Quantitative assessment of cell fate decision between autophagy and apoptosis

Abstract Autophagy and apoptosis are cellular processes that regulate cell survival and death, the former by eliminating dysfunctional components in the cell, the latter by programmed cell death. Stress signals can induce either process, and it is unclear how cells ‘assess’ cellular damage and make a ‘life’ or ‘death’ decision upon activating autophagy or apoptosis. A computational model of coupled apoptosis and autophagy is built here to analyze the underlying signaling and regulatory network dynamics. The model explains the experimentally observed differential deployment of autophagy and apoptosis in response to various stress signals. Autophagic response dominates at low-to-moderate stress; whereas the response shifts from autophagy (graded activation) to apoptosis (switch-like activation) with increasing stress intensity. The model reveals that cytoplasmic Ca2+ acts as a rheostat that fine-tunes autophagic and apoptotic responses. A G-protein signaling-mediated feedback loop maintains cytoplasmic Ca2+ level, which in turn governs autophagic response through an AMP-activated protein kinase (AMPK)-mediated feedforward loop. Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ) emerges as a determinant of the competing roles of cytoplasmic Ca2+ in autophagy regulation. The study demonstrates that the proposed model can be advantageously used for interrogating cell regulation events and developing pharmacological strategies for modulating cell decisions

β-Defensins Activate Human Mast Cells Via Mas-Related Gene X2

Human β-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells in vitro and cause increased vascular permeability in rats in vivo. In this study, we sought to determine whether hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells (BMMC) in vitro and had no effect on vascular permeability in vivo. By contrast, these peptides induced sustained Ca2+ mobilization and substantial degranulation in human mast cells, with hBD3 being more potent. Pertussis toxin (PTx) had no effect on hBD-induced Ca2+ mobilization, but La3+ and 2-aminoethoxydiphenyl borate (a dual inhibitor of inositol 1,4,5-triphosphate receptor and transient receptor potential channels) caused substantial inhibition of this response. Interestingly, degranulation induced by hBDs was substantially inhibited by PTx, La3+, or 2-aminoethoxydiphenyl borate. Whereas human mast cells endogenously express G protein-coupled receptor, Mas-related gene X2 (MrgX2), rat basophilic leukemia, RBL-2H3 cells, and murine BMMCs do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD-induced degranulation, but had no effect on anaphylatoxin C3a-induced response. Furthermore, ectopic expression of MrgX2 in RBL-2H3 and murine BMMCs rendered these cells responsive to hBDs for degranulation. This study demonstrates that hBDs activate human mast cells via MrgX2, which couples to both PTx-sensitive and insensitive signaling pathways most likely involving Gαq and Gαi to induce degranulation. Furthermore, murine mast cells are resistant to hBDs for degranulation, and this reflects the absence of MrgX2 in these cells. Copyright©2013 by The American Association of Immunologists, Inc

Cetuximab-Ag₂S quantum dots for fluorescence imaging and highly effective combination of ALA-based photodynamic/chemo-therapy of colorectal cancer cells

Colorectal cancer (CRC) has a poor prognosis and urgently needs better therapeutic approaches. 5-Aminolevulinic acid (ALA) induced protoprophyrin IX (PpIX) based photodynamic therapy (PDT) is already approved in the clinic for several cancers but not yet well investigated for CRC. Currently, systemic administration of ALA offers a limited degree of tumour selectivity, except for intracranial tumours, limiting its wider use in the clinic. Combination of effective ALA-PDT with chemotherapy may provide a promising alternative approach for CRC treatment. Herein, theranostic Ag2S quantum dots (AS-2MPA) optically trackable in near-infrared (NIR), conjugated with endothelial growth factor receptor (EGFR) targeting Cetuximab (Cet) and loaded with ALA for PDT monotherapy or ALA/5-fluorouracil (5FU) for the combination therapy is proposed for enhanced treatment of EGFR(+) CRC. AS-2MPA-Cet endowed excellent targeting of the high EGFR expressing cells and showed a strong intracellular signal for NIR optical detection in a comparative study performed on SW480, HCT116, and HT29 cells, which are high, medium and low EGFR expressers. Targeting provided enhanced uptake of the ALA loaded nanoparticles by strong EGFR expressing cells and formation of higher levels of PpIX. Cells also differ in their efficiency to convert ALA to PpIX, and SW480 was the best, followed by HT29, while HCT116 were determined as unsuitable for ALA-PDT. The therapeutic efficacy was evaluated in 2D cell cultures and 3D spheroids of SW480 and HT29 cells using AS-2MPA with either electrostatically loaded, hydrazone or amide linked ALA to achieve different levels of pH or enzyme sensitive release. Most effective phototoxicity was observed in SW480 cells using AS-2MPA-ALA-electrostatic-Cet due enhanced uptake of the particles, fast ALA release and effective ALA-to-PpIX conversion. Targeted delivery reduced the effective ALA concentration significantly which was further reduced with codelivery of 5FU. Delivery of ALA via covalent linkage was also effective for PDT, but required longer incubation time for the release of ALA in therapeutic doses. Phototoxicity was correlated with high levels of reactive oxygen species (ROS) and apoptotic/necrotic cell death. Hence, both AS-2MPA-ALA-Cet based PDT and AS-2MPA-ALA-Cet-5FU based Chemo/PDT combination therapy coupled with strong NIR tracking of the nanoparticles demonstrate an exceptional therapeutic effect on CRC cells and an excellent potential for synergistic multistage tumour targeting therapy

Highly Fluorescent Pyrene-Functional Polystyrene Copolymer Nanofibers for Enhanced Sensing Performance of TNT

A pyrene-functional polystyrene copolymer was prepared via 1,3-dipolar cycloaddition reaction (Sharpless-type click recation) between azide-functional styrene copolymer and 1-ethynylpyrene. Subsequently, nanofibers of pyrene-functional polystyrene copolymer were obtained by using electrospinning technique. The nanofibers thus obtained, found to preserve their parent fluorescence nature, confirmed the avoidance of aggregation during fiber formation. The trace detection of trinitrotoluene (TNT) in water with a detection limit of 5 nM was demonstrated, which is much lower than the maximum allowable limit set by the U.S. Environmental Protection Agency. Interestingly, the sensing performance was found to be selective toward TNT in water, even in the presence of higher concentrations of toxic metal pollutants such as Cd2+, Co2+, Cu2+, and Hg2+. The enhanced sensing performance was found to be due to the enlarged contact area and intrinsic nanoporous fiber morphology. Effortlessly, the visual colorimetric sensing performance can be seen by naked eye with a color change in a response time of few seconds. Furthermore, vapor-phase detection of TNT was studied, and the results are discussed herein. In terms of practical application, electrospun nanofibrous web of pyrene-functional polystyrene copolymer has various salient features including flexibility, reproducibility, and ease of use, and visual outputs increase their value and add to their advantage. © 2015 American Chemical Society

Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death

High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these “partial” intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented

Bench-to-bedside review : targeting antioxidants to mitochondria in sepsis

Peer reviewedPublisher PD

Clinical-pathological study on β-APP, IL-1β, GFAP, NFL, Spectrin II, 8OHdG, TUNEL, miR-21, miR-16, miR-92 expressions to verify DAI-diagnosis, grade and prognosis

Traumatic brain injury (TBI) is one of the most important death and disability cause, involving substantial costs, also in economic terms, when considering the young age of the involved subject. Aim of this paper is to report a series of patients treated at our institutions, to verify neurological results at six months or survival; in fatal cases we searched for βAPP, GFAP, IL-1β, NFL, Spectrin II, TUNEL and miR-21, miR-16, and miR-92 expressions in brain samples, to verify DAI diagnosis and grade as strong predictor of survival and inflammatory response. Concentrations of 8OHdG as measurement of oxidative stress was performed. Immunoreaction of β-APP, IL-1β, GFAP, NFL, Spectrin II and 8OHdG were significantly increased in the TBI group with respect to control group subjects. Cell apoptosis, measured by TUNEL assay, were significantly higher in the study group than control cases. Results indicated that miR-21, miR-92 and miR-16 have a high predictive power in discriminating trauma brain cases from controls and could represent promising biomarkers as strong predictor of survival, and for the diagnosis of postmortem traumatic brain injury