Intracellular C3 protects ß-cells from IL-1ß-driven cytotoxicity via interaction with Fyn-related kinase.

One of the hallmarks of type 1 but also type 2 diabetes is pancreatic islet inflammation, associated with altered pancreatic islet function and structure, if unresolved. IL-1ß is a proinflammatory cytokine which detrimentally affects ß-cell function. In the course of diabetes, complement components, including the central complement protein C3, are deregulated. Previously, we reported high C3 expression in human pancreatic islets, with upregulation after IL-1ß treatment. In the current investigation, using primary human and rodent material and CRISPR/Cas9 gene-edited ß-cells deficient in C3, or producing only cytosolic C3 from a noncanonical in-frame start codon, we report a protective effect of C3 against IL-1ß-induced ß-cell death, that is attributed to the cytosolic fraction of C3. Further investigation revealed that intracellular C3 alleviates IL-1ß-induced ß-cell death, by interaction with and inhibition of Fyn-related kinase (FRK), which is involved in the response of ß-cells to cytokines. Furthermore, these data were supported by increased ß-cell death in vivo in a ß-cell-specific C3 knockout mouse. Our data indicate that a functional, cytoprotective association exists between FRK and cytosolic C3.

Multi-criteria assignment problems for optimising the emergency medical services (EMS), considering non-homogeneous speciality of the emergency departments and EMS crews.

Dispatching of the EMS crews (ambulances) to awaiting patients and then directing the patients, that are already onboard, to appropriate Emergency Departments (ED), is a nontrivial decision problem. In many emergency medical systems it is handled by the Medical Dispatcher using various strategies-sometimes preferring the closest unit. However, applying a wrong strategy may result in transferring acute-state patients, who require very specialised medical aid, to low-speciality EDs with insufficient treatment capabilities. Then, they would need to be re-transferred to referential units, prolonging substantially the time to receive treatment. In some cases such a delay might make the treatment less effective or even impossible. In this work we propose two multi-criteria mathematical optimisation problems-the first one allows us to calculate the ambulance-to-patient assignment, the second one-to establish the patient-to-hospital assignment. These problems not only take the time-to-support criterion into consideration but also optimise for the speciality of care received by each patient. The ED dispatching problem proposed allows both for direct transfers of patients to referential units and for re-transferring them from non-referential EDs. The performance of the proposed approach is tested in simulations with real-life emergency cases from the NEMSIS data set and compared with classic assignment strategies. The tests showed the proposed approach is able to produce better and more fit-for-purpose dispatching results than other strategies tested. Additionally, we propose a framework for embedding the proposed optimisation problems in the current EMS/ED dispatching process.

Outside in: Roles of complement in autophagy.

The complement system is a well-characterized cascade of extracellular serum proteins that is activated by pathogens and unwanted waste material. Products of activated complement signal to the host cells via cell surface receptors, eliciting responses such as removal of the stimulus by phagocytosis. The complement system therefore functions as a warning system, resulting in removal of unwanted material. This review describes how extracellular activation of the complement system can also trigger autophagic responses within cells, up-regulating protective homeostatic autophagy in response to perceived stress, but also initiating targeted anti-microbial autophagy in order to kill intracellular cytoinvasive pathogens. In particular, we will focus on recent discoveries that indicate that complement may also have roles in detection and autophagy-mediated disposal of unwanted materials within the intracellular environment. We therefore summarize the current evidence for complement involvement in autophagy, both by transducing signals across the cell membrane, as well as roles within the cellular environment. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

Resuscitation of the patient with suspected/confirmed COVID-19 when wearing personal protective equipment: A randomized multicenter crossover simulation trial.

BACKGROUND: The aim of the study was to evaluate various methods of chest compressions in patients with suspected/confirmed SARS-CoV-2 infection conducted by medical students wearing full personal protective equipment (PPE) for aerosol generating procedures (AGP). METHODS: This was prospective, randomized, multicenter, single-blinded, crossover simulation trial. Thirty-five medical students after an advanced cardiovascular life support course, which included performing 2-min continuous chest compression scenarios using three methods: (A) manual chest compression (CC), (B) compression with CPRMeter, (C) compression with LifeLine ARM device. During resuscitation they are wearing full personal protective equipment for aerosol generating procedures. RESULTS: The median chest compression depth using manual CC, CPRMeter and LifeLine ARM varied and amounted to 40 (38-45) vs. 45 (40-50) vs. 51 (50-52) mm, respectively (p = 0.002). The median chest compression rate was 109 (IQR; 102-131) compressions per minute (CPM) for manual CC, 107 (105-127) CPM for CPRMeter, and 102 (101-102) CPM for LifeLine ARM (p = 0.027). The percentage of correct chest recoil was the highest for LifeLine ARM - 100% (95-100), 80% (60-90) in CPRMeter group, and the lowest for manual CC - 29% (26-48). CONCLUSIONS: According to the results of this simulation trial, automated chest compression devices (ACCD) should be used for chest compression of patients with suspected/confirmed COVID-19. In the absence of ACCD, it seems reasonable to change the cardiopulmonary resuscitation algorithm (in the context of patients with suspected/confirmed COVID-19) by reducing the duration of the cardiopulmonary resuscitation cycle from the current 2-min to 1-min cycles due to a statistically significant reduction in the quality of chest compressions among rescuers wearing PPE AGP.

Complement Component C3 Is Highly Expressed in Human Pancreatic Islets and Prevents ß Cell Death via ATG16L1 Interaction and Autophagy Regulation.

We show here that human pancreatic islets highly express C3, which is both secreted and present in the cytosol. Within isolated human islets, C3 expression correlates with type 2 diabetes (T2D) donor status, HbA1c, and inflammation. Islet C3 expression is also upregulated in several rodent diabetes models. C3 interacts with ATG16L1, which is essential for autophagy. Autophagy relieves cellular stresses faced by ß cells during T2D and maintains cellular homeostasis. C3 knockout in clonal ß cells impaired autophagy and led to increased apoptosis after exposure of cells to palmitic acid and IAPP. In the absence of C3, autophagosomes do not undergo fusion with lysosomes. Thus, C3 may be upregulated in islets during T2D as a cytoprotective factor against ß cell dysfunction caused by impaired autophagy. Therefore, we revealed a previously undescribed intracellular function for C3, connecting the complement system directly to autophagy, with a broad potential importance in other diseases and cell types.

The human serum protein C4b-binding protein inhibits pancreatic IAPP-induced inflammasome activation.

AIMS/HYPOTHESIS: Inflammasome activation and subsequent IL-1ß production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1ß production. METHODS: Macrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1ß secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP-C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot. RESULTS: C4BP significantly inhibited IAPP-mediated IL-1ß secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1ß-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1ß. CONCLUSIONS/INTERPRETATION: IAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.

C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Single-cell analysis of aneuploidy events using yeast whole chromosome painting probes (WCPPs).

Aneuploidy is considered a widespread genetic variation in such cell populations as yeast strains, cell lines and cancer cells, and spontaneous changes in the chromosomal copy number may have implications for data interpretation. Thus, aneuploidy monitoring is essential during routine laboratory practice, especially while conducting biochemical and/or gene expression analyses. In the present study, we constructed a panel of whole chromosome painting probes (WCPPs) to monitor aneuploidy in a single yeast Saccharomyces cerevisiae cell. The WCPP-based system was validated using "normal" haploid and diploid cells, as well as disomic cells both with and without cell synchronisation. FISH that utilised WCPPs was combined with DNA cell cycle analysis (imaging cytometry) to provide a detailed analysis of signal variability during the cell cycle. Chromosome painting can be utilised to detect spontaneously formed disomic chromosomes and study aneuploidy-promoting conditions. For example, the frequency of disomic chromosomes was increased in cells lacking NAD(+)-dependent histone deacetylase Sir2p compared with wild-type cells (p<0.05). In conclusion, WCPPs may be considered to be a powerful molecular tool to identify individual genomic differences. Moreover, the WCPP-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.

Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.

The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.