the studies related to the pathophysiological mechanisms of TNF signaling in neurodegenerative diseases, the use of TNF alpha-blockers in other autoimmune diseases, the adverse effects, and the efficacy of TNF alpha-blockers.Role of tumor necrosis factor and its receptors in neurodegenerative diseasesTNF is a regulatory cytokine and is a critical signaling protein involved in the pathophysiology of various immune-mediated and inflammatory disorders, including neurodegenerative diseases . WilliamAlthough TNF is a precise therapeutic target for the treatment of MS, a complete blockade of TNF alpha effects may not yield desired outcomes due to the heterogeneity of receptors. A better therapeutic strategy may be designed by being specific for either of the two anti pathways; antagonism of TNFR1 or agonizing the effects of TNFR2. Table Listeria monocytogenes [TNF-alpha inhibitors are remarkable in treating inflammatory bowel disease and rheumatological conditions including rheumatoid arthritis and ankylosing spondylitis . Other iytogenes . Severalytogenes . Other cytogenes . Fernándytogenes . They coytogenes . Mansourytogenes . The datytogenes . They suytogenes . Cohen eytogenes . They alytogenes . Singh eytogenes . Napolitytogenes . The litytogenes . Zhu et ytogenes . They coytogenes . Kopp coytogenes . They coytogenes . The absytogenes .We can deduce from the above studies that although adverse neurological outcomes rarely occur with the use of TNF alpha-blockers, physicians should still screen patients for risk factors before starting treatment so that iatrogenic harm is minimized. Most susceptible individuals are those who have a first-degree relative with MS. Moreover, if any neurological symptoms appear during treatment, the drug should be immediately discontinued, and proper evaluation of the patient should be done because the ultimate goal of using these biological agents is to benefit the patient and not to provide cure at the cost of other illnesses.Selective inhibition of TNFR1 improved MS symptoms A more targeted and selective approach toward TNF receptors might prove more beneficial in the treatment of MS than non-selective treatment. Experimental trials were redesigned to selectively inhibit TNFR1 to control pro-inflammatory effects or activate TNFR2 to promote anti-inflammatory and cell survival pathways.Caminero et al. hypothesized in their study that selective TNFR1 inhibition may lead to more favorable results in MS . Van HauThe above studies enlighten us that we can widen the therapeutic spectrum of these drugs by selectively targeting the TNF alpha receptors while ameliorating the neurological adverse effects reported with non-selective TNF alpha blockade. The success of selective TNFR1 blockers in preclinical trials further verified this hypothesis. These drugs can provide better treatment options in MS and other diseases where complete blockade of TNF effects was not effective. In addition, it can also be used as a revised strategy in other TNF-mediated diseases, that is, rheumatoid arthritis, inflammatory bowel disease, and psoriasis.Atrosab, a novel biological drug with anti-TNFR1 activityUnderstanding the effects of selective TNF receptors blockade and its success in preclinical trials led to the formulation of a novel biological agent with anti-TNFR1 activity, which can be tested and approved for the treatment of TNF-mediated diseases, especially those in which non-selective TNF alpha-blockers were proven to be detrimental . KontermThus we can see that after identifying the selective TNF receptor blockers, much work has been done to modify these biological molecules to increase their efficacy and safety so that they can be considered clinically appropriate therapeutic choices. Although atrosab has shown promise in preclinical trials, it has yet to pass clinical trials in human subjects before being recruited as an approved treatment option for MS and other TNF-mediated autoimmune disorders.These reformulated drugs can also be tested for use in other diseases such as psoriasis, inflammatory bowel disease, rheumatoid arthritis, and ankylosing spondylitis, where non-selective TNF alpha-blockers have been approved but their use is limited due to demyelinating side effects.LimitationsThis study is a traditional literature review, and the articles reviewed have not undergone any quality appraisal. Most of the studies included in this review are animal studies. Data search was not conducted systematically. Currently, we have no data of any clinical trials that depict how beneficial atrosab has been so far in reducing disability progression in patients with MS.The idea of receptor-selective modulation of TNF alpha appears promising based on the information currently available. While acknowledging the benefits of non-selective TNF alpha-blockers already in the market, we can apprehend that selective receptor blockade can be more rewarding in terms of therapeutic value and safety profile. The recent promise shown by atrosab, a premier selective TNFR1 blocker, gives us hope to discover a better drug for MS. We can also anticipate that this repurposing approach will open new horizons to discover more innovative drugs for many other diseases. Clinical trials are being conducted to validate the usage of selective TNFR1 blockers in human subjects. They may be a revolutionary advancement in the field of immunobiology if they demonstrate similar outcomes shown in preclinical trials. More studies should also be done by conducting extensive phase 3 and phase 4 clinical trials to evaluate the effectiveness of selective TNF alpha-blockers.


Cell‐to‐cell heterogeneity is a feature of the tumor necrosis factor (TNF)‐stimulated inflammatory response mediated by the transcription factor NF‐κB, motivating an exploration of the underlying sources of this noise. Here, we combined single‐transcript measurements with computational models to study transcriptional noise at six NF‐κB‐regulated inflammatory genes. In the basal state, NF‐κB‐target genes displayed an inverse correlation between mean and noise characteristic of transcriptional bursting. By analyzing transcript distributions with a bursting model, we found that TNF primarily activated transcription by increasing burst size while maintaining burst frequency for gene promoters with relatively high basal histone 3 acetylation (AcH3) that marks open chromatin environments. For promoters with lower basal AcH3 or when AcH3 was decreased with a small molecule drug, the contribution of burst frequency to TNF activation increased. Finally, we used a mathematical model to show that TNF positive feedback amplified gene expression noise resulting from burst size–mediated transcription, leading to a subset of cells with high TNF protein expression. Our results reveal potential sources of noise underlying intercellular heterogeneity in the TNF‐mediated inflammatory response. Single‐cell transcript measurements show that TNF stimulation primarily increases transcriptional burst size at NF‐κB target genes. Chromatin measurements and mathematical modeling are used to explore the sources and consequences of TNF‐mediated bursting. Transcriptional bursting at the HIV LTR has been shown to be influenced by chromatin environment both in the basal state  pause regulation were associated with differences in the regulation of transcriptional bursting in response to TNF; and reducing basal histone acetylation at the Tnf promoter prior to stimulation caused TNF to shift from increasing burst size to increasing burst frequency. Finally, we used a mathematical model to explore how TNF positive feedback affects cell‐to‐cell heterogeneity in Tnf transcription. We found that transcription mediated via a burst size increase, as compared to a burst frequency increase, resulted in more heterogeneous cell populations when amplified by positive feedback, with a small subset of high TNF producers. Overall, we conclude that TNF primarily increases transcriptional burst size for endogenous NF‐κB target promoters. Moreover, our results suggest that burst size–mediated transcription combined with positive feedback may contribute to the substantial cell‐to‐cell variability observed in the TNF‐mediated inflammatory response.In this study, we analyzed changes in gene expression noise and transcriptional bursting at six endogenous NF‐κB target promoters before and after TNF stimulation. We found that TNF stimulation increased mean transcription while maintaining noise for all but the most repressed NF‐κB‐target genes. We found that TNF stimulation primarily increased burst size while maintaining burst frequency, leading to highly skewed transcript distributions, especially for Nfkbia and Tnfaip3 encode the intracellular proteins IκB‐α and A20, respectively, which negatively regulate NF‐κB p65 . Following TNF stimulation, qPCR Fig . Increasters Fig .et al, Nfkbia to less than one mRNA on average per cell for Il6 and Csf2  in Jurkat T cells Fig   vs the log10(CV2) for the basal mRNA measurements of these six NF‐κB‐regulated genes in Jurkat cells  vs log10(CV2) for smFISH measurements of a subset of the same targets in HeLa cells . Noise decreased as mean increased along this trend line, but the slope of this inverse relationship was less steep than what would be expected from a Poisson distribution  that decreased after 2 h of TNF stimulation  and log10(CV2) of mRNA counts before and after TNF treatment. For the NF‐κB targets that increased mean without a significant reduction in noise , we observed that in some cases the points moved outside the basal trend line resulting in noise that further deviated from Poissonian behavior  and burst frequency mf = μ/(mb − 1) based on the sample moments are often used to describe deviation from Poisson distributions, for which mb = 1 and mf = ∞  of this distribution can be solved theoretically and then burst frequency (ka) and burst size  can be inferred by finding the optimum fit between the experimental and theoretical pdfs using maximum likelihood estimation (MLE)  to experimentally measured values when possible. Transcription of Il6 and Csf2 was too low to be measured accurately, and so we used the average decay rate measured for the other four targets, which displayed similar transcript stability (t1/2 ≈ 40 min) and is in line with previously reported values  and burst frequency .To further explore these differences, we fit our data to a two‐state model of promoter activity . These samples, which exhibit the largest transient increase in transcription induced by TNF, may not be well described by the random telegraph model. Model fits indicate that in the basal state, most genes share a low basal burst frequency of ˜ 1 transition per hour and a burst size of only a few transcripts  has been associated with changes in burst frequency for many genes  and heightened when looking at ser5‐p RNAPII (associated with initiation). Enrichment of NELF, which inhibits elongation, coupled with enrichment of ser5‐p RNAPII, is indicative of paused RNAPII at Tnfaip3, Tnf, and Il8, in contrast to the Il6 and Csf2 promoters. Taken together, the RNAPII ChIP shows that the Tnfaip3, Tnf, and Il8 promoters, which increase burst size after TNF treatment, accumulate more paused RNAPII than the Il6 and Csf2 promoters in response to TNF.We also measured total RNAPII, serine‐5 phosphorylated RNAPII (ser5‐p), serine‐2 phosphorylated RNAPII (ser2‐p), and negative elongation factor (NELF) before and at 2 and 4 h after TNF treatment Fig . We founIl6 and Csf2 and the more highly activated targets that show significant increases in burst size  vs burst size .Clustering our ChIP data, we found clear separation between size Fig . Within et al, Tnfaip3 and Tnf) would instead exhibit TNF‐mediated increases in burst frequency and a reduced burst size increase.Our ChIP data suggested an association between basal H3 acetylation (AcH3) at a target promoter and transcriptional bursting in response to TNF. Specifically, we observed that as basal AcH3 at the promoter increased, there was a shift toward a TNF‐mediated increase in burst size Fig . This isTnfaip3 and Tnf promoters by pretreating Jurkat cells with the histone acetyltransferase (HAT) inhibitor A‐485, a specific inhibitor of the HATs p300/CBP that are recruited by NF‐κB  in response to TNF stimulation for Tnf and Tnfaip3, although the effect was more pronounced for Tnf . Similarly, the reduction in AcH3 at the Tnf promoter resulted in transcriptional bursting that resembled Il6 and Csf2 (no change in burst size combined with an increased burst frequency). Thus, we conclude that decreasing basal AcH3 at target promoters shifts TNF‐induced transcription from increasing burst size to increasing burst frequency.We then compared transcription 1 h after TNF stimulation with and without A‐485 pretreatment. Overall, we found that A‐485 pretreatment significantly reduced mean mRNA expression for both  TNF Fig , green.  Tnf Fig . A‐485 paip3 Fig . Fitting Tnf Fig , green. et al, et al, et al, et al, et al, Our ChIP data also suggested an association between RNAPII pausing at target promoters and TNF‐mediated transcriptional bursting. Specifically, we observed that TNF‐mediated increases in burst size were associated with increased RNAPII promoter‐proximal pausing as measured by the accumulation of ser5‐p RNAPII Fig , perhapsTnfaip3 and Tnf promoters as measured by ChIP‐qPCR, but did not affect total RNAPII  in Jurkat T cells, the skewed HIV mRNA distributions produced by TNF activation of transcription via burst size resulted in viral activation when amplified by HIV‐mediated positive feedback, while transcription via burst frequency did not , which inhibits protein transport from the endoplasmic reticulum to the Golgi and thus blocks secretion. We found that BFA modestly reduced transcription at 2 h following TNF stimulation and also reduced the inferred burst size, while also increasing the inferred burst frequency  and simultaneously decreased burst size . By increasing burst frequency while simultaneously decreasing burst size, we were able to identify a region in which mean expression remains relatively constant but noise varies due to differences in burst behavior  and found that basal histone acetylation was associated with differences in TNF activation of transcriptional bursting  state was required to fit Il1b distributions. Zambrano et al similarly demonstrated that a promoter model with a third refractory state, combined with variability in upstream NF‐κB signaling, was necessary to explain their observation of a subset of “first responder” cells that produced higher levels of Nfkbia, Tnf, and HIV in HeLa cells . We expect that complex model configurations will be necessary to reproduce cell‐to‐cell heterogeneity of endogenous NF‐κB targets across cell types, stimulations, targets, and levels of expression.To model transcriptional bursting, we used the random telegraph model with one productive and one unproductive promoter state, and our results were largely consistent with our calculations of burst size and burst frequency based on the distribution moments. This simple promoter model sufficiently captured differences in transcriptional bursting “modes” following TNF treatment in Jurkat T cells that was a main focus of our study. However, two recent studies analyzing transcriptional bursting in response to stimulation of NF‐κB by TNF or LPS reported somewhat different results , 300 nM A‐485 , 62.5 nM JQ1 (Tocris), or Brefeldin A .Jurkat T‐cell clone E6‐1 was obtained from ATCC. Jurkat cells were cultured in Roswell Park Memorial Institute 1640 (RPMI) medium (Thermo Fisher Scientific). All media was supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific). Cells were maintained in 5% COGapdh. To calculate decay rates in Jurkat cells, we performed qRT–PCR after a 1‐h TNF treatment followed by 10 μg/ml actinomycin‐D treatment for varying times for Nfkbia, Tnfaip3, Tnf, and Il8 , including an on‐column DNase treatment. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific) and dT oligo primer. cDNA was diluted in nuclease‐free water and quantified using SsoAdvanced Universal SYBR Green Supermix on a CFX Connect Real‐Time System (Bio‐Rad) with the following amplification scheme: 95°C denaturation for 90 s followed by 40 cycles of 95°C for 15 s, 60°C annealing for 10 s, and 72°C elongation for 45 s with a fluorescence read at the end of each elongation step. This was followed by a 60–90°C melt‐curve analysis with 0.5°C increments to confirm product specificity. All samples were normalized to the house‐keeping gene Nfkbia, Tnfaip3, and Il8  available online (www.biosearchtech.com). All mRNAs were hybridized with Stellaris RNA FISH Probes labeled with Fluorescein  or Quasar 670  following the manufacturer's instructions. Briefly, Jurkat cells were treated under indicated conditions and then plated onto Cell‐Tak (Corning) coated Lab‐Tek #1.0 8‐well chambered coverglass (Thermo Fisher Scientific) or μ‐Slide 8‐well glass‐bottom coverslip (Ibidi). Cells were fixed in 3.7% formaldehyde (Thermo Fisher Scientific) for 10 min and then permeabilized overnight in 70% ethanol (Fisher Scientific). Cells were hybridized for 12 h overnight with the following probe set specific conditions: 250 nM probe for Tnf/Il6/Csf2 in 2× SSC (Thermo Fisher Scientific) with 10% formamide (Thermo Fisher Scientific) and 100 mg/ml dextran sulfate (Amresco) at 37°C, 50 nM probe for Tnfaip3 in 2× SSC with 10% formamide and 80 mg/ml dextran sulfate at 37°C, 250 nM probe for Nfkbia in 2× SSC with 12% formamide and 100 mg/ml dextran sulfate at 37°C, and 250 nM probe for Il8 in 2× SSC with 10% formamide and 100 mg/ml dextran sulfate at 25°C. For multiplex smFISH targeting Nfkbia and Tnf, probe concentrations were kept the same as for single gene smFISH and hybridization buffer for Tnfaip3 was used. After hybridization, cells were washed twice with 2× SSC and 10% formamide, counterstained with 100 ng/ml Hoechst 33342 (Thermo Fisher Scientific) for 15 min, and immersed in VectaShield mounting media (Vector Labs). Cells hybridized with Nfkbia, Tnfaip3, Tnf, and Il6 probes were imaged on an Axio Observer Zi inverted microscope (Zeiss) with an Orca Flash 4.0 V2 digital CMOS camera (Hamamatsu) and a 100× APO oil objective . Cells hybridized with Il8 and Csf2 probes were imaged on a Nikon Eclipse Ti spinning disk confocal microscope (Yokogawa CSU‐W1 spinning disk) with an Andor iXon Ultra888 EMCCD camera (Andor Technology) and a plan apochromatic 100× oil objective  after identification of regions of interest within the field of view with high cell numbers. In all cases, Z‐stacks of 30–80 images with 0.3 μm intervals were acquired. To avoid experimenter bias in selection of cells to image, only nuclear signal and not smFISH probe signal was used to select fields of view for imaging. All probe sequences are listed in Dataset The probe sets targeting et al, et al, We quantified mRNAs in individual cells using FISH‐Quant in MATLAB R2016B (Mathworks Inc.) (Mueller Chromatin immunoprecipitation was performed using the Upstate EZ‐Magna ChIP kit (Millipore). Briefly, 5 million cells per condition were fixed in 1% formaldehyde (Sigma) for 10 min, after which excess formaldehyde was quenched with 10× glycine at room temperature. Cells were washed three times with ice cold PBS and then lysed in 300 μl of 1% SDS lysis buffer with protease inhibitor cocktail (Roche). Lysates were sonicated with a Diagenode Bioruptor Plus with the following settings: 30 min of 30 s ON/30 s OFF at high power in a 4°C water bath. Sheared DNA was run on a 1% agarose gel (Lonza) to verify that sheared DNA was between 100 and 1,000 bp. Samples were pre‐cleared with PureProteome Protein G magnetic beads (Millipore) at 4°C and 5% of each sample was aliquoted as a percent input control. Samples were incubated with antibody at manufacturers' recommended concentrations overnight at 4°C. PureProteome beads were added and incubated for 1 h at 4°C. Beads were washed once each with low salt, high salt, and LiCl immune complex wash buffers, then washed twice with TE buffer, and then eluted with elution buffer at room temperature. Crosslinks were reverse by incubating samples with NaCl overnight at 65°C. DNA was purified using the QIAQuick PCR Cleanup kit (Qiagen). DNA was quantified using quantitative PCR using SsoAdvanced Universal SYBR Green Supermix on a CFX Connect Real‐Time System (Bio‐Rad). qPCR was run in triplicate, and melt curves were run to confirm product specificity. All primer sequences are listed in Dataset Cells were prepared for intracellular cytokine staining to detect TNF production using the BD Cytofix/Cytoperm kit (BD Biosciences). Briefly, 100,000 cells per condition were treated with TNF and Brefeldin A (BioLegend), then washed with PBS, and fixed with Fix/Perm for 20 min at 4°C. Fixed cells were washed twice with 1× Perm/Wash. Cells were stained with 4 μg/ml (1:125 dilution) anti‐TNF (eBioscience # 14‐7348‐81) for 1 h at 4°C, washed twice with 1× Perm/Wash, and then stained with 10 μg/ml (1:200 dilution) anti‐mouse‐AlexaFluor647 (Thermo Fisher Scientific A‐21235) for 1 h at 4°C. All data were acquired on an Attune NxT Flow Cytometer (Thermo Fish Scientific) analyzed with FlowJo .ka) and burst size (b = kt/ki) parameters that best fit the measured mRNA distributions to the full analytical solution to the two‐state stochastic gene expression model  was used to select burst frequency . All tests were performed with an alpha value of 0.05. All smFISH experiments included a sufficient number of cells to characterize the transcript distributions (n > 100 cells). A summary of all experimental conditions, biological replicates, and total cell numbers collected by smFISH is included in Table et al, et al, P < 0.05) if the 95% CI's were not overlapping . The mRNA produces TNF protein at the rate kp, is exported out of the cell at a rate kex, and degraded at a rate of gp. TNF is known to positively feedback onto its own production, and so a feedback loop was introduced into the model to increase the rate of mRNA production, km, as TNF increased. The reactions governing this model, along with accompany rate constants, are described in Table We modified an existing two‐state bursting parameter model . To replicate scenarios without positive feedback, amplification A was set to 0.To simulate treatment with exogenous TNF, we altered our burst size dynamically to reflect that TNF treatment increases experimental burst size in a time‐dependent curve. Our BFA experiment showed that cked Fig . We perfcked Fig . This phncy Fig. ; howeverncy Fig. . The oveet al, To stochastically simulate TNF protein and mRNA transcript production over time, we used network‐free stochastic simulator (NFSIM) (Sneddon To understand how the system behaves under basal conditions, we assumed equilibrium for the above equations. First, we examined promotor dynamics, and solved for steady‐state. By solving equation We can then examine TNF concentrations inside and outside the cell (equations Finally, we use EQ3, EQ7, and EQ8 to solve for mRNA values under steady‐state conditions, following:Tnf mRNA conditions. To understand how the unbounded feedback parameters influenced the model upon TNF treatment, we ran 2D parameter scans altering feedback parameters. Two parameters were chosen that qualitatively reproduced the time course of TNF‐activation experiments under deterministic simulation of the model (Fig Using this equation, we explored how the parameter space affects mRNA concentration before TNF treatment. By varying feedback parameters—the amplification rate, and the K half‐max—we recreated regions that matched basal odel Fig  and replki and ka. Four representative parameter combinations were chosen, each with an average of 10 mRNA transcripts per cell at 1 h (Fig To explore how changes in burst size and burst frequency influenced phenotypic outcomes, we stochastically simulated 1,000 cells using NFSIM, altering the parameters  1 h Fig .Study conception and experiment design: VLB, VCW, SG, and KM‐J. Experiments: VLB and VCW. Mathematical and stochastic modeling: MEB Data analysis: VLB, VCW, MEB, and KM‐J. Figure preparation and manuscript writing: VLB, MEB, and KM‐J. Manuscript editing: All authors. Funding and research supervision: KM‐J.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Review Process FileClick here for additional data file.