Clinical and virological profile of locally acquired acute hepatitis E in South Bulgaria.

INTRODUCTION: Acute hepatitis E virus (HEV) infection is recognized as a zoonosis in several European countries. We describe the characteristics and outcomes of locally acquired acute HEV hepatitis. METHODOLOGY: A prospective study was conducted among adult patients with acute HEV hepatitis at the University Hospital in Plovdiv, South Bulgaria between January 2020 and May 2022. An acute HEV infection case was a patient with acute hepatitis and laboratory-confirmed anti-HEV IgM antibodies and/or HEV RNA in serum. Demographic data, clinical manifestations, laboratory test results, and outcomes were recorded. RESULTS: A total of 46 patients were selected. Median age of 65 years (interquartile range [IQR] 50.8-74.3). 28 (60.87%) were male. 22 (47.83%) had comorbidities such as diabetes (15), liver cirrhosis (3), hepatitis B virus infection (2), and malignancies (2). Of the 46, 18 (39.13%) patients were viremic and, HEV genotype 3 was detected. The median (IQR) serum alanine aminotransferase, aspartate aminotransferase, bilirubin, platelet, and international normalized ratio levels were 992 (495.8-1714.3) U/L, 715 (262.5-1259.3) U/L, 204 (132.3-235.5) µmol/L, 204 (132.3-235.5) ×109 L, and 1.0 (0.89-1.19), respectively. Six patients with underlying liver diseases had severe hepatitis. A young patient with osteoarthritis progressed to acute liver failure and died. The persistent HEV infection was ruled out in 2 malignant patients who tested HEV RNA negative three months after discharge. CONCLUSIONS: Acute HEV hepatitis is a diagnosis to consider after excluding other causes of acute viral hepatitis. A diagnostic workup should include timely testing for HEV to identify the most vulnerable to severe consequences.

Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population.

The prevalence of hepatitis E virus (HEV) in the Bulgarian population remains underestimated. The aim of the present study was to evaluate age and gender trends in HEV prevalence in the heterogeneous Bulgarian population. Stored serum samples from blood donors and different patient sub-populations-kidney recipients (KR), patients with Guillain-Barre syndrome (GBS), Lyme disease (LD), patients with liver involvement and a clinical diagnosis other than viral hepatitis A and E (non-AE), hemodialysis (HD) and HIV-positive patients (HIV)-were retrospectively investigated for markers of past and recent/ongoing HEV infection. The estimated overall seroprevalence of past infection was 10.6%, ranging from 5.9% to 24.5% for the sub-populations evaluated, while the seroprevalence of recent/ongoing HEV infection was 7.5%, ranging from 2.1% to 20.4%. The analysis of the individual sub-populations showed a different prevalence with respect to sex. In regard to age, the cohort effect was preserved, as a multimodal pattern was observed only for the GBS sub-population. Molecular analysis revealed HEV 3f and 3e. The type of the population is one of the main factors on which the anti-HEV prevalence depends, highlighting the need for the development of guidelines related to the detection and diagnosis of HEV infection with regard to specific patient populations.

Novel biochemical, structural, and systems insights into inflammatory signaling revealed by contextual interaction proteomics.

Protein-protein interactions (PPIs) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components but mostly lack the context of cellular states. However, the generation of context-dependent PPI networks is essential for structural and systems-level modeling of biological processes-a goal that remains an unsolved challenge. Here we describe an experimental/computational strategy to achieve a modeling of PPIs that considers contextual information. This strategy defines the composition, stoichiometry, temporal organization, and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function.

Hepatitis of unknown aetiology in children - epidemiological overview of cases reported in Europe, 1 January to 16 June 2022.

Following the report of an excess in paediatric cases of severe acute hepatitis of unknown aetiology by the United Kingdom (UK) on 5 April 2022, 427 cases were reported from 20 countries in the World Health Organization European Region to the European Surveillance System TESSy from 1 January 2022 to 16 June 2022. Here, we analysed demographic, epidemiological, clinical and microbiological data available in TESSy. Of the reported cases, 77.3% were 5 years or younger and 53.5% had a positive test for adenovirus, 10.4% had a positive RT-PCR for SARS-CoV-2 and 10.3% were coinfected with both pathogens. Cases with adenovirus infections were significantly more likely to be admitted to intensive care or high-dependency units (OR = 2.11; 95% CI: 1.18-3.74) and transplanted (OR = 3.36; 95% CI: 1.19-9.55) than cases with a negative test result for adenovirus, but this was no longer observed when looking at this association separately between the UK and other countries. Aetiological studies are needed to ascertain if adenovirus plays a role in this possible emergence of hepatitis cases in children and, if confirmed, the mechanisms that could be involved.

Assessment of hepatitis E seropositivity among HIV-infected patients in Bulgaria.

It is debatable whether HIV-infected patients are at greater risk for hepatitis E virus (HEV) infection compared with healthy subjects. The reported anti-HEV seroprevalence among different groups in Bulgaria varied from 9.04% to 25.9%, but the information regarding the HIV population is still missing. The aim of the present study was to evaluate hepatitis E seroprevalence among HIV-infected patients in Bulgaria and to analyze demographic and immunological factors associated with HEV infection. Serum samples of 312 HIV-infected patients were analyzed retrospectively. Age, sex, residence and laboratory markers for HEV, HBV, HCV and HIV infection, and lymphocytes subpopulations were collected for all patients. None of the tested samples were positive for HEV RNA. HEV seroprevalence among HIV-infected patients was 10.9%. Males were more affected with the highest prevalence of positivity in the age group > 30 to ≤ 40 years. The documented HIV transmission routes in HIV/HEV co-infected group were heterosexual, homosexual, intravenous drug use (IDU), and vertical with predominace of the heterosexual route (z = 0.2; p = 0.804). There was a statistically significant trend of HIV mixed infection with routes of HIV transmission other than homosexual - heterosexual in HIV/HEV group and injection drug use in HIV/HBV/HCV co-infected group. The route of HIV transmission, in contexts of patients' behavior, was associated with HEV prevalence among HIV-infected patients.

Assessment of hepatitis E seropositivity among HIV-infected patients in Bulgaria

Abstract It is debatable whether HIV-infected patients are at greater risk for hepatitis E virus (HEV) infection compared with healthy subjects. The reported anti-HEV seroprevalence among different groups in Bulgaria varied from 9.04% to 25.9%, but the information regarding the HIV population is still missing. The aim of the present study was to evaluate hepatitis E seroprevalence among HIV-infected patients in Bulgaria and to analyze demographic and immunological factors associated with HEV infection. Serum samples of 312 HIV-infected patients were analyzed retrospectively. Age, sex, residence and laboratory markers for HEV, HBV, HCV and HIV infection, and lymphocytes subpopulations were collected for all patients. None of the tested samples were positive for HEV RNA. HEV seroprevalence among HIV-infected patients was 10.9%. Males were more affected with the highest prevalence of positivity in the age group > 30 to ≤ 40 years. The documented HIV transmission routes in HIV/HEV co-infected group were heterosexual, homosexual, intravenous drug use (IDU), and vertical with predominace of the heterosexual route (z = 0.2; p = 0.804). There was a statistically significant trend of HIV mixed infection with routes of HIV transmission other than homosexual - heterosexual in HIV/HEV group and injection drug use in HIV/HBV/HCV co-infected group. The route of HIV transmission, in contexts of patients' behavior, was associated with HEV prevalence among HIV-infected patients.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

Prevalence of hepatitis C in the adult population of Bulgaria: a pilot study.

OBJECTIVE: This study piloted a European technical protocol for conducting chronic hepatitis C prevalence surveys in the general population. The pilot study took place in the Bulgarian city of Stara Zagora in 2018, and results of setting up, conducting and evaluating the survey are presented. RESULTS: A probability-based sample of the general adult population was drawn from the local population registry, stratified by age and sex. A sample size of 999 was calculated, and accounting for 50% non-response, 1998 registered invitation letters were sent. Venous blood samples and questionnaire data were collected by the Regional Health Inspectorate in Stara Zagora. Blood samples were tested for anti-HCV, and if reactive for RNA. 252 (21.6%) of the participants were included in the study. Mean age and sex distribution differed between the participants (55.9 years, 60.3% females) and the total sample (48.9 years, 53.4%). The weighted chronic HCV prevalence among participants was 0.9% [95% CI 0.2-4.2%]. The approach of only sending registered letters contributed to a low response rate, and more efforts are needed to reduce non-response, especially among men and younger age groups. Results of the evaluation were integrated in the final technical protocol.

RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

SUMO-mediated regulation of NLRP3 modulates inflammasome activity.

The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1ß, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1ß release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1ß release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases.

Hepatitis E virus genotypes and subgenotypes causing acute hepatitis, Bulgaria, 2013-2015.

BACKGROUND: In industrialized areas of the world, including Europe, Hepatitis E Virus (HEV) is considered an emerging pathogen. In fact, autochthonous cases caused by HEV genotype 3 (HEV-3) are increasingly reported. Several studies described the human HEV-3 subtypes and strains circulating in West Europe countries; in contrast, very little is known about the HEV strains responsible for acute hepatitis E in countries of East Europe/Balkans, such as Bulgaria. METHODS AND FINDINGS: Anti-HEV IgM positive serum samples (n = 103) from acute hepatitis cases (2013-2015) from all over Bulgaria were analysed for HEV RNA by Real-Time PCR. Viremia was detected in 90/103 samples. A fragment of the viral genome (ORF-2 region) was amplified by nested PCR from 76/90 viremic samples, leading to a sequence in 64 of them. Genotyping by phylogenetic analysis with standard reference sequences showed HEV-1 in 1/64 cases, HEV-3 in 63/64. Subtyping of HEV-3 sequences showed 3e (39/63, 62%), 3f (n = 15/63, 24%) and 3c (n = 8/63, 13%) subtypes; in one case the sequence subtype was uncertain and classified as 3hi. In the phylogenetic tree, most 3e sequences grouped in two well distinct clusters (A and B), each one with very low intragroup genetic distances. In contrast, 3f and 3c were interspersed with reference sequences and showed lower tendency to cluster and/or higher intragroup distances. Geographically, while 3f and 3c were scattered throughout the country, 3e was restricted to the South-West area, with most cases in two towns about 40 kilometres apart from each other. CONCLUSIONS: Most acute hepatitis E cases in Bulgaria are caused by HEV-3, subtypes 3e, 3f and 3c. Circulation of 3e appears quite different from 3f and 3c, with 3e restricted to the South-West area while 3f and 3c diffused over the country. The factors underlying the observed molecular and geographical differences remain to be investigated.

Mind Bomb Regulates Cell Death during TNF Signaling by Suppressing RIPK1's Cytotoxic Potential.

Tumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2's E3 activity or RIPK1's ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF.

The developing human connectome project: A minimal processing pipeline for neonatal cortical surface reconstruction.

The Developing Human Connectome Project (dHCP) seeks to create the first 4-dimensional connectome of early life. Understanding this connectome in detail may provide insights into normal as well as abnormal patterns of brain development. Following established best practices adopted by the WU-MINN Human Connectome Project (HCP), and pioneered by FreeSurfer, the project utilises cortical surface-based processing pipelines. In this paper, we propose a fully automated processing pipeline for the structural Magnetic Resonance Imaging (MRI) of the developing neonatal brain. This proposed pipeline consists of a refined framework for cortical and sub-cortical volume segmentation, cortical surface extraction, and cortical surface inflation, which has been specifically designed to address considerable differences between adult and neonatal brains, as imaged using MRI. Using the proposed pipeline our results demonstrate that images collected from 465 subjects ranging from 28 to 45 weeks post-menstrual age (PMA) can be processed fully automatically; generating cortical surface models that are topologically correct, and correspond well with manual evaluations of tissue boundaries in 85% of cases. Results improve on state-of-the-art neonatal tissue segmentation models and significant errors were found in only 2% of cases, where these corresponded to subjects with high motion. Downstream, these surfaces will enhance comparisons of functional and diffusion MRI datasets, supporting the modelling of emerging patterns of brain connectivity.

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death.

TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production.

Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival.

Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.

Signal Integration by the IκB Protein Pickle Shapes Drosophila Innate Host Defense.

Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. We describe Pickle, a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-κB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.

Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting.

Fluorescence-activated cell sorting (FACS) is a laser-based, biophysical technology that allows simultaneous multiparametric analysis. For the analysis of dying cells, fluorescently labeled Annexin V (Annexin V(FITC)) and propidium iodide (PI) are the most commonly used reagents. Instead of PI, 4',6-diamidino-2-phenylindole (DAPI) can also be used. DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. DAPI can be combined with Annexin V(FITC)and the potentiometric fluorescent dye, tetramethylrhodamine methyl ester (TMRM), which measures mitochondrial permeability transition and mitochondrial membrane depolarization. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells. On apoptosis or necroptosis the TMRM signal is lost. The advantage of using Annexin V(FITC)/DAPI/TMRM is that the entire cell population is labeled, and it is easy to distinguish living (TMRM + /Annexin V(FITC)-/DAPI-) from dying or dead cells (apoptosis: TMRM-/Annexin V(FITC)+ /DAPI-; necrosis: TMRM-/Annexin V(FITC)+ /DAPI+). This is important because cell debris (fluorescent negative particles) must be avoided to establish the correct parameters for the FACS analysis, otherwise incorrect statistical values will be obtained. To obtain information on the cell concentration or absolute cell counts in a sample, it is recommended to add an internal microsphere counting standard to the flow cytrometric sample. This protocol describes the FACS analysis of cell death in HT1080 and L929 cells, but it can be readily adapted to other cell types of interest.

Techniques to Distinguish Apoptosis from Necroptosis.

The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis.