Resistance to apoptosis-inducing drugs frequently occurs in cancer cells, limiting their usefulness in ongoing cancer treatment. Despite ongoing efforts to overcome drug resistance, a definitive solution remains elusive. However, autophagy inhibition has been shown to enhance the effectiveness of some anticancer drugs and is a possible strategy for overcoming drug resistance. In this study, we demonstrate that chlorogenic acid (CGA), a natural antioxidant, significantly enhances beta-lapachone (β-Lap)-induced cell death in cancer cells. The augmented apoptosis induced by CGA is associated with activation of protein kinase A (PKA) in β-Lap-treated cells, independent of the antioxidant properties of CGA. As a result, PKA activation in cancer cells co-treated with β-Lap and CGA effectively inhibits autophagy. Notably, PKA activation leads to phosphorylation of microtubule-associated protein 1 A/1B-light chain 3 (LC3) at the serine 12 residue, causing autophagy suppression irrespective of mTORC activity. Importantly, the cell death induced by β-Lap and CGA in NQO1-overexpressing breast or lung cancers is closely linked to autophagy inhibition. These findings suggest that combining β-Lap and CGA might be a novel strategy for cancer therapy, particularly for overcoming drug resistance caused by autophagy induction in cancer cells.

Tumor tissues exhibit elevated oxidative stress, with the cystine-glutamate transporter xCT solute carrier family 7 member 11 (xCT/SLC7A11) protecting cancer cells from oxidative damage by facilitating cystine uptake for glutathione synthesis. Disulfidptosis, a newly identified form of programmed cell death (PCD), occurs in cells with high xCT/SLC7A11 expression under glucose-deprived conditions. Distinct from other PCD pathways, disulfidptosis is characterized by aberrant disulfide bond formation and cellular dysfunction, ultimately resulting in cancer cell death. This novel mechanism offers remarkable therapeutic potential by targeting the inherent oxidative stress vulnerabilities of rapidly growing cancer cells. Advances in nanotechnology enable the development of nanomaterials capable of inducing reactive oxygen species (ROS) generation, disrupting disulfide bonds. In addition, they are capable to deliver therapeutic agents directly to tumors, thereby improving therapeutic precision and minimizing off-target effects. Moreover, combining disulfidptosis with ROS-induced immunogenic cell death can remodel the tumor microenvironment and enhance anti-tumor immunity. This review explores the mechanisms underlying disulfidptosis, its therapeutic potential in cancer treatment, and the synergistic role of nanotechnology in amplifying its effects. Selective induction of disulfidptosis using nanomaterials represents a promising strategy for achieving more effective, selective, and less toxic cancer therapies.

Caspase-independent cell death (CICD) has recently become a very important mechanism in lung cancer, in particular, to overcome a critical failure in apoptotic cell death that is common to disease progression and treatment failures. The pathways involved in CICD span from necroptosis, ferroptosis, mitochondrial dysfunction, and autophagy-mediated cell death. Its potential therapeutic applications have been recently highlighted. Glutathione peroxidase 4 (GPX4) inhibition-driven ferroptosis has overcome drug resistance in non-small cell lung cancer (NSCLC). In addition, necroptosis involving RIPK1 and RIPK3 causes tumor cell death and modulation of immune responses in the tumor microenvironment (TME). Mitochondrial pathways are critical for CICD through modulation of metabolic and redox homeostasis. Ferroptosis is amplified by mitochondrial reactive oxygen species (ROS) and lipid peroxidation in lung cancer cells, and mitochondrial depolarization induces oxidative stress and leads to cell death. In addition, mitochondria-mediated autophagy, or mitophagy, results in the clearance of damaged organelles under stress conditions, while this function is also linked to CICD when dysregulated. The role of cell death through autophagy regulated by ATG proteins and PI3K/AKT/mTOR pathway is dual: to suppress tumor and to sensitize cells to therapy. A promising approach to enhancing therapeutic outcomes involves targeting mechanisms of CICD, including inducing ferroptosis by SLC7A11 inhibition, modulating mitochondrial ROS generation, or combining inhibition of autophagy with chemotherapy. Here, we review the molecular underpinnings of CICD, particularly on mitochondrial pathways and their potential to transform lung cancer treatment.

Programmed cell deaths (PCDs) are crucial for tumor progression. By analyzing 18 PCDs, we generated a robust multigene signature, Combined Cell Death Index (CCDI), comprising necroptosis and autophagy genes for non-small cell lung cancer (NSCLC). The CCDI accurately stratified patients by survival prognosis and predicted immunotherapy responses. We validated CCDI and prioritized CCDI genes using five single-cell RNA sequencing and two spatial transcriptomics datasets. CCDI positively correlates with tumor malignancy, invasiveness, and immunotherapy resistance. Four necroptosis genes (PTGES3, MYO6, CCT6A, and CTSH) may affect cancer cell evolution. In vitro, CTSH overexpression or PTGES3 knockdown inhibited NSCLC cell proliferation and migration while inducing necroptosis with necrosome formation. Moreover, we observed diminished CTSH, heightened PTGES3, and low necroptosis activity in 12 pairs of NSCLC tumors and normal tissues. CTSH overexpression or PTGES3 knockdown induced necroptosis and improved anti-PD1 therapy efficiency in syngeneic cancer mouse models. These findings indicate necroptosis genes as potential therapeutic targets in cancer treatments.

Mobilisation of Damage-Associated Molecular Patterns (DAMPs) determines the immunogenic properties of apoptosis, but the mechanisms that control DAMP exposure are still unclear. Here we describe an unconventional autophagic pathway that inhibits the release of ATP, a critical DAMP in immunogenic apoptosis, from dying cells. Mitochondrial BAK activated by BH3-only molecules interacts with prohibitins and stomatin-1 through its latch domain, indicating the existence of an interactome specifically assembled by unfolded BAK. This complex engages the WD40 domain of the autophagic effector ATG16L1 to induce unconventional autophagy, and the resulting LC3-positive vesicles contain ATP. Functional interference with the pathway increases ATP release during cell death, reduces ATP levels remaining in the apoptotic bodies, and improves phagocyte activation. These results reveal that an unconventional component of the autophagic burst that often accompanies apoptosis sequesters intracellular ATP to prevent its release, thus favouring the immunosilent nature of apoptotic cell death.

Lactylation, a newly identified post-translational modification, entails the attachment of lactate to lysine residues within proteins, profoundly modulating diverse cellular mechanisms underlying regulated cell death (RCD). This modification encompasses two primary categories: histone lactylation and non-histone lactylation. Histone lactylation assumes a pivotal regulatory function in the RCD process, primarily by modulating the transcriptional landscape of genes implicated in cell death. In contrast, non-histone lactylation exerts its influence by targeting transferases, transcription, cell cycle progression, death pathways, and metabolic processes that are intricately involved in RCD. This review provides a comprehensive overview of recent breakthroughs in understanding how lactylation regulates RCD, while also offering insights into potential avenues for future research, thereby deepening our comprehension of cellular fate determination.

Cancer cells possess high proliferative ability and usually override apoptosis and metastasize to distant lesions. Autophagy in cancer cells is a double-edged weapon where a cross-regulation postulation between apoptosis and autophagy exists. The aim of the present study was to investigate the effect of adding Thymoquinone (TQ) to Imatinib (IM) in HCT116 human colorectal cancer cell line model on various apoptotic and autophagy markers. The combination doses of IM and TQ were selected according to our previous study concerned with cytotoxicity and uptake/efflux genes modulation. In the current study, the combination induced autophagy in HCT116 cell line which in turn enhanced apoptosis. Moreover, early apoptosis was evidenced. The induction of both autophagy and apoptosis resulted in programmed cell death. The assessment of AMPK, Par-4, apoptosis markers, colony formation assays, flow cytometry and autophagy detection by acridine orange proved this rapport.

Adult hippocampal neurogenesis is inhibited by chronic psychological stress and impaired neurogenesis underlies stress-related psychological disorders. We previously reported that chronic restraint stress (CRS) evokes autophagic death of adult hippocampal neural stem cells (NSCs) while NSC-specific deletion of Atg7 prevents death of NSCs. Examination of cognitive ability and mood regulation next day of the termination of stress showed normal hippocampal function in mice deficient of Atg7. However, it was not investigated whether the preservation of NSC pool alleviates hippocampal neuronal alterations. Here, we show that CRS increased c-Fos-positive, activated neurons in the granule cell layer and decreased spine density of CA3 neurons in the hippocampus, and these hippocampal neuronal deficits were prevented by NSC-specific deletion of Atg7. Of note, our observation was conducted right after the termination of CRS. Therefore, our results suggest that the detrimental effects of stress on hippocampal neurons can be buffered by NSCs independent of neurogenesis and NSCs are essential to the hippocampal function both through the neurogenesis-dependent developmental process and by direct regulation of neural activation.

The global incidence and prevalence of cardiometabolic disorders have risen significantly in recent years. Although lifestyle choices in adulthood play a crucial role in the development of these conditions, it is well established that events occurring early in life can have an important effect. Recent research on cardiometabolic diseases has highlighted the influence of sexual dimorphism on risk factors, underlying mechanisms, and response to therapies. In this narrative review, we summarize the current understanding of sexual dimorphism in cardiovascular and metabolic diseases in the general population and within the framework of the Developmental Origins of Health and Disease (DOHaD) concept. We explore key risk factors and mechanisms, including the influence of genetic and epigenetic factors, placental and embryonic development, maternal nutrition, sex hormones, energy metabolism, microbiota, oxidative stress, cell death, inflammation, endothelial dysfunction, circadian rhythm, and lifestyle factors. Finally, we discuss some of the main therapeutic approaches, responses to which may be influenced by sexual dimorphism, such as antihypertensive and cardiovascular treatments, oxidative stress management, nutrition, cell therapies, and hormone replacement therapy.

Strong evidence suggests links between Parkinson's Disease (PD) and melanoma, as studies have found that people with PD are at an increased risk of developing melanoma and those with melanoma are at increased risk of developing PD. Although these clinical associations are well-established, the cellular and molecular pathways linking these diseases are poorly understood. Recent studies have found a previously unrecognized role for the neurodegeneration-associated protein alpha-synuclein (αSyn) in melanoma; the overexpression of αSyn promotes melanoma cell proliferation and metastasis. However, to our knowledge, no studies have investigated the role of αSyn in in vivo melanoma models outside of a xenograft paradigm.

Our study created and characterized Snca knockout in the spontaneously developing melanoma TG3 mouse line, TG3+/+Snca-/-.

We show that αSyn loss-of-function significantly delays melanoma onset and slows tumor growth in vivo in males. Furthermore, decreased tumor volume is correlated with a decreased DNA damage signature and increased apoptotic markers, indicating a role for αSyn in modulating the DNA damage response (DDR) pathway.

Overall, our study may suggest that targeting αSyn and its role in modulating the DDR and melanomagenesis could serve as a promising new therapeutic target.

It is well known that the host response to different human and animal coronaviruses infection is regulated by the aryl hydrocarbon receptor, a ligand-activated transcription factor. The present study investigates the expression of the aryl hydrocarbon receptor during bovine coronavirus infection, through in vitro and in silico investigations. The in vitro studies demonstrate that the aryl hydrocarbon receptor and as well as its targets, CYP1A1 and CYP1B1, were significantly activated by bovine coronavirus infection in bovine cells (MDBK). During infection, the pretreatment of cells with non-cytotoxic doses of CH223191, a selective inhibitor of the aryl hydrocarbon receptor, resulted in a significant reduction in virus yield and a downregulation in the viral spike protein expression. These findings occurred in the presence of the inhibition of aryl hydrocarbon receptor signaling. Our results reveal that the bovine coronavirus acts on viral replication, upregulating the aryl hydrocarbon receptor and its downstream target proteins, CYP1A1 and CYP1B1. In addition, following the in silico studies, the three-dimensional structural model of the bovine aryl hydrocarbon receptor in complex with the antagonist CH223191 indicates that the molecular mechanism, by which the PASB and TAD domains of the receptor interact with the inhibitor, is mainly driven by an extensive network of hydrophobic interactions, with a series of hydrogen bonds contributing to stabilizing the complex. Interestingly, bioinformatic analyses revealed that the PASB and TAD domains in the human and bovine aryl hydrocarbon receptor present high similarity at the primary sequence and three-dimensional structure levels. Taken together, these findings represent a fundamental step for the development of innovative drugs targeting AhR as a potential object for CoVs therapy.

Spermine (SPM) and spermidine (SPD) are polyamines found in all organisms, and their concentrations can be regulated by ingestion. We demonstrated that yeast-fermented garlic (YF) extract significantly increased autophag flux in OUMS-36T-1 and HeLa cells expressing the fluorescent probe (GFP-LC3-RFP-LC3ΔG). YF-induced increase of autophagy occurred independently of mTORC1 signaling, and RNA-sequencing analysis revealed that EGR1 was the most significantly altered gene in YF-treated OUMS-36T-1 cells. YF-treated EGR1-deficient HAP1 cells displayed reduced autophagic flux (p < 0.05). YF-induced increasing of autophagic flux occurred via a specific SPM/SPD ratio. HAP1 cells treated with equivalent amounts of SPD or SPM as that found in YF did not increase autophagic flux (p > 0.05); however, treatment with SPD and SPM in the same ratio as that found in YF increased autophagic flux (p < 0.05). This specific SPM/SPD ratio reduced MG132-induced proteostress via EGR1-dependent pathways (p < 0.05). Thus, the SPM/SPD balance may regulate autophagy via EGR1-dependent pathways, and controlling this balance may provide a strategy to maintain cellular homeostasis.

Feline coronavirus (FCoV) is an alphacoronavirus (αCoV) that causes moderate or chronic asymptomatic infection in cats. However, in a single infected cat, FCoV can modify its cellular tropism by acquiring the ability to infect macrophages, resulting in the development of feline infectious peritonitis (FIP). In this context, to restrain the impact of FCoV infection, scientific research has focused attention on the development of antiviral therapies involving novel mechanisms of action. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) signaling regulates the host response to different human and animal CoVs. Hence, the mechanism of action of AhR was evaluated upon FCoV infection in Crandell Feline Kidney (CRFK) and in canine fibrosarcoma (A72) cells. Following infection with feline enteric CoV (FECV), strain "München", a significant activation of AhR and of its target CYP1A1, was observed. The selective AhR antagonist CH223191 provoked a reduction in FCoV replication and in the levels of viral nucleocapsid protein (NP). Furthermore, the effect of the AhR inhibitor on the acidity of lysosomes in infected cells was observed. Our findings indicate that FCoV acts on viral replication that upregulates AhR. CH223191 repressed virus yield through the inhibition of AhR. In this respect, for counteracting FCoV, AhR represents a new target useful for identifying antiviral drugs. Moreover, in the presence of CH223191, the alkalinization of lysosomes in FCoV-infected CRFK cells was detected, outlining their involvement in antiviral activity.

Oncolytic virotherapy has emerged as a promising immunotherapy strategy against cancer. As the first picornavirus tested in humans for its oncolytic potential, Senecavirus A (SVA) possesses several advantageous features, including its small size, rapid replication, and ability to penetrate the vascular system of solid tumors, allowing for the specific targeting and lysis of tumor cells. Additionally, SVA does not integrate into the host genome, thus avoiding potential genomic damage, and it lacks oncogenes or other virulence genes. Importantly, no significant pathogenic effects have been observed in humans or companion animals. Due to its simple genetic structure, SVA is amenable to various genetic modifications, allowing it to carry exogenous genes to further enhance tumor therapy. This review summarizes current knowledge of SVA's mechanisms of action and its progress in oncolytic therapy research, while also addressing the challenges and future directions.

To review current knowledge of the various processes of programmed cell death and their roles in immunoregulation in periodontitis.

Relevant literature in the PubMed, Medline, and Scopus databases was searched, and a narrative review was performed. Programmed cell death and the regulation of its various pathways implicated in periodontal infection were reviewed.

Multicellular organisms dispose of unnecessary or damaged cells via programmed cell death. Programmed cell death lies at the core of the balance of cell death and survival in pathological progress and infection. Periodontitis is a complex infectious disease involving virulence factors of periodontal pathogens and tightly regulated immune responses of the host. Different types of programmed cell death can play opposite roles in periodontitis or exert their action combinatorially.

The coordinated system of various programmed cell death pathways and the extensive crosstalk among them play a fundamental role in the pathophysiology of periodontitis. Illuminating the precise roles and mechanisms of programmed cell death in periodontitis could open up novel therapeutic approaches.

Cellular vacuolization is a commonly observed phenomenon under physiological and pathological conditions. However, the mechanisms underlying vacuole formation remain largely unresolved.

LysoTracker Deep Red probes and Enhanced Green Fluorescent Protein-tagged light chain 3B (LC3B) plasmids were employed to differentiate the types of massive vacuoles. By confocal microscopy, lysosome-like massive vacuoles (LysoTracker Deep Red+), autophagosome-like massive vacuoles (LC3B-enhanced green fluorescent protein (EGFP+)), and autolysosome-like massive vacuoles (LC3B-EGFP+ LysoTracker Deep Red+) in starved HEK293T cells were observed.

In this study, we demonstrated that nutrient deficiency can induce the formation of massive vacuoles that appear highly electron-lucent in HEK293T cells. Additionally, these massive vacuoles, resulting from nutrient depletion, can originate from various organelles, including small vacuoles, autophagosomes, lysosomes, and autolysosomes. We found that massive vacuoles could form through two primary mechanisms: the accumulation of small vacuoles into larger vacuoles or the fusion of homogeneous or heterogeneous vacuoles. Further analysis revealed that the membranes of massive vacuoles, regardless of origin, were composed of a bilayer membrane structure. As the volume of the massive vacuoles increased, the cytoplasm and nucleus were displaced toward the periphery of the cells, leading to the formation of signet ring-like cells. Interestingly, we provided evidence that complete massive vacuoles or autophagosome-like massive vacuoles can be released and exist independently outside HEK293T cells.

Nutrient deprivation induces the formation of heterogeneous, massive vacuoles in human embryonic kidney cells, some of which contribute to the development of signet ring cells, while others lead to extracellular vacuole formation.

Autophagy is responsible for maintaining cellular balance and ensuring survival. Autophagy plays a crucial role in the development of diseases, particularly human cancers, with actions that can either promote survival or induce cell death. However, brain tumors contribute to high levels of both mortality and morbidity globally, with resistance to treatments being acquired due to genetic mutations and dysregulation of molecular mechanisms, among other factors. Hence, having knowledge of the role of molecular processes in the advancement of brain tumors is enlightening, and the current review specifically examines the role of autophagy. The discussion would focus on the molecular pathways that control autophagy in brain tumors, and its dual role as a tumor suppressor and a supporter of tumor survival. Autophagy can control the advancement of different types of brain tumors like glioblastoma, glioma, and ependymoma, demonstrating its potential for treatment. Autophagy mechanisms can influence metastasis and drug resistance in glioblastoma, and there is a complex interplay between autophagy and cellular responses to stress like hypoxia and starvation. Autophagy can inhibit the growth of brain tumors by promoting apoptosis. Hence, focusing on autophagy could offer fresh perspectives on creating successful treatments.

Oral cancer is a highly malignant disease characterized by recurrence, metastasis, and poor prognosis. Autophagy, a catabolic process induced under stress conditions, has been shown to play a dual role in oral cancer development and therapy. Recent studies have identified that autophagy activation in oral epithelial cells suppresses cancer cell survival by inhibiting key pathways such as the mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK), while activating the adenosine monophosphate-activated protein kinase (AMPK) pathway. Inducing autophagy promotes degradation of eukaryotic initiation factor 4E, thus reducing metastasis and enhancing the efficacy of chemotherapy, radiotherapy, and immunotherapy. Furthermore, autophagy induction can modulate the tumor immune microenvironment and enhance antitumor immunity. This review comprehensively summarizes the relationship between autophagy and oral cancer, focusing on its mechanisms and therapeutic potential when combined with conventional treatments. While promising, the precise mechanisms and clinical applications of autophagy inducers in oral cancer therapy remain to be elucidated, offering new directions for future research to improve treatment outcomes and reduce recurrence.

Cell death is an important process that supports morphogenesis during development and tissue homeostasis during adult life by removing damaged or unwanted cells and its dysregulation is associated with numerous disease states. There are different pathways through which a cell can undergo cell death, each relying on peculiar molecular mechanisms and morpho-ultrastructural features. To date, however, while molecular and genetic approaches have been successfully integrated into the field, cell death studies rarely incorporate ultrastructural data from electron microscopy. This review article reports a gallery of original transmission electron microscopy images to describe the ultrastructural features of cells undergoing different types of cell death programs, including necrosis, apoptosis, autophagy, mitotic catastrophe, ferroptosis, methuosis, and paraptosis. TEM has been an important technology in cell biology for well over 50 years and still continues to offer significant advantages in the area of cell death research. TEM allows detailed characterization of the ultrastructural changes within the cell, such as the alteration of organelles and subcellular structures, the nuclear reorganization, and the loss of membrane integrity that enable a distinction between the different forms of cell death based on morphological criteria. Possible pitfalls are also described.

Macroautophagy/autophagy is a highly conserved evolutionary mechanism involving lysosomes for the degradation of cytoplasmic components including organelles. The constitutive, basal level of autophagy is fundamental for preserving cellular homeostasis; however, alterations in autophagy can cause disease pathogenesis, including cancer. The role of autophagy in cancer is particularly complicated, since this process acts both as a tumor suppressor in precancerous stages but facilitates tumor progression during carcinogenesis and later stages of cancer progression. This shift between anti-tumor and pro-tumor roles may be influenced by genetic and environmental factors modulating key pathways such as those involving autophagy-related proteins, the PI3K-AKT-MTOR axis, and AMPK, which often show dysregulation in tumors. Autophagy regulates various cellular functions, including metabolism of glucose, glutamine, and lipids, cell proliferation, metastasis, and several types of cell death (apoptosis, ferroptosis, necroptosis and immunogenic cell death). These multifaceted roles demonstrate the potential of autophagy to affect DNA damage repair, cell death pathways, proliferation and survival, which are critical in determining cancer cells' response to chemotherapy. Therefore, targeting autophagy pathways presents a promising strategy to combat chemoresistance, as one of the major reasons for the failure in cancer patient treatment. Furthermore, autophagy modulates immune evasion and the function of immune cells such as T cells and dendritic cells, influencing the tumor microenvironment and cancer's biological behavior. However, the therapeutic targeting of autophagy is complex due to its dual role in promoting survival and inducing cell death in cancer cells, highlighting the need for strategies that consider both the beneficial and detrimental effects of autophagy modulation in cancer therapy. Hence, both inducers and inhibitors of autophagy have been introduced for the treatment of cancer. This review emphasizes the intricate interplay between autophagy, tumor biology, and immune responses, offering insights into potential therapeutic approaches that deploy autophagy in the cancer suppression.

Patients with hypoxemia require high-concentration oxygen therapy. However, prolonged exposure to oxygen concentrations 21% higher than physiological concentrations (hyperoxia) may cause oxidative cellular damage. Pulmonary alveolar epithelial cells are major targets for hyperoxia-induced oxidative stress. In this study, we evaluated the therapeutic potential of the antioxidant N-acetyl-L-cysteine (NAC) for preventing hyperoxia-induced cell death. In vitro experiments were performed using the human lung cancer cell line A549. In brief, NAC-treated and untreated cells were exposed to various concentrations of oxygen (hyperoxia) for different durations. The results indicated that hyperoxia inhibited proliferation and caused cell cycle arrest in A549 cells. It also induced necrosis and autophagy. Furthermore, hyperoxia increased intracellular reactive oxygen species levels and altered mitochondrial membrane potential. Co-treatment with NAC improved the survival of cells exposed to 95% oxygen for 24 h. Experiments performed using a neonatal rat model of acute lung injury confirmed that hyperoxia induced an autophagic response. This study provides evidence for hyperoxia-induced autophagy both in vitro and in vivo. NAC can protect A549 cells from death induced by short-term hyperoxia. Our findings may inform protective strategies against hyperoxia-induced injury in developing lungs-for example, bronchopulmonary dysplasia in premature infants.

Strong evidence suggests links between Parkinson's Disease (PD) and melanoma, as studies have found that people with PD are at an increased risk of developing melanoma and those with melanoma are at increased risk of developing PD. Although these clinical associations are well-established, the cellular and molecular pathways linking these diseases are poorly understood. Recent studies have found a previously unrecognized role for the neurodegeneration-associated protein alpha-synuclein (αSyn) in melanoma; the overexpression of αSyn promotes melanoma cell proliferation and metastasis. However, to our knowledge, no studies have investigated the role of αSyn in in vivo melanoma models outside of a xenograft paradigm. Our study created and characterized Snca knockout in the spontaneously developing melanoma TG3 mouse line, TG3+/+Snca-/-. We show that αSyn loss-of-function significantly delays melanoma onset and slows tumor growth in vivo. Furthermore, decreased tumor volume is correlated with a decreased DNA damage signature and increased apoptotic markers, indicating a role for αSyn in modulating the DNA damage response (DDR) pathway. Overall, our study provides evidence that targeting αSyn and its role in modulating the DDR and melanomagenesis could serve as a promising new therapeutic target.

Polycyclic aromatic hydrocarbons (PAHs), including the Group 1 human carcinogen benzo[a]pyrene (BaP), are produced by the incomplete combustion of organic matter and thus are present in tobacco smoke, charbroiled food and diesel exhaust. The nematode Caenorhabditis elegans is an established model organism, however it lacks the genetic components of the classical mammalian cytochrome P450 (CYP)-mediated BaP-diol-epoxide metabolism pathway. We therefore introduced human CYP1A1 or CYP1A2 together with human epoxide hydrolase (EPHX) into the worm genome by Mos1-mediated Single Copy Insertion (MosSCI) and evaluated their response to BaP exposure via toxicological endpoints. Compared to wild-type control, CYP-humanised worms were characterised by an increase in pharyngeal pumping rate and a decrease in volumetric surface area. Furthermore, BaP exposure reduced reproductive performance, as reflected in smaller brood size, which coincided with the downregulation of the nematode-specific major sperm protein as determined by transcriptomics (RNAseq). BaP-mediated reproductive toxicity was exacerbated in CYP-humanised worms at higher exposure levels. Collagen-related genes were downregulated in BaP-exposed animals, which correlate with the reduction in volumetric size. Whole genome DNA sequencing revealed a higher frequency of T > G (A > C) base substitution mutations in worms expressing human CYP1A1;EPHX which aligned with an increase in DNA adducts identified via an ELISA method (but not classical 32P-postlabelling). Overall, the CYP-humanised worms provided new insights into the value of genome-optimised invertebrate models by identifying the benefits and limitations within the context of the (3Rs) concept which aims to replace, reduce and refine the use of animals in research.

Specifically controlling cell pyroptosis is advantageous for oncotherapy as it allows simultaneous ablation of primary tumors and activation of immunogenicity of tumor environment. Herein, a facile and robust strategy is presented to construct efficient NIR-activated helical pyroptosis agents (PyroAs) with negligible dark cytotoxicity. It is demonstrated that the construction of four intramolecular B-X bonds (X = O or N) within the BODIPY chromophore enforces a significant twisting of its π-conjugation, yielding a variety of helical HBD molecules with desired high photosensitivity and negligible dark toxicity. A robust approach is established to extend HBD into the near-infrared (NIR) region through site-selective incorporation of an electron-withdrawing ester moiety. It is also proved that targeted delivery of the NIR-activated HBD-ER to the endoplasmic reticulum (ER) specifically activates pyroptosis pathway by equipping it with an ER-targeting moiety. Finally, the favorable biocompatibility, excellent antitumor efficacy, and remarkable systematic immune response of this unique NIR-activated helical PyroAs are shown in vivo, demonstrating its potential application in solid tumor immunotherapy.

Hepatocellular carcinoma (HCC) ranks as the fourth most common cause of mortality globally among all cancer types. Programmed cell death (PCD) is a crucial biological mechanism governing cancer progression, tumor expansion, and metastatic dissemination. Furthermore, the tumor microenvironment (TME) is critical in influencing overall survival (OS) and immune responses to immunotherapeutic interventions. From a multi-omics perspective, the combination of PCD and TME could help to predict the survival of HCC patient survival and immunotherapy response. Our study analyzed variations in the PCD- and TME-classifier used in the classification of HCC patients into two subgroups: PCD high-TME low and PCD low-TME high. In the following step, we compared the tumor somatic mutation (TMB), immunotherapy response, and functional annotation of both groups of patients. Lastly, Western Blot (WB) were conducted. The immunohistochemistry (IHC) was performed on the Human Protein Atlas (HPA). In the PCD-TME classifier, 23 PCD-related genes and three immune cell types were identified. Patients' prognoses and responses to therapy could be accurately predicted using this model. The findings of this study provide a new instrument for the clinical management of HCC patients, and they contribute to the development of accurate treatment strategies for these patients.

A paradigm shift in the study of cell death is currently occurring: whereas previously we had always considered that there were "points of no return" in any cell death pathway, we now realize that in many types of active, regulated cell death, this is not the case. We are also learning that cells that "almost die," but nevertheless survive, can transiently take on an altered state, with potential implications for understanding cancer therapies and relapse. In this perspective, we parse the many forms of cell death by analogy to suicide, sabotage, and murder, and consider how cells that might be "instructed" to engage a cell death pathway might nevertheless survive.

Arterial hypertension (HTN) is one of the major global contributors to cardiovascular diseases and premature mortality, particularly due to its impact on vital organs and the coexistence of various comorbidities such as chronic renal disease, diabetes, cerebrovascular diseases, and obesity. Regardless of the accessibility of several well-established pharmacological treatments, the percentage of patients achieving adequate blood pressure (BP) control is still significantly lower than recommended levels. Therefore, the pharmacological and non-pharmacological management of HTN is currently the major focus of healthcare systems. Various strategies are being applied, such as the development of new pharmacological agents that target different underlying physiopathological mechanisms or associated comorbidities. Additionally, a novel group of interventional techniques has emerged in recent years, specifically for situations when blood pressure is not properly controlled despite the use of multiple antihypertensives in maximum doses or when patients are unable to tolerate or desire not to receive antihypertensive medications. Nonetheless, reducing the focus on antihypertensive medication development by the pharmaceutical industry and increasing recognition of ineffective HTN control due to poor drug adherence demands ongoing research into alternative approaches to treatment. The aim of this review is to summarize the potential novel pharmacological targets for the treatment of arterial hypertension as well as the future perspectives of the treatment strategy.

Myocardial ischemia-reperfusion injury (MI/RI) is an unavoidable risk event for acute myocardial infarction, with ferroptosis showing close involvement. We investigated the mechanism of MI/RI inducing myocardial injury by inhibiting the ferroptosis-related SLC7A11/glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathway and activating mitophagy.

A rat MI/RI model was established, with myocardial infarction area and injury assessed by TTC and H&E staining. Rat cardiomyocytes H9C2 were cultured in vitro, followed by hypoxia/reoxygenation (H/R) modeling and the ferroptosis inhibitor lipoxstatin-1 (Lip-1) treatment, or 3-Methyladenine or rapamycin treatment and overexpression plasmid (oe-SLC7A11) transfection during modeling. Cell viability and death were evaluated by CCK-8 and LDH assays. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial membrane potential was detected by fluorescence dye JC-1. Levels of inflammatory factors, reactive oxygen species (ROS), Fe2+, malondialdehyde, lipid peroxidation, GPX4 enzyme activity, glutathione reductase, GSH and glutathione disulfide, and SLC7A11, GPX4, LC3II/I and p62 proteins were determined by ELISA kit, related indicator detection kits and Western blot.

The ferroptosis-related SLC7A11/GSH/GPX4 pathway was repressed in MI/RI rat myocardial tissues, inducing myocardial injury. H/R affected GSH synthesis and inhibited GPX4 enzyme activity by down-regulating SLC7A11, thus promoting ferroptosis in cardiomyocytes, which was averted by Lip-1. SLC7A11 overexpression improved H/R-induced cardiomyocyte ferroptosis via the GSH/GPX4 pathway. H/R activated mitophagy in cardiomyocytes. Mitophagy inhibition reversed H/R-induced cellular ferroptosis. Mitophagy activation partially averted SLC7A11 overexpression-improved H/R-induced cardiomyocyte ferroptosis. H/R suppressed the ferroptosis-related SLC7A11/GSH/GPX4 pathway by inducing mitophagy, leading to cardiomyocyte injury.

Increased ROS under H/R conditions triggered cardiomyocyte injury by inducing mitophagy to suppress the ferroptosis-related SLC7A11/GSH/GPX4 signaling pathway activation.

Cardiac hypertrophy is characterized by an increased volume of individual cardiomyocytes rather than an increase in their number. Myocardial hypertrophy due to pathological stimuli encountered by the heart, which reduces pressure on the ventricular walls to maintain cardiac function, is known as pathological hypertrophy. This eventually progresses to heart failure. Certain varieties of regulated cell death (RCD) pathways, including apoptosis, pyroptosis, ferroptosis, necroptosis, and autophagy, are crucial in the development of pathological cardiac hypertrophy. This review summarizes the molecular mechanisms and signaling pathways underlying these RCD pathways, focusing on their mechanism of action findings for pathological cardiac hypertrophy. It intends to provide new ideas for developing therapeutic approaches targeted at the cellular level to prevent or reverse pathological cardiac hypertrophy.

As obligate parasites, viruses need to hijack resources from infected cells to complete their lifecycle. The interaction between the virus and host determines the viral infection process, including viral propagation and the disease's outcome. Understanding the interaction between the virus and host factors is a basis for unraveling the intricate biological processes in the infected cells and thereby developing more efficient and targeted antivirals. Among the various fundamental virus-host interactions, autophagy plays vital and also complicated roles by directly engaging in the viral lifecycle and functioning as an anti- and/or pro-viral factor. Autophagy thus becomes a promising target against virus infection. Since the COVID-19 pandemic, there has been an accumulation of studies aiming to investigate the roles of autophagy in SARS-CoV-2 infection by using different models and from distinct angles, providing valuable information for systematically and comprehensively dissecting the interplay between autophagy and SARS-CoV-2. In this review, we summarize the advancements in the studies of the interaction between SARS-CoV-2 and autophagy, as well as detailed molecular mechanisms. We also update the current knowledge on the pharmacological strategies used to suppress SARS-CoV-2 replication through remodeling autophagy. These extensive studies on SARS-CoV-2 and autophagy can advance our understanding of virus-autophagy interaction and provide insights into developing efficient antiviral therapeutics by regulating autophagy.

Intracranial aneurysm (IA), is a localized dilation of the intracranial arteries, the rupture of which is catastrophic. Hypertension is major IA risk factor that mediates endothelial cell damage. Sox17 is highly expressed in intracranial vascular endothelial cells, and GWAS studies indicate that its genetic alteration is one of the major genetic risk factors for IA. Vascular endothelial cell injury plays a vital role in the pathogenesis of IA. The genetic ablation of Sox17 plus hypertension induced by AngII can lead to an increased incidence of intracranial aneurysms had tested in the previous animal experiments. In order to study the underlying molecular mechanisms, we established stable Sox17-overexpressing and knockdown cell lines in human brain microvascular endothelial cells (HBMECs) first. Then flow cytometry, western blotting, and immunofluorescence were employed. We found that the knockdown of Sox17 could worsen the apoptosis and autophagy of HBMECs caused by AngII, while overexpression of Sox17 had the opposite effect. Transmission electron microscopy displayed increased autophagosomes after the knockdown of Sox17 in HBMECs. The RNA-sequencing analysis shown that dysregulation of the Sox17 gene was closely associated with the autophagy-related pathways. Our study suggests that Sox17 could protect HBMECs from AngII-induced injury by regulating autophagy and apoptosis.

Korean mistletoe (Viscum album L. var. coloratum) is renowned for its medicinal properties, including anti-cancer and immunoadjuvant effects. This study aimed to elucidate the mechanisms by which Korean mistletoe lectin (V. album L. var. coloratum agglutinin; VCA) modulates breast cancer cell apoptosis and macrophage polarization. The specific objectives were to (1) investigate the direct effects of VCA on MCF-7 breast cancer cells and THP-1-derived M1/M2 macrophages; (2) analyze the impact of VCA on the paracrine interactions between these cell types; and (3) compare the efficacy of VCA in 2D vs. 3D co-culture models to bridge the gap between in vitro and in vivo studies. We employed both 2D and 3D models, co-culturing human M1/M2 macrophages with human MCF-7 breast cancer cells in a Transwell system. Our research demonstrated that M1 and M2 macrophages significantly influenced the immune and apoptotic responses of breast cancer cells when exposed to VCA. M1 macrophages exhibited cytotoxic characteristics and enhanced VCA-induced apoptosis in both 2D and 3D co-culture models. Conversely, M2 macrophages initially displayed a protective effect by reducing apoptosis in breast cancer cells, but this protective effect was reversed upon exposure to VCA. Furthermore, our findings illustrate VCA's ability to modulate M1 and M2 polarization in breast cancer cells. Finally, the use of magnetic 3D cell cultures suggests their potential to yield results comparable to conventional 2D cultures, bridging the gap between in vitro and in vivo studies.

Macroautophagy/autophagy has previously been regarded as simply a way for cells to deal with nutrient emergency. But explosive work in the last 15 years has given increasingly new knowledge to our understanding of this process. Many of the functions of autophagy that are unveiled from recent studies, however, cannot be reconciled with this conventional view of cell survival but, instead, point to autophagy being integrally involved at a deeper level of cell biology, playing a critical role in maintaining homeostasis and promoting an integrated stress/immune response. The new appreciation of the role of autophagy in the evolutionary trajectory of cancer and cancer interaction with the immune system provides a mechanistic framework for understanding the clinical benefits of autophagy-based therapies. Here, we examine current knowledge of the mechanisms and functions of autophagy in highly plastic and aggressive melanoma as a model disease of human malignancy, while highlighting emerging dimensions indicating that autophagy is at play beyond its classical face.Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; ATG: autophagy related; BRAF: B-Raf proto-oncogene, serine/threonine kinase; CAFs: cancer-associated fibroblasts; CCL5: C-C motif chemokine ligand 5; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTLA4: cytotoxic T-lymphocyte associated protein 4; CTL: cytotoxic T lymphocyte; DAMPs: danger/damage-associated molecular patterns; EGFR: epidermal growth factor receptor; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FITM2: fat storage inducing transmembrane protein 2; HCQ: hydroxychloroquine; ICB: immune checkpoint blockade; ICD: immunogenic cell death; LDH: lactate dehydrogenase; MAPK: mitogen-activated protein kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NDP52: nuclear dot protein 52; NFKB/NF-κ B: nuclear factor kappa B; NBR1: the neighbor of BRCA1; NK: natural killer; NRF1: nuclear respiratory factor 1; NSCLC: non-small-cell lung cancer; OPTN: optineurin; PDAC: pancreatic ductal adenocarcinoma; PDCD1/PD-1: programmed cell death 1; PPT1: palmitoyl-protein thioesterase 1; PTEN: phosphatase and tensin homolog; PTK2/FAK1: protein tyrosine kinase 2; RAS: rat sarcoma; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGFB/TGF-β: transforming growth factor beta; TMB: tumor mutational burden; TME: tumor microenvironment; TSC1: TSC complex subunit 1; TSC2: TSC complex subunit 2; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated.

Spinal cord injury (SCI) is a severe condition that often leads to persistent damage of nerve cells and motor dysfunction. Autophagy is an intracellular system that regulates the recycling and degradation of proteins and lipids, primarily through lysosomal-dependent organelle degradation. Numerous publications have highlighted the involvement of autophagy in the secondary injury of SCI. Therefore, gaining a comprehensive understanding of autophagy research is crucial for designing effective therapies for SCI.

Dates were obtained from Web of Science, including articles and article reviews published from its inception to October 2023. VOSviewer, Citespace, and SCImago were used to visualized analysis. Bibliometric analysis was conducted using the Web of Science data, focusing on various categories such as publications, authors, journals, countries, organizations, and keywords. This analysis was aimed to summarize the knowledge map of autophagy and SCI.

From 2009 to 2023, the number of annual publications in this field exhibited wave-like growth, with the highest number of publications recorded in 2020 (44 publications). Our analysis identified Mei Xifan as the most prolific author, while Kanno H emerged as the most influential author based on co-citations. Neuroscience Letters was found to have published the largest number of papers in this field. China was the most productive country, contributing 232 publications, and Wenzhou Medical University was the most active organization, publishing 39 papers.

We demonstrated a comprehensive overview of the relationship between autophagy and SCI utilizing bibliometric tools. This article could help to enhance the understanding of the field about autophagy and SCI, foster collaboration among researchers and organizations, and identify potential therapeutic targets for treatment.

By replacing and removing defective or infected cells, programmed cell death (PCD) contributes to homeostasis maintenance and body development, which is ubiquitously present in mammals and can occur at any time. Besides apoptosis, more novel modalities of PCD have been described recently, such as necroptosis, pyroptosis, ferroptosis, and autophagy-dependent cell death. PCD not only regulates multiple physiological processes, but also participates in the pathogenesis of diverse disorders, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is mainly classified into metabolic dysfunction-associated steatotic liver (MASL) and metabolic dysfunction-associated steatohepatitis (MASH), and the latter putatively progresses to cirrhosis and hepatocellular carcinoma. Owing to increased incidence and obscure etiology of MASH, its management still remains a tremendous challenge. Recently, hepatocyte PCD has been attracted much attention as a potent driver of the pathological progression from MASL to MASH, and some pharmacological agents have been proved to exert their salutary effects on MASH partly via the regulation of the activity of hepatocyte PCD. The current review recapitulates the pathogenesis of different modalities of PCD, clarifies the mechanisms underlying how metabolic disorders in MASLD induce hepatocyte PCD and how hepatocyte PCD contributes to inflammatory and fibrotic progression of MASH, discusses several signaling pathways in hepatocytes governing the execution of PCD, and summarizes some potential pharmacological agents for MASH treatment which exert their therapeutic effects partly via the regulation of hepatocyte PCD. These findings indicate that hepatocyte PCD putatively represents a new therapeutic point of intervention for MASH.

The involvement of cell death in acute kidney injury (AKI) is linked to multiple factors including energy depletion, electrolyte imbalance, reactive oxygen species, inflammation, mitochondrial dysfunction, and activation of several cell death pathway components. Since our review in 2003, discussing the relative contributions of apoptosis and necrosis, several other forms of cell death have been identified and are shown to contribute to AKI. Currently, these various forms of cell death can be fundamentally divided into accidental cell death and regulated or programmed cell death based on functional aspects. Several death initiator and effector molecules switch molecules that may act as signaling components triggering either death or protective mechanisms or alternate cell death pathways have been identified as part of the machinery. Intriguingly, several of these cell death pathways share components and signaling pathways suggesting complementary or compensatory functions. Thus, defining the cross talk between distinct cell death pathways and identifying the unique molecular effectors for each type of cell death may be required to develop novel strategies to prevent cell death. Furthermore, depending on the multiple forms of cell death simultaneously induced in different AKI settings, strategies for combination therapies that block multiple cell death pathways need to be developed to completely prevent injury, cell death, and renal function. This review highlights the various cell death pathways, cross talk, and interactions between different cell death modalities in AKI.

There is a propensity for synthetic cannabinoid abuse to spread worldwide. CP-55,940, a synthetic cannabinoid having the ability to activate both CB1 and CB2 receptors, has been shown to induce cell death in neurons as well as other cells. Here we investigate molecular events underling the adverse effects of CP-55,940 on neuronal cells. Exposure of mouse neuroblastoma Neuro2a cells to 10-50 µM CP-55,940 results in concentration-dependent cell death that is not accompanied by an induction of apoptosis. CP-55,940 also stimulates autophagy, but the stimulation is not followed by an increase in autophagic degradation. Transcriptome analysis using DNA microarray revealed the increased expression of genes for the cholesterol biosynthesis pathway that is associated with the activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis. However, free cholesterol is localized mainly to cytoplasmic structures, although it is localized to the plasma membrane in healthy cells. Thus, cellular trafficking of cholesterol seems to be somewhat disrupted in CP-55,940 stimulated cells. These results show for the first time that CP-55,940 stimulates autophagy as well as cholesterol biosynthesis, although not all the processes involved in the cellular response to CP-55,940 seem to be complete in these cells.

Seed aging, a natural and inevitable process occurring during storage. Oats, an annual herb belonging to the Gramineae family and pooideae. In addition to being a healthy food, oats serve as ecological pastures, combating soil salinization and desertification. They also play a role in promoting grassland agriculture and supplementing winter livestock feed. However, the high lipid and fat derivatives contents of oat seeds make them susceptible to deterioration, as fat derivatives are prone to rancidity, affecting oat seed production, storage, development, and germplasm resource utilization. Comparative studies on the effects of aging on physiology and cytological structure in covered and naked oat seeds are limited. Thus, our study aimed to determine the mechanism underlying seed deterioration in artificially aged 'LongYan No. 3' (A. sativa) and 'BaiYan No. 2' (A. nuda) seeds, providing a basis for the physiological evaluation of oat seed aging and serving as a reference for scientifically safe storage and efficient utilization of oats.

In both oat varieties, superoxide dismutase and catalase activities in seeds showed increasing and decreasing trends, respectively. Variance analysis revealed significant differences and interaction in all measured indicators of oat seeds between the two varieties at different aging times. 'LongYan No. 3' seeds, aged for 24-96 h, exhibited a germination rate of < 30%, Conductivity, malondialdehyde, soluble sugar, and soluble protein levels increased more significantly than the 'BaiYan No. 2'. With prolonged aging leading to cell membrane degradation, reactive oxygen species accumulation, disrupted antioxidant enzyme system, evident embryo cell swelling, and disordered cell arrangement, blocking the nutrient supply route. Simultaneously, severely concentrated chromatin in the nucleus, damaged mitochondrial structure, and impaired energy metabolism were noted, resulting in the loss of 'LongYan No. 3' seed vitality and value. Conversely, 'BaiYan No. 2' seeds showed a germination rate of 73.33% after 96 h of aging, consistently higher antioxidant enzyme activity during aging, normal embryonic cell shape, and existence of the endoplasmic reticulum.

ROS accumulation and antioxidant enzyme system damage in aged oat seeds, nuclear chromatin condensation, mitochondrial structure damage, nucleic acid metabolism and respiration weakened, oat seed vigor decreased. 'LongYan No. 3' seeds were more severely damaged under artificial aging than 'BaiYan No. 2' seeds, highlighting their heightened susceptibility to aging effects.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.