Reduced Cell Death Research Articles

MicroRNA-based genetic therapy in leukemia: properties, delivery, and experimental models.

Leukemia is a type of cancer that affects white blood cells. In this disease, immature blood cells undergo genetic mutations, leading to excessive replication and reduced cell death compared to healthy cells. In cancer, there may be the activation of oncogenes and the deactivation of tumor suppressor genes that control certain cellular functions. Despite the undeniable contribution to the patient's recovery, conventional cancer treatments may have some not-so-beneficial effects. In this case, gene therapy appears as an alternative to classical treatments. Gene therapy delivers genetic material to cells to replace or modify dysfunctional genes, a safe method for neoplasms. One of the types of nucleic acids explored in gene therapy is microRNA (miRNA), a group of endogenous, non-proteincoding, small single-stranded RNA molecules involved in the regulation of gene expression, cell division, differentiation, angiogenesis, migration, apoptosis, and carcinogenesis. This review aims to bring together the most recent advances found in the literature on cancer gene therapy based on microRNAs in the oncological context, focusing on leukemia.

SERCA2 phosphorylation at the heart of the disease

Gonnot et al. [1] thoroughly investigated the regulatory role of glycogen synthase kinase 3 beta (GSK3β) in modulating cardiac isoform 2 of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) activity. They have found that in ischemic hearts of patients and mouse-GSK3β -mediated SERCA2 phosphorylation at serine 663 dampens the SERCA2 pump activity and induces Ca2+ overload which sensitizes towards myocardial ischemia-reperfusion (I/R) injury. The inhibition of serine 663 phosphorylation significantly increases SERCA2 activity and, by preventing cytosolic and mitochondrial Ca2+ overload, reduces cell death during reperfusion. Augmented SERCA2 activity also substantially improves excitation-contraction coupling in cardiomyocytes upon recovery from reperfusion injury. This study provides valuable insights into pathophysiological relevance of GSK3β -mediated SERCA2 phosphorylation in the context of heart diseases and paves the way for designing novel clinical therapeutic approaches to alleviate post infartion heart failure.

A novel organotypic cortical slice culture model for traumatic brain injury: molecular changes induced by injury and mesenchymal stromal cell secretome treatment.

Traumatic brain injury (TBI) is a major worldwide neurological disorder with no neuroprotective treatment available. Three-dimensional (3D) in vitro models of brain contusion serving as a screening platform for drug testing are lacking. Here we developed a new in vitro model of brain contusion on organotypic cortical brain slices and tested its responsiveness to mesenchymal stromal cell (MSC) derived secretome. A focal TBI was induced on organotypic slices by an electromagnetic impactor. Compared to control condition, a temporal increase in cell death was observed after TBI by propidium iodide incorporation and lactate dehydrogenase release assays up to 48 h post-injury. TBI induced gross neuronal loss in the lesion core, with disruption of neuronal arborizations measured by microtubule-associated protein-2 (MAP-2) immunostaining and associated with MAP-2 gene down-regulation. Neuronal damage was confirmed by increased levels of neurofilament light chain (NfL), microtubule associated protein (Tau) and ubiquitin C-terminal hydrolase L1 (UCH-L1) released into the culture medium 48 h after TBI. We detected glial activation with microglia cells acquiring an amoeboid shape with less ramified morphology in the contusion core. MSC-secretome treatment, delivered 1 h post-injury, reduced cell death in the contusion core, decreased NfL release in the culture media, promoted neuronal reorganization and improved microglia survival/activation. Our 3D in vitro model of brain contusion recapitulates key features of TBI pathology. We showed protective effects of MSC-secretome, suggesting the model stands as a tractable medium/high throughput, ethically viable, and pathomimetic biological asset for testing new cell-based therapies.

Increased mitochondrial free Ca2+ during ischemia is suppressed, but not eliminated by, germline deletion of the mitochondrial Ca2+ uniporter

Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex-vivo-perfused hearts from germline MCU-knockout (KO) and wild-type (WT) mice using transmural spectroscopy. Matrix Ca2+ levels are measured with a genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and the known fall in pH during ischemia, hearts are glycogen depleted to decrease the ischemic fall in pH. At 20min of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared with MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts, suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU.

Effects of rivaroxaban on myocardial mitophagy in the rat heart.

This study aims to demonstrate the efficacy of rivaroxaban's pharmacokinetic effects on myocardial mitophagy in rats by inducing apoptosis. In this double-blind experiment, Wistar albino male rats were randomly divided into three groups for an experimental ischemia model: the sham group (Group 1; n=7), the control group (Group 2; n=7), and the drug group (Group 3; n=7). Rivaroxaban was perorally administered with gavage at 2 mg/ kg/day for 28 days in Group 3. The heart was surgically exposed, and ischemia was achieved by compressing the vessel around the proximal part of the left anterior descending coronary artery for 10 min. The heart tissue was then transected, removed, and morphologically and immunohistochemically examined under a light microscope. Heart sections were immunohistochemically marked with caspase 3, caspase 9, APAF1, and Bcl-2 antibodies. Group 1 was compared to the rivaroxaban-treated group, and the pathways inducing apoptosis was increased (caspase 3, caspase 9, APAF1; p<0.015, p<0.004, and p<0.01, respectively) and Bcl-2, the molecule that inhibits apoptosis, was decreased (p<0.01) in Group 3. The present study provides an evidence that the mitophagy response is less in rivaroxaban-treated rats, showing the protective effect of rivaroxaban against acute ischemia. Rivaroxaban-treated rats may have reduced cell death in cardiomyocytes during myocardial infarction and thus have reduced damage to the heart tissue caused by myocardial infarction.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

Can l-ascorbic acid and trans-resveratrol protect HaCaT cells from fine particulate matter toxicity?

Continuous exposure of human skin to air pollution can result in a range of undesirable skin conditions. In our recent study, UV and visible light were found to increase cytotoxicity of fine particulate matter (PM2.5 ) against human keratinocytes. Since it is impossible to avoid exposure of human skin to PM2.5 , effective strategies are needed to reduce their damaging effects. l-ascorbic acid and resveratrol were tested as potential topical agents against pollution-related skin impairment. Although these agents were previously found to ameliorate PM-dependent damage, the effect of light and seasonal variation of particles were not previously studied. EPR spin-trapping, DPPH assay, and singlet oxygen phosphorescence were used to determine the scavenging activities of the antioxidants. MTT, JC-10 and iodometric assays were used to analyze the effect on PM2.5 -induced cytotoxicity, mitochondrial damage and oxidation of lipids. Live-cell imaging was employed to examine wound-healing properties of cells. Light-induced, PM2.5 -mediated oxidative damage was examined by immunofluorescent staining. Both antioxidants effectively scavenged free radicals and singlet oxygen produced by PM2.5 , reduced cell death and prevented oxidative damage to HaCaT cells. l-ascorbic acid and resveratrol, especially when applied in combination, can protect HaCaT cells against the dark and light induced toxicity of PM2.5 .

MiR-210-5p promotes the differentiation of human induced pluripotent stem cells into dopaminergic neural precursors by targeting SMAD4 and SUFU and treats parkinsonian rats

The differentiation of human induced pluripotent stem cells (hiPSCs) into functional dopaminergic neural precursors is the basis of cell therapy for Parkinson's disease (PD). However, the use of small molecule inhibitors/activators in the differentiation of hiPSCs in vitro leads to cell death and low differentiation efficiency. Moreover, the mechanism of differentiation remains unclear. MiR-210-5p was increased during hiPSCs differentiation. Whether it promotes hiPSCs differentiation and transplantation needs further study. Here, we overexpressed miR-210-5p in hiPSCs to study its roles and mechanisms. We found that miR-210-5p promoted the differentiation of hiPSCs into dopaminergic neural precursors and reduced the expression of SMAD4 and SUFU meanwhile. Luciferase assays showed that miR-210-5p binded to SMAD4 and SUFU, which are key molecules in the key signals (TGF-β and SHH) of hiPSCs differentiation. Furthermore, in the effect evaluation of cell transplantation into parkinsonian rats, the degree of behavioral recovery and the growth of transplanted cells in the group overexpressed miR-210-5p were similar to those in the positive group with all small molecule inhibitors/activators. Therefore, we conclude that miR-210-5p promotes the differentiation of hiPSCs into dopaminergic neural precursors by targeting SMAD4 and SUFU. In the therapeutic evaluation of cell transplantation, miR-210-5p can replace the use of corresponding small molecule inhibitors/activators to reduce cell death. This study provides an experimental basis and a new target for the miRNA-modified differentiation of hiPSCs and cell transplantation in clinical treatment of PD in the future.

Antioxidant, Antiviral, and Anti-Inflammatory Activities of Lutein-Enriched Extract of Tetraselmis Species.

Microalgae are proposed to have powerful applications for human health in the pharmaceutical and food industries. Tetraselmis species (sp.), which are green microalgae, were identified as a source of broad-spectrum health-promoting biological activities. However, the bioactivity of these species has not been elucidated. We aimed to confirm the antioxidant, antiviral, and anti-inflammatory effects of Tetraselmis sp. extract (TEE). TEE showed 2,2-diphenyl-1-picryl-hydrazyl-hydrate radical and hydrogen peroxide scavenging activities and reduced plaque formation in Vero E6 cells infected with vaccinia virus. TEE treatment also significantly inhibited nitric oxide (NO) production and improved cell viability in lipopolysaccharide (LPS)-induced RAW264.7 cells. These anti-inflammatory effects were further analyzed in LPS-induced RAW 264.7 cells and the zebrafish model. Further, TEE reduced induced NO synthase expression and proinflammatory cytokine release, including tumor necrosis factor-α, interleukin-6, and interleukin-1β, through MAPKs and NF-κB-dependent mechanisms. Further analysis revealed that TEE increased the survival rate and reduced cell death and NO production in an LPS-stimulated zebrafish model. Further, high-performance liquid chromatography revealed a strong presence of the carotenoid lutein in TEE. Overall, the results suggest that lutein-enriched TEE may be a potent antioxidant, antiviral, and anti-inflammatory agent that could be sustainably utilized in industrial applications.

Ectopic over-expression of HaFT-1, a 14-3-3 protein from Haloxylon ammodendron, enhances acquired thermotolerance in transgenic Arabidopsis.

Haloxylon ammodendron, an important shrub utilized for afforestation in desert areas, can withstand harsh ecological conditions such as drought, high salt and extreme heat. A better understanding of the stress adaptation mechanisms of H. ammodendron is vital for ecological improvement in desert areas. In this study, the role of the H. ammodendron 14-3-3 protein HaFT-1 in thermotolerance was investigated. qRT-PCR analysis showed that heat stress (HS) priming (the first HS) enhanced the expression of HaFT-1 during the second HS and subsequent recovery phase. The subcellular localization of YFP-HaFT-1 fusion protein was mainly detected in cytoplasm. HaFT-1 overexpression increased the germination rate of transgenic Arabidopsis seeds, and the survival rate of HaFT-1 overexpression seedlings was higher than that of wild-type (WT) Arabidopsis after priming-and-triggering and non-primed control treatments. Cell death staining showed that HaFT-1 overexpression lines exhibited significantly reduced cell death during HS compared to WT. Transcriptome analysis showed that genes associated with energy generation, protein metabolism, proline metabolism, autophagy, chlorophyll metabolism and reactive oxygen species (ROS) scavenging were important to the thermotolerance of HS-primed HaFT-1 transgenic plants. Growth physiology analysis indicated that priming-and-triggering treatment of Arabidopsis seedlings overexpressing HaFT-1 increased proline content and strengthened ROS scavenging activity. These results demonstrated that overexpression of HaFT-1 increased not only HS priming but also tolerance to the second HS of transgenic Arabidopsis, suggesting that HaFT-1 is a positive regulator in acquired thermotolerance.

1723-P: Dual-Label Kinetic Assay to Monitor ß-Cell Death and Proliferation

A deficit of β-cell mass is common to all types of diabetes. Thus, many research efforts are dedicated to developing therapies to preserve and expand β-cell mass. Current methods assessing β-cell proliferation and cell death are limited due to reliance on a single time point, and usually cannot measure proliferation and death simultaneously. Here we have developed and optimized a novel method, based on the single-cell and population-level analyses using real-time kinetic labeling (SPARKL), to study kinetics of cell proliferation and death in β cells. SPARKL uses two fluorescent dyes - SYTO21 labels all cells in green, and YOYO3, a red cell-impermeable viability dye that stains dead cells. We continuously imaged cells every few hours over numerous days and have optimized growth conditions and dye concentrations to monitor INS1 or primary rodent or human islet cells on single cell and population levels. As a first step, we compared growth under 6, 11 and 20 mM glucose and glucolipotoxicity (20 mM glucose and 1 mM palmitate). We found a doubling time of 52±2, 48±4, 44±3 and &amp;gt;120 h, respectively, in INS1 cells. At 48 h, fewer than 16% of the cells are dead in varying glucose concentrations compared to 69±5% dead cells in the glucolipotoxic condition (N=8). Harmine, a known mitogen of β-cells, enhanced proliferation in all glucose concentrations by 5-15% and protected from glucolipotoxic cell death (N=3). In primary islets, as expected, the proliferation rates were extremely low and often masked by cell death in varying glucose concentrations. Glucolipotoxicity led to &amp;gt;80% cell death in mouse and human islets (N=3). Finally, we screened a library of 73 epigenetic modifiers to screen for compounds that rescued INS1 cells from glucolipotoxic-like stress (overexpression of ChREBPβ). Our screen yielded four epigenetic modifiers that reduced cell death to less than 2% at 48 h. In conclusion, SPARKL is a novel and useful method that is accurate, unbiased, and requires minimal handling to simultaneously measure β-cell proliferation and death. Disclosure L.S.Katz: None. P.Wang: None. H.U.Kaniskan: None. J.D.Gelles: None. J.Chipuk: None. A.F.Stewart: None. D.Scott: None. Funding National Institutes of Health (R01DK130300)

B-cell capacity for expansion and differentiation into plasma cells are altered in osteoarthritis

ObjectiveAutoantibody (autoAbs) production in osteoarthritis (OA), coupled with evidence of disturbed B-cell homoeostasis, suggest a potential role for B-cells in OA. B-cells can differentiate with T-cell help (T-dep) or using alternative Toll like recptor (TLR) co-stimulation (TLR-dep). We analysed the capacity for differentiation of B-cells in OA versus age-matched healthy controls (HCs) and compared the capacity of OA synovitis-derived stromal cells to provide support for plasma cell (PC) maturation. MethodsB-cells were isolated from OA and HC. Standardised in vitro models of B-cell differentiation were used comparing T-dep (CD40 (cluster of differentiation-40/BCR (B-cell receptor)-ligation) versus TLR-dep (TLR7/BCR-activation). Differentiation marker expression was analysed by flow-cytometry; antibody secretion (immunnoglobulins IgM/IgA/IgG) by ELISA (enzyme-linked immunosorbent assay), gene expression by qPCR (quantitative polymerase chain reaction). ResultsCompared to HC, circulating OA B-cells showed an overall more mature phenotype. The gene expression profile of synovial OA B-cells resembled that of PCs. Circulating B-cells differentiated under both TLR-dep and T-dep, however OA B-cells executed differentiation faster in terms of change in surface marker and secreted more antibody at Day 6, while resulting in similar PC numbers at Day 13, with an altered phenotype at Day 13 in OA. The main difference was reduced early B-cells expansion in OA (notably in TLR-dep) and reduced cell death. Stromal cells support from OA-synovitis allowed better PC survival compared to bone marrow, with an additional population of cells and higher Ig-secretion. ConclusionOur findings suggest that OA B-cells present an altered capacity for proliferation and differentiation while remaining able to produce antibodies, notably in synovium. These findings may partly contribute to autoAbs development as recently observed in OA synovial fluids.

Does Sinapic Acid Provide Neuroprotection Against Cisplatin-Induced Toxicity in HT-22 Cells?

Objective: The aim of this study was to explain the benefits and possible protective mechanisms of sinapic acid (SA) against cisplatin-induced oxido-inflammatory damage in HT-22 rat hippocampal cells by biochemical and molecular methods. Materials and methods: Sinapic acid (SA) was applied at different concentrations (100, 400, and 800 μM) before cisplatin treatment on HT-22 cells under in vitro conditions to elicit neuroprotective activity. Half an hour after SA treatment, 5.5 μM cisplatin was added to all wells except the control group and incubated for 24 hours. Cell viability was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cytotoxicity was determined by lactate dehydrogenase (LDH) assays. Oxidative stress was evaluated by total antioxidant capacity (TAC), catalase (CAT), glutathione reductase (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) assays. In addition, the effect of SA on Caspase-3 gene regulation in HT-22 cells was investigated by real-time PCR. Results: Cisplatin decreased cell viability by approximately 40% and increased LDH level in HT-22 cells. In SA-treated groups, cell viability increased and LDH level decreased dose-independently. SA showed neuroprotective activity by inhibiting the cytotoxic activity of cisplatin and increasing the antioxidant activity in cells. Similarly, Caspase-3, which was up-regulated by cisplatin, approached the control value upon SA administration. SA eliminated the neurotoxicity of cisplatin and significantly reduced cell death and oxidative stress. Conclusion: The results of this study indicate that SA protects HT-22 cells against cisplatin by inhibiting both the formation of oxidative stress and induction of cell apoptosis.

Autophagy Regulation Using Multimodal Chlorin e6-Loaded Polysilsesquioxane Nanoparticles to Improve Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising anticancer noninvasive technique that relies on the generation of reactive oxygen species (ROS). Unfortunately, PDT still has many limitations, including the resistance developed by cancer cells to the cytotoxic effect of ROS. Autophagy, which is a stress response mechanism, has been reported as a cellular pathway that reduces cell death following PDT. Recent studies have demonstrated that PDT in combination with other therapies can eliminate anticancer resistance. However, combination therapy is usually challenged by the differences in the pharmacokinetics of the drugs. Nanomaterials are excellent delivery systems for the efficient codelivery of two or more therapeutic agents. In this work, we report on the use of polysilsesquioxane (PSilQ) nanoparticles for the codelivery of chlorin-e6 (Ce6) and an autophagy inhibitor for early- or late-stage autophagy. Our results, obtained from a reactive oxygen species (ROS) generation assay and apoptosis and autophagy flux analyses, demonstrate that the reduced autophagy flux mediated by the combination approach afforded an increase in the phototherapeutic efficacy of Ce6-PSilQ nanoparticles. We envision that the promising results in the use of multimodal Ce6-PSilQ material as a codelivery system against cancer pave the way for its future application with other clinically relevant combinations.

Exogenous hydrogen sulfide alleviates chromium toxicity by modulating chromium, nutrients and reactive oxygen species accumulation, and antioxidant defence system in mungbean (Vigna radiata L.) seedlings

Chromium (Cr), a highly toxic redox-active metal cation in soil, seriously threatens global agriculture by affecting nutrient uptake and disturbing various physio-biochemical processes in plants, thereby reducing yields. Here, we examined the effects of different concentrations of Cr alone and in combination with hydrogen sulfide (H2S) application on the growth and physio-biochemical performance of two mungbeans (Vigna radiata L.) varieties, viz. Pusa Vishal (PV; Cr tolerant) and Pusa Ratna (PR; Cr sensitive), growing in a pot in hydroponics. Plants were grown in the pot experiment to examine their growth, enzymatic and non-enzymatic antioxidant levels, electrolyte balance, and plasma membrane (PM) H+-ATPase activity. Furthermore, root anatomy and cell death were analysed 15 days after sowing both varieties in hydroponic systems. The Cr-induced accumulation of reactive oxygen species caused cell death and affected the root anatomy and growth of both varieties. However, the extent of alteration in anatomical features was less in PV than in PR. Exogenous application of H2S promoted plant growth, thereby improving plant antioxidant activities and reducing cell death by suppressing Cr accumulation and translocation. Seedlings of both cultivars treated with H2S exhibited enhanced photosynthesis, ion uptake, glutathione, and proline levels and reduced oxidative stress. Interestingly, H2S restricted the translocation of Cr to aerial parts of plants by improving the nutrient profile and viability of root cells, thereby relieving plants from oxidative bursts by activating the antioxidant machinery through triggering the ascorbate-glutathione cycle. Overall, H2S application improved the nutrient profile and ionic homeostasis of Cr-stressed mungbean plants. These results highlight the importance of H2S application in protecting crops against Cr toxicity. Our findings can be utilised to develop management strategies to improve heavy metal tolerance among crops.

Sirtuins Modulators Counteract Mitochondrial Dysfunction in Cellular Models of Hypoxia: Relevance to Schizophrenia

Schizophrenia (SZ) is a neurodevelopmental-associated disorder strongly related to environmental factors, such as hypoxia. Because there is no cure for SZ or any pharmacological approach that could revert hypoxia-induced cellular damages, we evaluated whether modulators of sirtuins could abrogate hypoxia-induced mitochondrial deregulation as a neuroprotective strategy. Firstly, astrocytes from control (Wistar) and Spontaneously Hypertensive Rats (SHR), a model of both SZ and neonatal hypoxia, were submitted to chemical hypoxia. Then, cells were exposed to different concentrations of Nicotinamide (NAM), Resveratrol (Resv), and Sirtinol (Sir) for 48hrs. Our data indicate that sirtuins modulation reduces cell death increasing the acetylation of histone 3. This outcome is related to the rescue of loss of mitochondrial membrane potential, changes in mitochondrial calcium buffering capacity, decreased O2−rad levels and increased expression of metabolic regulators (Nrf-1 and Nfe2l2) and mitochondrial content. Such findings are relevant not only for hypoxia-associated conditions, named pre-eclampsia but also for SZ since prenatal hypoxia is a relevant environmental factor related to this burdensome neuropsychiatric disorder.

Burn wound conversion: clinical implications for the treatment of severe burns.

The identification of novel treatments for severe burn wounds relies on accurate clinical assessments of the extent of injury. However, evaluation of burn wound depth can be challenging due to the tendency for burn wounds to progress over time in a little-understood process known as 'burn wound conversion'. Local factors affecting the burn wound, such as inflammation, oxidative stress-induced tissue damage, vasostasis and bacterial infections, lead to increased cell death by apoptosis or oncosis, while systemic events may promote burn wound conversion. Acute shock, metabolic derangements, age or immunomodulation can modify cytokine secretion, lower immune responses, decrease blood flow or cause bacterial infection at the burn wound site. Therefore, therapeutic approaches targeting specific mechanisms that reduce cell death, improve wound reperfusion and promote tissue regrowth should favourably enhance burn wound healing, and long-term functional and aesthetic outcomes. Our current understanding of these mechanisms mostly comes from animal studies, underscoring the need for extensive research in humans. A streamlined approach would be to investigate the parallels in other disease states that exhibit ischaemia and potential reperfusion, such as ischaemic stroke and myocardial infarction. Moreover, in view of the limited knowledge available on the subject, the need exists for further clinical research into burn wound conversion and novel target pathways to ameliorate its effects. This review describes events that affect the viability of cells at the burn wound site resulting in burn wound conversion, and identifies potential targets for clinical interventions that may diminish burn wound conversion.

Impact of Resolvin-D1 in the resolution of atrial inflammation and arrhythmogenic cardiac remodelling in a rat model of myosin light chain 4 (MYL-4) mutation

Atrial fibrillation (AF) is the most common heart rhythm disorder. AF risk factors include age, hypertension, obesity, or myocardial infarction. AF can cause severe complications such as stroke and sudden death. Hence, AF is a major public health issue. Proteins called actins and myosins interact to allow myocardial contractions. A mutation affecting the gene encoding a myosin light chain type 4 (MYL-4) protein is associated with a genetically inheritable form of AF. Furthermore, we have recently demonstrated that MYL-4 rats develop atrial inflammation and fibrosis. Conventional anti-inflammatory medications are ineffective against AF. Bioactive endogenous autacoids, called specialized proresolving mediators, including resolvin-D1: RvD1, have been described to promoting inflammation-resolution and potentially reduce AF occurrence. To elucidate the impact of a triggered resolution of inflammation in the prevention of AF. Resolution of atrial inflammation by daily treatment with RvD1 prevents the development of AF in a rat model of MYL-4 mutation. We have developed a colony of male and female Sprague-Dawley Knock-In rats, mutated on the Myl-4 gene. A dose of 2 μg/kg/d RvD1 (or equivalent dose of vehicle) was administered to wild-type (WT) and MYL-4 rats for 21 days. AF was assessed by in vivo electrophysiological study. The structure of the atria was studied by echocardiography, histology and electron microscopy. The expression levels of proteins (by western blotting) and genes (by qPCR) involved in inflammation and cellular apoptosis have been assessed in the right and left atria of MYL-4 rats compared to WT. Compared to WT rat, MYL-4 mutation causes an increased risk of AF, associated with an inflammatory and apoptotic profile in the atria. We observed that daily treatment with RvD1 prevents atrial inflammation (IL-1β), reduces cell death (Bax, Bcl2) and attenuates atrial structural remodelling (dilation, hypertrophy). Inflammation plays a crucial role in the development of arrhythmogenic atrial remodeling observed in rats carrying MYL4 mutation. In addition, this project contributes to highlight the potential therapeutic effects of the RvD1 treatment, to resolve inflammation and prevent AF in the context of a rhythm disturbances provoked by cardiac mutations.

Abstract 360: Role Of Mitochondrial Dynamics In Vascular Smooth Muscle Cell Death

Background: Vascular smooth muscle cell (VSMC) death has been linked to the pathogenesis of various vascular diseases. Excessive mitochondrial fission in response to pathological stimuli contributes to cell death, thus, inhibition of mitochondrial fission can be a promising therapeutic target in vascular diseases. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission by attenuation of its GTPase activity. As association between fission inhibition &amp; VSMCs cell death has been implicated in vascular diseases, the underlying mechanism warrants further investigation. We assessed the effects of mdivi-1 (an inhibitor of Drp1-mediated mitochondrial fission) on staurosporine (STS)-induced mitochondrial dysfunction, impaired autophagy, &amp; cell death in aortic VSMC. Methods &amp; Results: The flow cytometry analysis demonstrated reduced cell death in VSMCs-treated with mdivi-1. The mitochondrial fission was evaluated using Mitotracker Red staining. mdivi-1-treated VSMCs showed significantly higher mitochondrial networks, mean branch length, &amp; mitochondrial footprint compared to STS-treated cells indicating reduced mitochondrial fragmentation. Inhibitory effects of mdivi-1 on fission were exerted by reduced Drp1 phosphorylation at 616. Mitochondrial function via evaluation of mitochondrial permeability transition pore opening, mitochondrial membrane potential &amp; bioenergetic profile showed restored mitochondrial function &amp; metabolic potential in the mdivi-1-treated cells compared to the STS group. The mdivi-1-treated VSMCs showed significantly reduced ROS generation indicating suppressed oxidative stress. The improved mitochondrial structural integrity due to inhibition of fission was associated with reduced mitophagy &amp; cell death-related proteins. Conclusion: In response to apoptotic stimuli, excessive Drp1-dependent mitochondrial fission leads to mitochondrial damage, playing a key role in VSMC death. Inhibition of Drp1-dependent mitochondrial fission by mdivi-1 attenuated mitochondrial fission &amp; prevented cell death in VSMCs. Pharmacological inhibition of mitochondrial fission provides a potential therapeutic target in vascular diseases associated with loss of VSMC.

New in vitro screening system to detect drug-induced liver injury using a culture plate with low drug sorption and high oxygen permeability

Drug-induced liver injury (DILI) is a major factor underlying drug withdrawal from the market. Therefore, it is important to predict DILI during the early phase of drug discovery. Metabolic activation and mitochondrial toxicity are good indicators of the potential for DILI. However, hepatocyte function, including drug-metabolizing enzyme activity and mitochondrial function, reportedly decreases under conventional culture conditions; therefore, these conditions fail to precisely detect metabolic activation and mitochondrial toxicity-induced cell death. To resolve this issue, we employed a newly developed cell culture plate with high oxygen permeability and low drug sorption (4-polymethyl-1-pentene [PMP] plate). Under PMP plate conditions, cytochrome P450 (CYP) activity and mitochondrial function were increased in primary rat hepatocytes. Following l-buthionine-sulfoximine-induced glutathione depletion, acetaminophen-induced cell death significantly increased under PMP plate conditions. Additionally, 1-aminobenzotriazole reduced cell death. Moreover, mitochondrial toxicity due to mitochondrial complex inhibitors (ketoconazole, metformin, and phenformin) increased under PMP plate conditions. In summary, PMP plate conditions could improve CYP activity and mitochondrial function in primary rat hepatocytes and potentially detect metabolic activation and mitochondrial toxicity.