Release Of Intracellular Contents Research Articles

Abstract PO4-06-10: Targeting necroptosis in breast cancer induced by Ophiobolin A

Abstract Abstract Necroptosis or programmed necrosis is a form of cell death distinct from apoptosis. Necroptotic cells display the character of rounding of the cell, gain in cell volume, rupture of plasma membrane and release of intra-cellular contents. Although few key necroptosis regulators including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) has been identified, their role/mechanism in inducing cell death in triple-negative breast cancer (TNBC) which lack RIPK3 expression remains elusive. Ophiobolin A is a compound naturally made by fungus and potently kills cancer cells especially TNBC. Our current study investigated the cell death mechanism(s) induced by Ophiobolin A (natural product) in breast cancer cell lines. The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathways including apoptosis, necroptosis, ferroptosis and paraptosis, by analyzing markers of cell death using flow cytometry and western blotting. The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with green fluorescence protein (GFP) in HeLa, MDA-MB-231, MDA-MB-468 cell lines which lacks endogenous RIPK3 expression and in MCF7 estrogen receptor (ER) positive cell line. OpA induced necrosome formation was identified by formation of puncta in TNBC and HeLa cells which lack RIP3 expression, however, necrosomes were not detectable in ER positive cells. Clinical relevance of RIPK3 and MLKL has been analyzed using bioinformatic tools. Effect of OpA In vivo has been analyzed using Immune compromised SCID mice. The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBC compared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT were shown to have more sensitivity towards OpA when compared to uninduced cell lines, which is sensitive to Necrostatin-1, an inhibitor of necroptosis. However, apoptosis, ferroptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms that OpA induces necroptosis cell death pathway in EMT undergone cells. OpA treatment significantly increased puncta in TNBC compared non-TNBC cells, further confirming the necroptotic cell death in TNBC. Bioinformatic analysis revealed that RIPK3 is negatively corelated with breast cancer and has least over-all survival in patients who lack RIPK3 expression. OpA treatment showed a significant tumor regression in tumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enriched TNBC. The study further needs an in-depth analysis of molecular mechanism induced by OpA and involvement of EMT in necroptotic cell death. Citation Format: Tolulope Ojo, Santhalakshmi Ranganathan. Targeting necroptosis in breast cancer induced by Ophiobolin A [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-10.

Identification, distribution, bactericidal and immunoregulatory activities of NK-lysins in grass carp (Ctenopharyngodon idella)

NK-lysins, which are antimicrobial peptide mainly produced by cytotoxic T lymphocytes and natural killer cells, play an important role in immune defense against infection. In this study, using CiNkla gene as template, 2 highly homologs CiNklb and CiNklc were further identified in grass carp. The three NK-lysin transcripts were ubiquitously distributed in various tissues, and CiNkla and CiNklb have the highest transcripts in spleen while CiNklc has the highest transcript in gill. Upon challenge with Aeromonas hydrophila, the transcripts of CiNkla and CiNklb were significantly increased in intestine, whereas transcript of CiNklc increased in liver. The concentration of steady-state CiNkla protein was highest in spleen in healthy fish, while upon challenge with A. hydrophila, highest level of CiNkla protein was detected in head kidney at 6 h post infection (hpi), and in liver at either 24 hpi or 48 hpi. The recombinant CiNkla and CiNklb purified from Pichia pastoris show direct bactericidal activity against both Gram-negative and Gram-positive bacterial pathogens. The A. hydrophila treated with CiNkla showed morphological change, release of intracellular contents and genome DNA degradation. Supplementation of CiNkla or CiNklb to cultured head kidney derived macrophages (HKDMs) of grass carp changes the transcription of inflammatory factors and chemokines. In vivo stimulation of fish with recombinant CiNkla or CiNklb confirmed both molecules could increase transcription of chemokines and cytokines in tissues, and liver was sensitive to stimulation and induced into an inflammation condition at earlier timepoints. Our results indicate that transcription and expression of NK-lysins are modulated by bacterial infection, and CiNkla and CiNklb could modulate host immune defense.

Necroptosis and Its Involvement in Various Diseases.

Necroptosis is a regulated form of cell death involved in the development of various pathological conditions. In contrast to apoptosis, plasma membrane rupture (PMR) occurs in cells in the relatively early stage of necroptosis; therefore, necroptosis induces a strong inflammatory response. Stimuli, including tumor necrosis factor (TNF), interferon (IFN)α/β, lipopolysaccharide, polyI:C, and viral infection, induce the formation of necrosomes that lead to membrane rupture and the release of intracellular contents, termed danger-associated molecular patterns (DAMPs). DAMPs are the collective term for molecules that normally reside in the cytoplasm or nucleus in living cells without inducing inflammation but induce strong inflammatory responses when released outside cells. Recent studies have provided a better understanding of the mechanisms underlying PMR and the release of DAMPs. Moreover, necroptosis is involved in various pathological conditions, and mutations in necroptosis-related genes can cause hereditary autoinflammatory syndromes. Thus, manipulating necroptosis signaling pathways may be useful for treating diseases involving necroptosis.

Caspase-Linked Programmed Cell Death in Prostate Cancer: From Apoptosis, Necroptosis, and Pyroptosis to PANoptosis.

Prostate cancer (PCa) is a complex disease and the cause of one of the highest cancer-related mortalities in men worldwide. Annually, more than 1.2 million new cases are diagnosed globally, accounting for 7% of newly diagnosed cancers in men. Programmed cell death (PCD) plays an essential role in removing infected, functionally dispensable, or potentially neoplastic cells. Apoptosis is the canonical form of PCD with no inflammatory responses elicited, and the close relationship between apoptosis and PCa has been well studied. Necroptosis and pyroptosis are two lytic forms of PCD that result in the release of intracellular contents, which induce inflammatory responses. An increasing number of studies have confirmed that necroptosis and pyroptosis are also closely related to the occurrence and progression of PCa. Recently, a novel form of PCD named PANoptosis, which is a combination of apoptosis, necroptosis, and pyroptosis, revealed the attached connection among them and may be a promising target for PCa. Apoptosis, necroptosis, pyroptosis, and PANoptosis are good examples to better understand the mechanism underlying PCD in PCa. This review aims to summarize the emerging roles and therapeutic potential of apoptosis, necroptosis, pyroptosis, and PANoptosis in PCa.

Dynamics of necroptosis in kidney ischemia-reperfusion injury.

Necroptosis, a pathway of regulated necrosis, involves recruitment and activation of RIPK1, RIPK3 and MLKL, leading to cell membrane rupture, cell death and release of intracellular contents causing further injury and inflammation. Necroptosis is believed to play an important role in the pathogenesis of kidney ischemia-reperfusion injury (IRI). However, the dynamics of necroptosis in kidney IRI is poorly understood, in part due to difficulties in detecting phosphorylated MLKL (pMLKL), the executioner of the necroptosis pathway. Here, we investigated the temporal and spatial activation of necroptosis in a mouse model of unilateral warm kidney IRI, using a robust method to stain pMLKL. We identified the period 3-12 hrs after reperfusion as a critical phase for the activation of necroptosis in proximal tubular cells. After 12 hrs, the predominant pattern of pMLKL staining shifted from cytoplasmic to membrane, indicating progression to the terminal phase of necroptotic cell death. Mlkl-ko mice exhibited reduced kidney inflammation at 12 hrs and lower serum creatinine and tubular injury at 24 hrs compared to wild-type littermates. Interestingly, we observed increased apoptosis in the injured kidneys of Mlkl-ko mice, suggesting a relationship between necroptosis and apoptosis in kidney IRI. Together, our findings confirm the role of necroptosis and necroinflammation in kidney IRI, and identify the first 3 hrs following reperfusion as a potential window for targeted treatments.

Current View on the Use of Extracorporeal Detoxification Methods for the Treatment of Rhabdomyolysis (Review)

Rhabdomyolysis is a syndrome caused by destruction and necrosis of muscle tissue, which is accompanied by the release of intracellular contents into the systemic circulation. The etiology of rhabdomyolysis is multifaceted, however, regardless of the etiological factor, the central element of its pathophysiology is systemic endotoxemia with multiple organ failure syndrome. Acute renal failure is one of the most common manifestations of organ dysfunction. Considering the pathogenetic model of the development of systemic endotoxemia, the timely use of extracorporeal therapy, which reduces mortality in organ failure, seems promising. All the current types of extracorporeal therapy can be divided into convection (hemofiltration), diffusion (hemodialysis), convection/diffusion (hemodiafiltration), sorption (hemoperfusion) and plasma exchange (plasmapheresis, plasma exchange, plasma sorption, etc.) methods based on physical principle.The aim of the reviewwas to summarize the available clinical data on extracorporeal treatments for rhabdomyolysis and to assess the feasibility and best indications for these methods based on the current pathogenetic model of rhabdomyolysis.Material and methods.The search for information was carried out in the Web of Science, Scopus, Medline, PubMed, RSCI, E-library and other databases. Eighty-one sources were identified containing current therapeutic approaches and relevant data of clinical and scientific research on the subject of this review.Results.In this review, the main etiological, epidemiological and pathogenetic models of acute renal injury in rhabdomyolysis have been discussed. The main methods of extracorporeal therapy have been reviewed and evaluated based on current understanding, and latest clinical data on their effectiveness have been summarized.Conclusion.The choice of the optimal extracorporeal treatment method, the time of initiation and duration of the procedure still remain controversial. The solution to this issue can potentially help to better correct the electrolyte disturbances and could protect against organ dysfunction, which would improve the outcome in patients with rhabdomyolysis.

Rational design and characterization of cell-selective antimicrobial peptides based on a bioactive peptide from Crocodylus siamensis hemoglobin

Antimicrobial resistance is a growing health concern. Antimicrobial peptides are a potential solution because they bypass conventional drug resistance mechanisms. Previously, we isolated a peptide from Crocodylus siamensis hemoglobin hydrolysate, which has antimicrobial activity and identified the main peptide from this mixture (QL17). The objective of this work was to evaluate and rationally modify QL17 in order to: (1) control its mechanism of action through bacterial membrane disruption; (2) improve its antimicrobial activity; and (3) ensure it has low cytotoxicity against normal eukaryotic cells. QL17 was rationally designed using physicochemical and template-based methods. These new peptide variants were assessed for: (1) their in vitro inhibition of microbial growth, (2) their cytotoxicity against normal cells, (3) their selectivity for microbes, and (4) the mode of action against bacteria using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. The results indicate that all designed peptides have more potent antimicrobial efficacy than QL17 and IL15 peptides. However, only the most rationally modified peptides showed strong antimicrobial activity and minimal toxicity against normal cells. In particular, IL15.3 (hydrophobicity of 47% and net charge of + 6) was a potent antimicrobial agent (MIC = 4–12 μg/mL; MBC = 6–25 μg/mL) and displayed excellent selectivity for microbes (cf. human cells) via FACS assays. Microscopy confirmed that IL15.3 acts against bacteria by disrupting the cell membrane integrity and penetrating into the membrane. This causes the release of intracellular content into the outer environment leading to the death of bacteria. Moreover, IL15.3 can also interact with DNA suggesting it could have dual mode of action. Overall, a novel variant of QL17 is described that increases antimicrobial activity by over 1000-fold (~ 5 μg/mL MIC) and has minimal cytotoxicity. It may have applications in clinical use to treat and safeguard against bacteria.

Effect of Thermal Inactivation on Antioxidant, Anti-Inflammatory Activities and Chemical Profile of Postbiotics.

Inactivation is a crucial step in the production of postbiotics, with thermal inactivation being the prevailing method employed. Nevertheless, the impact of thermal treatment on bioactivity and chemical composition remains unexplored. The objective of this study was to assess the influence of heating temperature on the antioxidant, anti-inflammatory properties and the chemical composition of ET-22 and BL-99 postbiotics. The findings revealed that subjecting ET-22 and BL-99 to thermal treatment ranging from 70 °C to 121 °C for a duration of 10 min effectively deactivated them, leading to the disruption of cellular structure and release of intracellular contents. The antioxidant and anti-inflammatory activity of ET-22 and BL-99 postbiotics remained unaffected by mild heating temperatures (below 100 °C). However, excessive heating at 121 °C diminished the antioxidant activity of the postbiotic. To further investigate the impact of thermal treatments on chemical composition, non-targeted metabolomics was conducted to analyze the cell-free supernatants derived from ET-22 and BL-99. The results revealed that compared to mild inactivation at temperatures below 100 °C, the excessive temperature of 121 °C significantly altered the chemical profile of the postbiotic. Several bioactive components with antioxidant and anti-inflammatory properties, including zomepirac, flumethasone, 6-hydroxyhexanoic acid, and phenyllactic acid, exhibited a significant reduction in their levels following exposure to a temperature of 121 °C. This decline in their abundance may be associated with a corresponding decrease in their antioxidant and anti-inflammatory activities. The cumulative evidence gathered strongly indicates that heating temperatures exert a discernible influence on the properties of postbiotics, whereby excessive heating leads to the degradation of heat-sensitive active constituents and subsequent diminishment of their biological efficacy.

The Many Faces of MLKL, the Executor of Necroptosis.

Necroptosis is a recently discovered form of regulated cell death characterized by the disruption of plasma membrane integrity and the release of intracellular content. Mixed lineage kinase domain-like (MLKL) protein is the main player of this cell death pathway as it mediates the final step of plasma membrane permeabilization. Despite the significant progress in our knowledge of the necroptotic pathway and MLKL biology, the precise mechanism of how MLKL functions remain unclear. To understand in what way MLKL executes necroptosis, it is crucial to decipher how the molecular machinery of regulated cell death is activated in response to different stimuli or stressors. It is also indispensable to unveiling the structural elements of MLKL and the cellular players that are required for its regulation. In this review, we discuss the key steps that lead to MLKL activation, possible models that explain how it becomes the death executor in necroptosis, and its emerging alternative functions. We also summarize the current knowledge about the role of MLKL in human disease and provide an overview of existing strategies aimed at developing new inhibitors that target MLKL for necroptosis intervention.

Pyroptosis and its role in cancer

Programmed cell death (PCD) is mediated by specific genes that encode signals. It can balance cell survival and death. Pyroptosis is a type of inflammatory, caspase-dependent PCD mediated by gasdermin proteins, which function in pore formation, cell expansion, and plasma membrane rupture, followed by the release of intracellular contents. Pyroptosis is mediated by caspase-1/3/4/5/11 and is primarily divided into the classical pathway, which is dependent on caspase-1, and the non-classical pathway, which is dependent on caspase-4/5/11. Inflammasomes play a vital role in these processes. The various components of the pyroptosis pathway are related to the occurrence, invasion, and metastasis of tumors. Research on pyroptosis has revealed new options for tumor treatment. This article summarizes the recent research progress on the molecular mechanism of pyroptosis, the relationship between the various components of the pyroptosis pathway and cancer, and the applications and prospects of pyroptosis in anticancer therapy.

Uncoupled pyroptosis and IL-1β secretion downstream of inflammasome signaling.

Inflammasomes are supramolecular platforms that organize in response to various damage-associated molecular patterns and pathogen-associated molecular patterns. Upon activation, inflammasome sensors (with or without the help of ASC) activate caspase-1 and other inflammatory caspases that cleave gasdermin D and pro-IL-1β/pro-IL-18, leading to pyroptosis and mature cytokine secretion. Pyroptosis enables intracellular pathogen niche disruption and intracellular content release at the cost of cell death, inducing pro-inflammatory responses in the neighboring cells. IL-1β is a potent pro-inflammatory regulator for neutrophil recruitment, macrophage activation, and T-cell expansion. Thus, pyroptosis and cytokine secretion are the two main mechanisms that occur downstream of inflammasome signaling; they maintain homeostasis, drive the innate immune response, and shape adaptive immunity. This review aims to discuss the possible mechanisms, timing, consequences, and significance of the two uncoupling preferences downstream of inflammasome signaling. While pyroptosis and cytokine secretion may be usually coupled, pyroptosis-predominant and cytokine-predominant uncoupling are also observed in a stimulus-, cell type-, or context-dependent manner, contributing to the pathogenesis and development of numerous pathological conditions such as cryopyrin-associated periodic syndromes, LPS-induced sepsis, and Salmonella enterica serovar Typhimurium infection. Hyperactive cells consistently release IL-1β without LDH leakage and pyroptotic death, thereby leading to prolonged inflammation, expanding the lifespans of pyroptosis-resistant neutrophils, and hyperactivating stimuli-challenged macrophages, dendritic cells, monocytes, and specific nonimmune cells. Death inflammasome activation also induces GSDMD-mediated pyroptosis with no IL-1β secretion, which may increase lethality in vivo. The sublytic GSDMD pore formation associated with lower expressions of pyroptotic components, GSDMD-mediated extracellular vesicles, or other GSDMD-independent pathways that involve unconventional secretion could contribute to the cytokine-predominant uncoupling; the regulation of caspase-1 dynamics, which may generate various active species with different activities in terms of GSDMD or pro-IL-1β, could lead to pyroptosis-predominant uncoupling. These uncoupling preferences enable precise reactions to different stimuli of different intensities under specific conditions at the single-cell level, promoting cooperative cell and host fate decisions and participating in the pathogen "game". Appropriate decisions in terms of coupling and uncoupling are required to heal tissues and eliminate threats, and further studies exploring the inflammasome tilt toward pyroptosis or cytokine secretion may be helpful.

Anticandidal Activity of Capsaicin and Its Effect on Ergosterol Biosynthesis and Membrane Integrity of Candida albicans.

Oral candidiasis is an infection of the oral cavity commonly caused by Candida albicans. Endodontic treatment failure has also been found to be persistent from C. albicans in the root canal system. Despite the availability of antifungal drugs, the management of Candida oral infection is difficult as it exhibits resistance to a different class of antifungal drugs. Therefore, it is necessary to discover new antifungal compounds to cure fungal infections. This study aimed to examine the antifungal susceptibility of Capsaicin, an active compound of chili pepper. The susceptibility of Capsaicin and Fluconazole was tested against the Candida species by the CLSI (M27-A3) method. The effect of Capsaicin on the fungal cell wall was examined by the ergosterol inhibitory assay and observed by the scanning electron micrograph. The MIC range of Capsaicin against Candida isolates from oral (n = 30), endodontic (n = 8), and ATCC strains (n = 2) was 12.5-50 µg/mL. The MIC range of Fluconazole (128- 4 µg/mL) significantly decreased (2- to 4-fold) after the combination with Capsaicin (MIC/4) (p < 0.05). Capsaicin (at MIC) significantly reduced the mature biofilm of C. albicans by 70 to 89% (p < 0.01). The ergosterol content of the cell wall decreased significantly with the increase in the Capsaicin dose (p < 0.01). Capsaicin showed high sensitivity against the hyphae formation and demonstrated a more than 71% reduction in mature biofilm. A fluorescence microscopy revealed the membrane disruption of Capsaicin-treated C. albicans cells, whereas a micrograph of electron microscopy showed the distorted cells' shape, ruptured cell walls, and shrinkage of cells after the release of intracellular content. The results conclude that Capsaicin had a potential antifungal activity that inhibits the ergosterol biosynthesis in the cell wall, and therefore, the cells' structure and integrity were disrupted. More importantly, Capsaicin synergistically enhanced the Fluconazole antifungal activity, and the synergistic effect might be helpful in the prevention of Fluconazole resistance development and reduced drug-dosing.

Long-Term Examination of Water Chemistry Changes Following Treatment of Cyanobacterial Bloom with Coagulants and Minerals.

The abundant growth in cyanobacterial blooms poses severe ecological threats with a high risk to aquatic organisms and global public health. Control of cyanobacterial blooms involves spraying cyanobacteria removal materials, including coagulants. However, little is known about the fate of the coagulated-cyanobacteria-laden water. Here, we examined long-term changes in water quality following treatment with various coagulants and minerals for cyanobacterial removal when the coagulated cyanobacterial cells were not removed from the water. An experiment in a controlled water system tested the effects of six different compounds, one conventional coagulant, two natural inorganic coagulants, and three minerals. All tested coagulants and minerals exhibited >75% of cyanobacterial removal efficiency. However, compared to the control, higher concentrations of nitrogen were observed from some samples treated during the experimental period. After 20 months, the final total phosphorus concentration of the raw water increased 20-fold compared to the initial concentration to 11.82 mg/L, indicating significant nutrient release over time. Moreover, we observed that the decomposition of sedimented cyanobacterial cells caused the release of intracellular contents into the supernatant, increasing phosphorous concentration over time. Therefore, cyanobacterial cells should be removed from water after treatment to prevent eutrophication and maintain water quality.

Ferroptosis and Autoimmune Diseases.

Adequate control of autoimmune diseases with an unclear etiology resulting from autoreactivation of the immune system remains a major challenge. One of the factors that trigger autoimmunity is the abnormal induction of cell death and the inadequate clearance of dead cells that leads to the exposure or release of intracellular contents that activate the immune system. Different from other cell death subtypes, such as apoptosis, necroptosis, autophagy, and pyroptosis, ferroptosis has a unique association with the cellular iron load (but not the loads of other metals) and preserves its distinguishable morphological, biological, and genetic features. This review addresses how ferroptosis is initiated and how it contributes to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel diseases. The mechanisms responsible for ferroptosis-associated events are discussed. We also cover the perspective of targeting ferroptosis as a potential therapeutic for patients with autoimmune diseases. Collectively, this review provides up-to-date knowledge regarding how ferroptosis occurs and its significance in autoimmune diseases.

Evaluating the Antibacterial Activity and Mode of Action of Thymol-Loaded Chitosan Nanoparticles Against Plant Bacterial Pathogen Xanthomonas campestris pv. campestris.

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in cruciferous crops, resulting in severe yield loss worldwide. The excessive use of chemical pesticides in agriculture to control diseases has raised significant concern about the impact on the environment and human health. Nanoparticles have recently gained significant attention in agriculture owing to their promising application in plant disease control, increasing soil fertility and nutrient availability. In the current study, we synthesized thymol-loaded chitosan nanoparticles (TCNPs) and assessed their antibacterial activity against Xcc. The synthesis of TCNPs was confirmed by using ultraviolet–visible spectroscopy. Fourier-transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy analysis revealed the functional groups, size, and shape of TCNPs, with sizes ranging from 54 to 250 nm, respectively. The antibacterial activity of TCNPs against Xcc was investigated in vitro by liquid broth, cell viability, and live dead staining assay, and all of them demonstrated the antibacterial activity of TCNPs. Furthermore, TCNPs were found to directly inhibit the growth of Xcc by suppressing the growth of biofilm formation and the production of exopolysaccharides and xanthomonadin. The ultrastructure studies revealed membrane damage in TCNP-treated Xcc cells, causing a release of intracellular contents. Headspace/gas chromatography (GC)–mass spectrometry (MS) analysis showed changes in the volatile profile of Xcc cells treated with TCNPs. Increased amounts of carbonyl components (mainly ketones) and production of new volatile metabolites were observed in Xcc cells incubated with TCNPs. Overall, this study reveals TCNPs as a promising antibacterial candidate against Xcc.

P–452 The challenge of ovarian tissue culture: 2D versus 3D

Abstract Study question Does an alginate matrix scaffold improve ovarian tissue culture? Summary answer Ovarian tissue culture within an alginate scaffold has no advantage over conventional culture, being more time consuming and less reproductible What is known already Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. Several studies have been carried out aiming to increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells. For in vitro follicle culture, recent research has shifted from two dimensional (2D) toward the use of three-dimensional (3D) structures. The use of a matrix maintains the architecture and mimics in vivo conditions, with a variable access to oxygen and nutrients. This bridges the gap between conventional cell culture and animal models. Study design, size, duration Ovarian tissue fragments were divided into 2 groups: conventional culture (2D culture) and culture using an alginate matrix scaffold (3D culture). Tissue was evaluated at four time-points: immediately after thawing and after 24, 48 and 72 hours of culture. Participants/materials, setting, methods Rat ovarian tissue was cryopreserved and thawed with validated protocols. Follicular analysis was conducted after haematoxylin and eosin staining, regarding density, classification and degeneration. Tissue viability was assessed using lactate dehydrogenase (LDH) levels in supernatants and histological score. Three parameters were considered, namely, interstitial oedema, follicular cell degeneration and percentage of tissue in necrosis. Apoptosis was assessed by caspase 3 immunostaining. Proliferating cells were identified using Ki67 immunohistochemical labelling. Main results and the role of chance Follicular density, cell proliferation and apoptosis both in follicles and stroma was similar in both culture conditions. Stromal cells proliferation was stable in conventional culture but decreased in 3D culture (p = 0.001), which can be explained by the rigidity of alginate matrix. At 24 hours of culture, cytotoxicity was lower in the 3D model (p = 0.006), due to low levels of LDH in the supernatant, that may be related to retention within the matrix. As culture time increased cytotoxicity seemed to be similar. Degradation of the tissue was suggested by the histological score analysis of tissue during 72 hours of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). In the interior of the alginate scaffold, the bioavailability of oxygen and nutrients may be limited, affecting cell survival over time and conditioning higher level of necrosis and release of intracellular content. Limitations, reasons for caution There are two major limitations that should be addressed in future research, namely the study of the tissue-matrix interactions and culture medium supplementation to decrease follicular atresia. Wider implications of the findings: There is no advantage in the use of an alginate matrix scaffold for ovarian tissue culture, as it is more time consuming, difficult to perform and less reproductible. Trial registration number Not applicable

Cellular Responses to the Efferocytosis of Apoptotic Cells.

The rapid and efficient phagocytic clearance of apoptotic cells, termed efferocytosis, is a critical mechanism in the maintenance of tissue homeostasis. Removal of apoptotic cells through efferocytosis prevents secondary necrosis and the resultant inflammation caused by the release of intracellular contents. The importance of efferocytosis in homeostasis is underscored by the large number of inflammatory and autoimmune disorders, including atherosclerosis and systemic lupus erythematosus, that are characterized by defective apoptotic cell clearance. Although mechanistically similar to the phagocytic clearance of pathogens, efferocytosis differs from phagocytosis in that it is immunologically silent and induces a tissue repair response. Efferocytes face unique challenges resulting from the internalization of apoptotic cells, including degradation of the apoptotic cell, dealing with the extra metabolic load imposed by the processing of apoptotic cell contents, and the coordination of an anti-inflammatory, pro-tissue repair response. This review will discuss recent advances in our understanding of the cellular response to apoptotic cell uptake, including trafficking of apoptotic cell cargo and antigen presentation, signaling and transcriptional events initiated by efferocytosis, the coordination of an anti-inflammatory response and tissue repair, unique cellular metabolic responses and the role of efferocytosis in host defense. A better understanding of how efferocytic cells respond to apoptotic cell uptake will be critical in unraveling the complex connections between apoptotic cell removal and inflammation resolution and maintenance of tissue homeostasis.

Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration

Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and release of intracellular contents, the execution of which depends on the membrane-disrupting activity of the Mixed Lineage Kinase Domain-Like protein (MLKL) upon its phosphorylation. Here we found myofibers committed MLKL-dependent necroptosis after muscle injury. Either pharmacological inhibition of the necroptosis upstream kinase Receptor Interacting Protein Kinases 1 (RIPK1) or genetic ablation of MLKL expression in myofibers led to significant muscle regeneration defects. By releasing factors into the muscle stem cell (MuSC) microenvironment, necroptotic myofibers facilitated muscle regeneration. Tenascin-C (TNC), released by necroptotic myofibers, was found to be critical for MuSC proliferation. The temporary expression of TNC in myofibers is tightly controlled by necroptosis; the extracellular release of TNC depends on necroptotic membrane rupture. TNC directly activated EGF receptor (EGFR) signaling pathway in MuSCs through its N-terminus assembly domain together with the EGF-like domain. These findings indicate that necroptosis plays a key role in promoting MuSC proliferation to facilitate muscle regeneration.

SAT-372 A Case of Hypercalcemia Secondary to Rhabdomyolysis Induced by Heroin Use

Background: Rhabdomyolysis is a potential life threatening condition defined as injury of the skeletal muscle, which results in the release of intracellular contents into the circulation. This muscle injury is often associated with the development of myoglobinuria, electrolyte abnormalities, and often ARF; it can be caused by diverse mechanisms including drugs and toxins. We present a case of rhabdomyolysis complicated by hypercalcemia and ARF in a patient with a history of polysubstance abuse after using intravenous heroin. Case: A 28 y/o male with h/o polysubstance comes to the ED with c/o fatigue, nausea, vomiting and decreased urine output for about 5 days; patient acknowledged using heroin and afterwards he developed weakness and tenderness in upper and lower extremities. Additionally he admitted cocaine and marijuana abuse. On physical exam vital signs were unremarkable and he had some mild tenderness to palpation mostly of the quadriceps bilaterally. Laboratory data was significant for acute kidney injury (cr 11.33 mg/dl, n: 0.7–1.5 mg/dl) and CPK 171920 u/l (n: 22–269 u/l). He received 2 L of NS in the ED and was started on NS at 100cc/hour.He underwent hemodialysis on day 2; initially he was treated for hypocalcemia with calcium and vitamin D supplementation until day 11 were hypercalcemia (calcium 12.7 mg/dl, n: 8.7–10.3 mg/dl; ionized calcium 1.7 mmol/l, n: 1.12–1.32) was noted; this was associated with concomitant suppression of PTH (5 pg/ml, n: 10–65 pg/ml). He remained asymptomatic from calcium abnormalities during his hospitalization, his urine output recovered progressively, hemodialysis was discontinued on day 13. Upon discharge was recommended to f/u with nephrology. Discussion: Various neurological and neuromuscular complications of heroin abuse have been described; one of these is rhabdomyolysis; its pathophysiology in heroin abuse is thought to be multifactorial; including acidosis, hypoxia, muscle compression and adulterants found in heroin. Narcotics may also have direct cell toxicity and alter membrane transport. Usually upon initial presentation hypocalcemia is one of the most common electrolyte imbalances seen with rhabdomyolysis. The proposed mechanism is precipitation of serum calcium salts in necrotic muscle. This may be followed by hypercalcemia during the diuretic phase of ARF which appears to be a relatively unusual complication associated with the presence of severe muscle damage due to metastatic calcium salts that are liberated from the necrotic muscle and the return to the serum. Conclusion: This case report highlights the importance of recognizing potential electrolyte imbalances in patients with rhabdomyolysis; it appears, that concomitant rhabdomyolysis and ARF are needed for a patient to develop hypercalcemia.Serum calcium should always be routinely measured and the appropriate treatment should be implemented to improve outcomes.

Partners in Crime: The Interplay of Proteins and Membranes in Regulated Necrosis.

Pyroptosis, necroptosis, and ferroptosis are well-characterized forms of regulated necrosis that have been associated with human diseases. During regulated necrosis, plasma membrane damage facilitates the movement of ions and molecules across the bilayer, which finally leads to cell lysis and release of intracellular content. Therefore, these types of cell death have an inflammatory phenotype. Each type of regulated necrosis is mediated by a defined machinery comprising protein and lipid molecules. Here, we discuss how the interaction and reshaping of these cellular components are essential and distinctive processes during pyroptosis, necroptosis, and ferroptosis. We point out that although the plasma membrane is the common target in regulated necrosis, different mechanisms of permeabilization have emerged depending on the cell death form. Pore formation by gasdermins (GSDMs) is a hallmark of pyroptosis, while mixed lineage kinase domain-like (MLKL) protein facilitates membrane permeabilization in necroptosis, and phospholipid peroxidation leads to membrane damage in ferroptosis. This diverse repertoire of mechanisms leading to membrane permeabilization contributes to define the specific inflammatory and immunological outcome of each type of regulated necrosis. Current efforts are focused on new therapies that target critical protein and lipid molecules on these pathways to fight human pathologies associated with inflammation.