Nasopharyngeal carcinoma (NPC) is an uncommon type of malignancy/cancer worldwide. However, NPC is an endemic disease in southeast Asia and southern China and the reasons behind the underlying for such changes are unclear. Even though the Epstein–Barr infection (EBV) has been suggested as an important reason for undistinguishable NPC, the EBV itself is not adequate to source this type of cancer. The risk factors, for example, genetic susceptibility, and environmental factors might be associated with EBV to undertake a part in the NPC carcinogenesis. Normal healthy people have a memory B cell pool where the EBV persists, and any disturbance of this connection leads to virus-associated B cell malignancies. Less is known about the relationship between EBV and epithelial cell tumors, especially the EBV-associated nasopharyngeal carcinoma (EBVaNPC) and EBV-associated gastric carcinoma (EBVaGC). Currently, it is believed that premalignant genetic changes in epithelial cells contribute to the aberrant establishment of viral latency in these tumors. The early and late phases of NPC patients’ survival rates vary significantly. The presence of EBV in all tumor cells presents prospects for the development of innovative therapeutic and diagnostic techniques, despite the fact that the virus’s exact involvement in the carcinogenic process is presently not very well known. EBV research continues to shed light on the carcinogenic process, which is important for a more comprehensive knowledge of tumor etiology and the development of targeted cancer therapeutics. In order to screen for NPC, EBV-related biomarkers have been widely used in a few high-incidence locations because of their close associations with the risks of NPC. The current review highlights the scientific importance of EBV and its possible association with NPC. The Epstein–Barr virus (EBV), which is sometimes also referred to as human herpesvirus 4, is a member of the herpes virus family . It is oChildren are most often infected with EBV . Most ofBoth the eradication of viral infections and the inhibition of tumor development depend critically on the activation of inflammasomes and the subsequent inflammatory response . ChronicThe genome of EBV is 172 kb long which encodes almost 100 open reading frames (ORFs) . These OA very rare type of carcinoma, which belongs to head and neck cancers, is nasopharyngeal carcinoma (NPC) . It was Based on how the tumor cells look under a light microscope, the World Health Organization (WHO) has classified NPC into two basic histological types. One is keratinizing squamous cell carcinoma (type I) and the other is non-keratinizing squamous cell carcinoma (type II and type III). Differentiated non-keratinizing carcinoma (type II) and undifferentiated carcinoma (type III), which are mostly EBV-positive tumors, are additional subtypes of the non-keratinizing type . Less thThe American Joint Committee on Cancer (AJCC) re-evaluated the staging procedure in 2018 due to the variations in imaging technology and better results linked to optimal medication . Recent The early detection of NPC is challenging, possibly because it is difficult to inspect the nasopharynx and because the symptoms of NPCs might be mistaken for those of other, more prevalent illnesses ,19. A moIn view of the obvious explicitness of the connection between the degrees of IgA of VCA and NPC, a mass serological location program in the city of Wuzhou, China was conducted in 1980, where it was shown that EBV-explicit antibodies are valuable for NPC’s initial distinguishing proof . The expUsing electronic microscopy, the particles of herpes virus in a subset of tumor cells derived from Burkitt lymphoma (BL) were detected ,38. AfteThe discovery of EBV in UCNT has motivated many organizations to investigate undifferentiated NPCs . StomachEBV is a two-fold strand DNA infection with a genome from 170 to 180 kB which contains almost 100 qualities for a dormant or lytic disease of host cells . The virEBV is the main human oncogenic infection to be recognized. It is connected to classic Hodgkin’s lymphoma (HL), Burkitt lymphoma (BL), B cell lymphoma (BcL), NK/T cell nasal lymphoma, two sorts of epithelial carcinoma, gastric disease , and cellular breakdown in the lung’s cells  . For GC ➢Neck lymph node enlargement.➢Bleeding from the mouth or nose.➢Ear-related issues and hearing loss caused by the tumor’s closeness to the Eustachian tube, which causes obstruction and fluid collection in the middle ear.➢Sore throat.➢Difficulty breathing through the nose.➢Headache.NPC may not cause any symptoms in its early stages . PossiblOncogenic viral infections are thought to play a key role in the development of 20% of all types of human cancers, or about 2 million cases every year . InfectiAn IgA antibody against Epstein-1 (EBNA1/IgA) and VCA/IgA assessed by enzyme-linked immunosorbent assay (ELISA) has higher specificity, sensitivity, and positive predictive value than other techniques, according to a recent study . Accordi➢The most common method of EBV transmission is via bodily fluids, notably saliva. EBV may, however, be spread by blood and sperm during sexual activity, blood transfusions, and organ transplants.➢EBV may be transmitted by touching an infected individual’s recently used drinking glass or toothbrush. The virus is likely to live on a surface for as long as it stays moist.Regarding transmission:When infected with EBV for the first time (primary EBV infection), the virus may spread for many weeks prior to the onset of symptoms . The virBoth of the EBV latent gene products (the EBERs and LMP1) and homogeneous lengths of TRs were previously discovered in NPC and precancerous lesions . It was While the EBV genome is lost during the long-term passage of different NPC in vitro cell lines, the dormant EBV disease is ceaselessly distinguished in every growth cell in xenograft models from patients and the NPC clinical tissues in both the fundamental and principal repeating textures in essential and repeating cases ,70,71. TEBNA1 is only one of the numerous non-polyadenylated RNAs found in certain EBV malignant growth cells, which incorporate EBER1 and EBER2. In latent infected epithelial cells, the EBERs self-replicate and bind to the ribosomal protein L22 to deliver ribonucleoprotein particles ,77. The The interaction of EBV with B cells is essential to understand the biology of the virus. EBV can easily infect and modify B cells to a normal state in vitro tests, making them a useful model system for understanding the biology and behavior of the virus ,81. The EBV latent gene expression investigations in malignant tumors associated with the virus and cell lines created from BL biopsies have uncovered two extra EBV inertness qualities ,86. The The preferred method to identify EBV infection in cells and tissues is in situ hybridization to the many EBV-encoded short RNA (EBER) transcripts  . NPC’s iThe many types of EBV latent infections reflect the cellular milieu as well as the intricate interactions and cooperation between host regulatory elements and viral promoters that result in EBV latent features . DiffereThe LMP2A promotor is additionally controlled by EBNA2. The transcripts require the circularization of the genome on the grounds that LMP2A and LMP2B cross the TRs in the U1 region . The cirIn the relatively limited EBV latency found in NPC and HL, the promoter QP Sans tata promotes transcription of EBNA1. The TRs that govern the expression of LMP1 include another promoter called L1-TT-TR . The numVirally coded proteins LMP1, LMP2, and EBNA1 could be taken advantage of as restorative focuses in NPC. The capacity of EBNA1 has been studied because of its ceaseless articulation in every growth cell and the basic function in keeping up with the EBV episomal genome . In lighIt is currently realized that EBNA1 connects with explicit host cell parts to cause viral inactivity and prompt host cell oncogenic change. With an ongoing leap forward in how we might interpret EBNA1′s primary science, new proof proposes that both EBNA1 dimers and oligomers assume a part in viral latency instructions . In prevAnalysts should initially comprehend the capacity of EBV latent qualities communicated in NPC to assess the role of viral disease in the malignant growth process . LikewisThe EBNA1 protein is essential for the separation and replication of the EBV epi-some, but it has also been shown to protect apoptotic cells, promote cell endurance, and directly contribute to the tumorigenic aggregate . The desIn rodent fibroblast transformation tests, LMP1 is a regular oncogene, and it is important for EBV-prompted B cell change in vitro. When LMP1 is generated in cells, it up-regulates antiapoptotic proteins Bcl-2, A20) and increases cytokine production (interleukin (IL)-6, IL-8), among other things  and incr.Two non-coding or non-polyadenylated RNAs, known as EBERs 1 and 2, are highly expressed in all kinds of EBV latency and act as sensitive indicators of EBV infection in cells and tissues . The proWhen the genomes of EBV isolated from diverse parts of the globe or individuals with various virus-associated diseases are examined, restriction fragment length polymorphism analysis reveals striking similarities . DiffereA subset of latent genes, particularly those encoding EBNA3A, EBNA3B, EBNA3C, and EBNA-LP, display allelic polymorphisms that are connected to the EBV type (with a succession homology of 50–80% depending upon the area) . EBV-2 pThe coinfection of the host with many virus strains can help the evolution of EBV by allowing genetic recombination and variation . In HIV-EBV infects B lymphocytes at the start of primary infection, but it is not clear where these cells are infected or if the infection also affects the higher respiratory epithelial cells. EBV colonizes the pool of memory B cells to develop long-term persistence in vivo. A latent infection is created, which is identified by the expression of a small subset of viral genes known as latent genes . DiffereWhen looking at tumor types connected to the virus, the dual tropism of EBV infection is clear. The in vivo memory B cell pool may be infected by EBV. In vitro, inactive B cells can be quickly transformed into infected lymphoblastoid cell lines (LCLs), which resemble several malignant B cell tumors closely associated with EBV . HoweverThe adoption of cytotoxic T cells specific to EBV as a complement to standard treatment to prevent and effectively treat NPC cells positive for EBV expressing subdominant EBV antigens  has been proposed  and, theThe principal cytotoxic T cell was well defined for EBV and was used to develop the cytotoxic cells for patients with HLs. As per the outcomes, the mixture of these cells in patients prompted clinical antiviral action in vivo and brought down EBV DNA levels in their blood . RecentlA stage I clinical preliminary trial uncovered that the treatment of recurrent NPC patients with ATB-explicit TB cells prompted the reduction of tumoral clinical reactions in 6 out of 10 patients. Meanwhile, scientists have observed that the receptive exchange of CTL well defined for autolysis was protected and connected to huge anticancer action in patients with cutting-edge NPC in a review containing ten patients . The utiThe utilization of receptive cell treatment has been carried out previously which designated the LMP1/2 and EBNA1 antigens that are communicated in NPC. The process made explicit LMP/EBNA1 T cells utilizing the ADE1-LMP poly adenovirus vector, which worked in the ideal improvement of viral T cells from low-recurrence antecedents . Most paThe etiology of NPCs has been a mystery for more than a century since it was initially reported in 1901. Studies of southern Chinese migrants demonstrate that the incidence of NPCs is 10 to 30 times higher than that of other races when they settle in other countries, an unusual trend among malignant tumors that suggests a heredity component of the risk of NPC . ContrasA family history of NPCs and some natural variables appear to assume a vital role in NPCs, as shown by the most recent examinations. The risk factors include Cantonian ethnicity, male sex, EBV disease, routinely eating fish preserved in salt, low utilization of new vegetables and natural products, smoking, and certain antigen classes of human leukocytes (HLA) . Other HThe prevalence of EBV infection demonstrates the close relationship between the virus and immune system, which is characterized by a persistent, asymptomatic infection that resides in the memory B cell compartment. EBV-associated B cell malignancies originate from perturbing this connection, which happens in different types of immunosuppression. Although it seems to be a consequence of the virus’s improper establishment of latency in epithelial cells that have previously undergone premalignant genetic modifications, the virus’s function in the link between EBVaNPC and EBVaGC is less clear. Regardless of the intricacies of these interactions and the precise role that EBV plays in the carcinogenic process, there is certainly an opportunity to exploit this relationship for the therapeutic benefit of patients. We may successfully target EBV-associated carcinomas if we use novel treatment strategies including viral reactivation, gene therapy, or therapeutic vaccination.


Unfortunately, she died from fulminant pneumonia. Conclusion: Despite identical genetic disposition, WD symptom presentations may develop differently in monozygotic twins, and they may need to be placed on a very different therapeutical regimen. The underlying gene-environment interaction is unclear so far.Background: Wilson’s disease (WD) is an autosomal-recessive disorder of copper deposition caused by pathogenic variants in the copper-transporting ATP7B gene. There is not a clear correlation between genotype and phenotype in WD regarding symptom manifestations. This is supported by the presentation of genetically identical WD twins with phenotypic discordance and different response behavior to WD-specific therapy. Case Presentation: One of the female homozygous twins (age: 26 yrs) developed writing, speaking, swallowing and walking deficits which led to in-patient examination without conclusive results but recommended genetic testing. Both sisters were tested and were heterozygous for the C.2304dupC;p(Met769Hisf*26) and the C.3207C>A;p(His1069Gln) mutation. Self-medication of the affected sibling with 450 mg D-penicillamine (DPA) did not prevent further deterioration. She developed a juvenile parkinsonian syndrome and became wheelchair-bound and anarthric. A percutaneous endoscopic gastrostomy was applied. Her asymptomatic sister helped her with her daily life. Despite the immediate increase of the DPA dose (up to 1800 mg within 3 weeks) in the severely affected patient and the initiation of DPA therapy (up to 600 mg within 2 weeks) in the asymptomatic patient after the first visit in our institution, liver function tests further deteriorated in both patients. After 2 months, the parkinsonian patient started to improve and walk again, but experienced several falls, broke her right shoulder and underwent two necessary surgical interventions. With further consequent copper elimination therapy, liver dysfunction improved in both patients, without need for orthotopic liver transplantation (LTX) in the severely affected patient. Her excellent recovery of liver and brain dysfunction was only transiently interrupted by the development of a nephrotic syndrome which disappeared after switching to Cuprior Wilson´s disease (WD) is an autosomal recessively inherited disorder of copper metabolism caused by a defect of the large ATP7B gene localized on chromosome 13 ,3 which Hepatic and systemic overload of toxic copper is the major cause of tissue pathology and clinical symptoms in many different organs including the central nervous system (CNS) in the later stages of WD-symptom presentation ,12,13. HThe challenging task of finding a correlation between the ATP7B genotype and the WD phenotype has been attempted in different studies without much success ,22,23,24Several case reports of homozygous WD twins with different disease phenotypes and also studies in animal models indicate and support the involvement of epigenetic changes in the pathogenesis of WD and its phenotypic presentation, suggesting mostly that environmental or nutritional factors may affect WD ,28,29,30Here, we describe another pair of monozygotic WD twins with different clinical presentations, but in contrast to previous reports on monozygotic WD-twins, we will focus more on the difficulties of treatment and management and also demonstrate the differences of disease course and therapy response.The history of these monozygotic sisters before their first visit in our institution is summarized in After her divorce, sister 1 progressively developed neuropsychiatric symptoms which were initially interpreted to be of psychogenic origin. Then, hemochromatosis was suspected, and an inpatient evaluation was performed over three months. Finally, during a further neurological inpatient evaluation, WD was diagnosed because of the presence of Kayser-Fleischer rings. Treatment with 300 mg d-penicillamine (DPA) was initiated and the patient was dismissed with the recommendation of genetic testing, which confirmed WD in both sisters.With a significant time lag of about 4 months after the diagnosis of WD in sister 1, the two patients finally presented themselves in our institution on their own initiation.Sister 1 had increased the DPA dose to 450 mg. Nevertheless, she had experienced a rapid deterioration during the previous 4 months. At the first presentation , sister ® 150 mg within 6 days by exchanging 1 tablet DPA to 1 tablet Cuprior®. After this switch, the protein loss and the general condition, especially the dyspnea and the neurological symptoms, further improved  application.Clinically and neurologically, the second sister was asymptomatic at the first presentation  as well as three non-motor symptoms  were scored as to whether these symptoms were absent (0) or only mildly (1), moderately (2) or severely (3) present. The motor scores were summed up to yield a Wilson motor score (MotS: 0–21), the three non-motor items were summed up to give a non-motor score (N-MotS: 0–9) and the sum of MotS and N-MotS yielded the total score (TSC: 0–30). These scores have been used in our department to monitor therapy in WD for about 30 years now  and are Out of a long list of laboratory parameters being determined during our routine therapy monitoring of WD the following parameters were selected to demonstrate efficacy in copper chelating therapy and the difference in response to therapy of the twins: the liver enzymes ALT (circles), GGT (triangles) and CHE (squares) B and theBoth sisters underwent further investigations which confirmed severe impairment of brain and liver in sister 1 and normal brain imaging and mild liver involvement in sister 2 .The optical coherence tomography (OCT) examination revealed a normal size optic disc with almost normal retinal nerve fiber layer (RNFL) in both eyes of the two patients. The specific pattern of change in WD with the reduction in diameter of RNFL, GCIP and INL could not be seen clearly in segmented layers. There were slight differences between the OCT parameters of the two patients, but they were still in the normal range in comparison with normal control .Several case reports on homozygous WD twins with a different clinical phenotype have been presented ,28,29,30Clinical symptoms in WD result from tissue damage due to copper intoxication. Copper intoxication is dependent on duration of exposure to and the amount of toxic copper which is penetrated into the tissue of an organ. However, damage of an organ in WD is not only dependent on the transport of copper to the organ, but also on the ability of the organ to regenerate. The liver, e.g., is exposed to high amounts of copper in WD long before other organs are involved, but it has a high capability for regeneration. On the other hand, the central nervous system is well protected against copper for a long time by the blood brain barrier. Nevertheless, it is highly vulnerable to heavy metal intoxication .Differences in time to diagnosis and differences in nutrition and lifestyle all add to the variability of the phenotype. More than 1000 different mutations of the ATP7B gene cause a high genotype variation ,5. ThereIn our WD twins no difference in their disease development is obvious until their marriages. After marriage, both sisters lived at different places and sister 2 became pregnant soon after her marriage, which  may have been an unusual treatment of beginning WD. We therefore think that differences in lifestyle and pregnancy may have led to a difference in the clinical manifestation of WD. That WD might also have become manifest in sister 2 can be seen from the elevated liver enzyme levels which further deteriorated even when WD-specific treatment was initiated  hydrolase  has a crucial role in methionine metabolism as it is responsible for metabolizing SAH to homocysteine. If the expression or activity of SAHH are decreased, the level of SAH, which acts as an inhibitor of DNA methylation reactions, will increase. Importantly, SAHH enzyme activity and gene (AHCY) transcript levels are decreased in the presence of hepatic copper accumulation with consequent downstream changes in methionine metabolism parameters ,22.Notably, the toxic milk mouse from the Jackson Laboratory (tx-j mouse), which has a spontaneous point mutation affecting the second transmembrane region of the copper transporter, showed dysregulation of methionine metabolism and global DNA hypomethylation in hepatocytes ,24, withSo far, WD is thought of as a monogenic disease. However, additional involvement of modifier genes cannot be excluded, as long as whole genome analysis has not been performed in larger cohorts of WD patients. In a recent analysis of clinical findings in a large cohort of long-term-treated WD patients, a broad spectrum of comorbidities has been described with the possibility of mutual interaction with WD . This isThere is no clear-cut geno/phenotyp correlation in WD, either before or after WD-specific therapy. This is supported by the observation that homozygotic WD twins may present with totally discordant phenotypes and a totally different response behavior to therapy as in the cases presented above. The reasons are poorly understood and range from differences in lifestyle and compliance to a complex metabolic influence on gene regulation.


This perspective examines the proposition that chronically elevated blood glucose levels caused by type 2 diabetes (T2D) harm body tissues by locally generating reactive oxygen species (ROS). A feed-forward scenario is described in which the initial onset of defective beta cell function T2D becomes sustained and causes chronic elevations in blood glucose, which flood metabolic pathways throughout the body, giving rise to abnormally high local levels of ROS. Most cells can defend themselves via a full complement of antioxidant enzymes that are activated by ROS. However, the beta cell itself does not contain catalase or glutathione peroxidases and thereby runs a greater risk of ROS-induced damage. In this review, previously published experiments are revisited to examine the concept that chronic hyperglycemia can lead to oxidative stress in the beta cell, how this relates to the absence of beta cell glutathione peroxidase (GPx) activity, and whether this deficiency might be ameliorated by genetic enrichment of beta cell GPx and by oral antioxidants, including ebselen, a GPx mimetic. Much more is known about the clinical phenotype of type 2 diabetes (T2D) than is known about its primary genetic causes. Accordingly, it is accurate to consider T2D as a syndrome of polygenic origin rather than a single genetic illness. The prevalence and incidence of diabetes in the world continue to climb to unprecedented heights, so it is important to consider the possible oxidative features of the internal and external environments that impact mammals and humans who have developed T2D. Two important variables are the oxidative stress that can be caused by chronic hyperglycemia and the competency of the consequent innate antioxidant response. In this perspective, I revisit experiments designed to evaluate the success of endogenous Nrf2 and antioxidant gene expression to repair damaged beta cells in T2D.Islets comprise approximately 2–3% of the pancreatic mass and consist primarily of beta cells and alpha cells, amongst others. Beta cells synthesize and secrete insulin in response to rising blood glucose levels, whereas alpha cells synthesize and secrete glucagon in response to hypoglycemia. The primary defect in T2D is defective glucose-induced insulin secretion, which is made worse in the presence of general insulin resistance, a condition that frequently accompanies the development of the syndrome of T2D, obesity, and hyperlipidemia.Physiologic levels of ROS from various sources contribute positively to many cellular processes. However, chronic hyperglycemia can lead to excessive formation of ROS via several metabolic pathways, including oxidative phosphorylation, glyceraldehyde autoxidation, PKC activation, and metabolism of hexosamine, glucosamine, dihydroxyacetone, enediol, and glyceraldehyde ,2; Figur. If the In the last half of the 20th century, a great deal was learned about abnormalities in beta cell function in humans and animals with T2D. Much of this information came from clinical research that involved studies such as the oral and intravenous glucose tolerance tests . T2D was characterized metabolically as a state involving not only hyperglycemia but paradoxically also one in which fasting circulating insulin levels could be in the normal or even in the distinctly abnormally high range. Over several decades it became appreciated that high basal insulin levels were common in T2D and were, for the most part, associated with individuals who were obese and, to a lesser extent, lean. It was also learned from intravenous glucose tolerance testing that the initial burst of insulin release within minutes of injecting glucose that was normally present in non-diabetic humans was totally absent in subjects with T2D. This loss was unique to glucose because the first-phase responses to other agonists, such as amino acids, glucagon, and isoproterenol, were fully intact. These features strongly suggested that T2D was clearly distinct from T1D, an immunologic disease that involved the death of beta cells and the total disappearance of agonist-induced insulin responses.In roughly the same time frame, there was intense research attention on exploring the causes of elevations in blood insulin levels in obese individuals with and without T2D. The primary in vivo methodology initially involved glucose clamp studies, which can be performed under normoglycemic or hyperglycemic conditions. The overall study design has several different variations, but the overall goal is to ascertain the quantitative relationship between blood glucose and insulin levels as one or the other is manipulated independently. For one example, it was observed that obese and aged individuals with elevated basal insulin levels were resistant to the effects of exogenous insulin such that more infused insulin was required to maintain a given clamped glucose value. Such findings gave rise to the term insulin resistance and led to a great deal of in vitro research examining molecular mechanisms of insulin action and insulin resistance in various tissues, including the liver, muscle, and adipose tissue.Research into the regulation of beta cell function revealed that insulin gene expression in cell lines was adversely affected if the cells were chronically cultured in media containing high concentrations of glucose. For example, experiments using the beta cell line HIT-T15 showed that chronic culturing of these cells in media containing supraphysiologic concentrations over many weeks caused progressive declines in levels of insulin mRNA, insulin content, and glucose-induced insulin secretion . Loss ofExtension of these in vitro experiments to in vivo experiments with a rodent model of T2D, the male Zucker Diabetic Rat (ZDF) fed a high-fat diet, were conducted by comparing data from control rats and rats treated with troglitazone, a drug that lowers blood glucose in these rodents. The control animals developed progressive hyperglycemia and an associated loss of PDx-1 and insulin mRNAs and diminution of glucose-stimulated insulin secretion. Treatment with troglitazone prevented hyperglycemia and the adverse effects on PDX-1 and insulin gene expression and improved insulin secretion .Clinical reports of elevated levels of oxidative stress markers in patients with T2D are numerous. Relevantly, the islet is among the least well-endowed tissues in terms of intrinsic antioxidant enzyme expression. Essentially, the beta cell expresses only superoxide dismutases and hemoxygenases with very little, if any, glutathione peroxidase or catalase ,4,5,15, These observations led to an assessment of the importance of the absence of glutathione peroxidase activity in beta cells during periods of oxidative stress. These experiments involved both HIT-T15 cells and isolated islets from male Wistar rats and from humans . We obseEbselen -one, a nontoxic seleno-organic drug, is a lipid-soluble, orally bioavailable small molecule classified as a GPx mimetic ,33,34 thdb/db-GPx(+) mice was initially ameliorated by ten weeks of age and then by 20 weeks almost completely reversed (compare the red line in db/db-GPx-1(+) overexpressed animals compared to db/db-GPx-1(−) control animals. The control animals lost intranuclear MafA, which was prevented by GPx-1 overexpression. We concluded that it was a transgenic enhancement of intrinsic antioxidant defenses of mouse beta cells that protected them against the deterioration of beta cells that occurs during the development of hyperglycemia. These results led us to examine more closely whether animals just beginning to develop spontaneous T2D and whose beta cells were just on the verge of being exposed to initial hyperglycemia and oxidative stress might evince evidence for intrinsic antioxidant expression via the Nrf2/antioxidant pathway as an attempt to initiate beta cell repair [We studied whether an increase in GPx-1 in beta cells specifically would protect them from the adverse effects of chronic hyperglycemia . For thel repair .The strategy of these studies was to examine the evidence for endogenous antioxidant-mediated repair mechanisms via activation of the Nrf2/antioxidant pathway ,42,43,44 that migThe main emphasis of this perspective is that oxidative stress caused by chronic hyperglycemia is an important force in the continued deterioration of beta cell function in T2D and is relentless in the face of inadequately treated chronic hyperglycemia. When hyperglycemia becomes a constant feature of T2D, beta cell damage is amplified and sustained by its own failure to correct excessive levels of glucose and reactive oxygen species. This begets increasingly more beta cell damage, a vicious self-perpetuating cycle that represents a feed-forward mechanism for even more cellular damage. A caveat to this logic is that animal models of T2D also develop high levels of blood triglyceride, which raises the possibility lipids and not necessarily glucose, may play a role in the progressive demise of beta cell function, i.e., via lipotoxicity . We asseThe evidence cited in this perspective and brief review suggest that incomplete resolution of hyperglycemia characteristic of medical treatment of T2D is itself a factor in the continued development of beta cell deterioration over time and that chronic oxidative stress plays a major role in this pathogenesis. It seems possible that clinical strategies involving antioxidants should enhance protection from persistent hyperglycemia and that this might allow the completion of beta cell repair and resolution of hyperglycemia after the onset of the disease.


The current theme issue is intended to focus on the advances and the development of drug repurposing and strategies for cancer treatment. The issue contains the contributions from researchers studying various aspects of drug repurposing and screening for anticancer management with a particular interest in metformin (dimethylbiguanide) as well as other small marketed drugs.Metformin is the recommended first line of treatment of type 2 Diabetes Mellitus. Recently, metformin is gaining international interest for its potential use to treat/prevent different types of cancer, cardiovascular disease, ageing, and neurological disorders. Several studies reported the potential anticancer activity of metformin that promoted the initiation of tens of clinical trials that aimed to investigate its potential against endometrial, prostate, pancreas, lung and breast cancer. Nonetheless, uncertainties still exist with respect to understanding the cellular mode of action of metformin, its antiproliferative ability as well as the effective antiproliferative concentration.Luo et al.) that evaluated the scientific literature on the efficacy of the combinational use of metformin and anticancer agents on lung cancer survival in both diabetic and non-diabetic patients. Briefly, the study is a comprehensive meta-analyses of randomized controlled trials (RCTs) and observational cohort studies with two outcome indicators, namely overall survival (OS) and progression-free survival (PFS). The clinical evidence obtained from the current analysis suggested that metformin, in combination with antineoplastic agents, may improve the PFS and OS compared to standard antineoplastic agents alone. While this finding is in line with our previously published work that metformin may provide synergistic effect to chemotherapy .In their article “Metformin Adjunct With Antineoplastic Agents for the Treatment of Lung Cancer: A Meta-Analysis of Randomized Controlled Trials and Observational Cohort Studies”  claimed that disulfiram complexed with Copper (DSF/Cu) has a potential in the treatment of colorectal cancer. The study claimed that DSF/Cu inhibits cell viability of colorectal cancer cells in a dose dependent manner both in vitro and in vivo (subcutaneous xenograft mice model). The authors suggested that the DSF/Cu complex induced inhibition of cell viability was due to cellular autophagy rather than apoptosis. To confirm the potential mechanism, the authors employed a (CRISP-Cas9)library screening to identify genes, expression of which was either up or down regulated by DSF/Cu complex exposure. Screening resulted in identification of five –autophagy related genes , out of which expression of ULK1 was significantly upregulated. ULK1 is a kinase that participates in autophagosome assembly. Effect of DSF/Cu treatment on induction of autophagy was abolished in ULK1 knockdown cells, which was showed by suppressed expression of LC3 (marker of autophagosomes) after DSF/Cu treatment of ULK1 knockdown cells. This suggests that autophagy can at least partially play role in DSF/Cu induced cytotoxicity. Moreover, DSF/Cu induced cytotoxicity could be partially overcome by pretreatment of cells with autophagy inhibitor—choloroquine (CQ).Interestingly, another study in this theme titled “The Disulfiram/Copper Complex Induces Autophagic Cell Death in Colorectal Cancer by Targeting ULK1”  upon exposure to triclabendazole displayed typical signs of pyroptosis including annexin V staining, regulating the apoptoic protein levels of Bax, Bcl-2, and enhanced cleavage of caspase-8/9/3/7 and PARP. Furthermore, cellular treatment with triclabendazole resulted in an enhanced cleavage of gasdermin E (GSDME), which is a key molecule in the pyroptosis process, indicating that triclabendazole is a potent inducer of pyroptosis.Nevertheless, activation of apoptosis or programmed cell death is considered as a major drug target for most anti-cancer therapies, particularly breast cancer. While chemotherapy is considered as the main treatment for breast cancer, there is a poor overall response in breast cancer patients due to the development and activation of anti-apoptotic systems that facilitate the escape of drug-induced apoptosis . In theiDrug repurposing involves finding new therapeutic benefits of marketed drugs rather than searching for entirely new therapeutic agents. The process has become especially important for developing anticancer therapies, as the majority of currently available oncology drugs have numerous side effects, low efficacy and high cost. In this themed issue, we assembled a collection of various studies that investigated drugs with potential anticancer activity including metformin, ivermectin, triclabendazole, and disulfiram copper complex. While these drugs appear to have different potency and mechanism of actions, they all showed positive anticancer activity warranting further research to evaluate their potential repurposing for anticancer uses.


In recent years, the biological role of changes in physical factors in carcinogenesis and progression has attracted increasing attention. Matrix stiffness, also known as ECM stress, is a critical physical factor of tumor microenvironment and remains alternating during carcinogenesis as a result of ECM remodeling through activation of cancer-associated fibroblasts and extracellular collagen accumulation, crosslinking and fibrosis. Different content and density of extracellular collagen in ECM endows matrix with varying stiffness. Physical signals induced by matrix stiffness are transmitted to tumor cells primarily by the integrins receptor family and trigger a series of mechanotransduction that result in changes in tumor cell morphology, proliferative capacity, and invasive ability. Importantly, accumulating evidence revealed that changes in matrix stiffness in tumor tissues greatly control the sensitivity of tumor cells in response to chemotherapy, radiotherapy, and immunotherapy through integrin signaling, YAP signaling, and related signaling pathways. Here, the present review analyzes the current research advances on matrix stiffness and tumor cell behavior with a view to contributing to tumor cell growth and treatment, with the hope of improving the understanding of the biological role of matrix stiffness in tumors. During tumorigenesis, solid stress and extracellular matrix (ECM) stress have been interacting as tumor external and internal forces, respectively. Solid stress refers to the stress harbored by the solid phase of tumors, including the stress exerted by the surrounding normal tissue to inhibit tumor expansion as the tumor expands , 2. ECM The cross-linking of ECM proteins and collagen deposits gives the ECM different stiffness and elasticity, which in turn creates differential physical stresses on tumors , 16. ECMRecently, cell stiffness, which represents the elasticity of individual cells, has also attracted much attention . In subcIn spite of the fact that the importance of the physical factors in the tumor stroma that interact with tumor cells has been gradually recognized, physical factors in the tumor, including the matrix stiffness, remain a relatively novel concept for oncologists. Whether the matrix stiffness has a similar effect on tumor growth across different tumors and between different subtypes of the same tumor as the tumor progresses, and the mechanisms by which it affects tumor growth and possible implications for cancer therapy is not yet conclusive. Matrix stiffness has been observed to have differential effects on tumor cell morphology, proliferation, apoptosis, and migration, and these effects make tumor cells differentially sensitive to chemotherapy, radiotherapy, and immunotherapy. Recent advances in technology for the analysis of stiffness have also created a unique research platform to uncover the effects of stiffness on the biological characteristics of tumor cells. In this review, we focus on elucidating the role of matrix stiffness in the functions and behaviors of tumor cells and the potential impact of matrix stiffness on cancer therapy. We summarize the effects of matrix stiffness on cell morphology, proliferation, motility and invasion, and sensitivity to chemoradiotherapy in different tumor cells, and also propose some points that are still controversial and inconclusive, and discuss possible mechanisms and causes, in order to gain a comprehensive understanding of the current role of matrix stiffness in tumors. Overall, the results of these studies suggest that matrix stiffness is not only involved in tumor cell growth, but also is an important mediator of response to chemotherapy and radiotherapy.Matrix stiffness, also known as the elastic modulus (Young’s modulus) of a substance, is mainly caused by rearrangement, cross-linking, and deposit along with degradation of specific ECM proteins . The aggAtomic force microscopy (AFM) is one of the main instruments for measuring matrix stiffness in the laboratories, which identifies the alterations of elastic-mechanical properties at a nanoscale . Widely Matrix stiffness and biological forces are the main internal force and external biological force that control cell morphology. The rigid substrate provides better physical support for cell growth and increases cell adhesion and cytoskeleton construction . A posit2, 1673um2 and 3920um2, respectively, and the cell proliferation activity also increased with progressive stiffness [However, the controversial effect of matrix stiffness on tumor cell morphology was observed in several research reports. On 1 kPa substrate, cervical cancer cells were spherical with few pseudopodia, whereas cells cultured on 20 kPa substrate were rich in pseudopodia, indicating a greater ability to migrate on a stiffer stiffness . In ovartiffness . Hepatoctiffness . In conttiffness . Human btiffness .Cancer cell shapes have been associated with cell proliferative capacity . IncreasProliferative capacity is an important criterion for evaluating cell viability. Given the role of ECM in maintaining structural basis and mechanical integrity of tumor tissues, increasing stiffness of ECM including collagen deposition promotes focal adhesion assembly and enhances cytoskeletal function in cancer cell, ultimately favoring cell proliferation and invasion , 57. CirDifferently, human glioblastoma cells presented lower DNA content, spreading area and cell proliferation in 26.6 kPa substrate compared to 1 kPa, and 26.6 kPa substrate led to a 70% decrease in DNA content on day 14 compared to day 1. Increased matrix stiffness induced cell apoptosis by upregulating HIF1α expression, which can lead to activation of downstream apoptotic pathways. Using temozolomide (TMZ) as a model drug, which causes DNA base pair mismatch and triggers downstream apoptotic pathways that exerts antitumor effects depending on active cell cycle progression and extensive cell proliferation, tumor cells showed increasing drug resistance with increasing matrix stiffness because of the lower cell proliferation rate in 26.6 kPa . Given tChanges in matrix stiffness have a striking effect on the properties of ECM. Cells sense and respond to a variety of mechanical information including the stiffness by altering their cytoskeletal structure, which significantly affect the ability of cell motility and invasion . Matrix In culture environments with different matrix stiffness , breast cancer cells showed enhanced migratory ability with increased stiffness through activating integrin β1 and FAK directly, accelerating focal adhesion maturation and inducing the downstream cascades of intracellular signaling of the RhoA/ROCK pathway . In animAggressive breast cancer often shows an abnormal peritumoral stiff area, which results from desmoplastic reaction and tumor cell infiltration into the peritumoral stroma , 75. Com2+ preferred membrane ion channel and activation of TRPV4 channel could promote matrix biosynthesis by mediating calcium influx and activating phosphatidylinotol 3-kinase (PI3K) [Epithelial to mesenchymal transition (EMT) is one of the basic biological processes of cancer cell invasion and metastasis. During the processes of EMT, cells lose their epithelial characteristics, including cell junctions and polarity, and acquire a mesenchymal morphology and invasive capacity , 79. Mate (PI3K) , 84. TGFe (PI3K) . Overalle (PI3K) . To dateDrug insensitivity and chemoresistance are two leading causes of tumor progression, recurrence and cancer death. Understanding the mechanisms by which cancer cells overcome chemotherapy-induced cell death and increase sensitivity to chemotherapeutic drugs is critical for improving cancer patient survival. Almost all antineoplastic drugs are dose dependent, and drugs enter the tumor tissue from blood vessels, penetrate the tumor stroma and achieve an effective concentration of the drug, which is the prerequisite and key to the destruction of tumor cells. The increased volume of the tumor increases the fixed tension of the host tissue and results in compression of vascular tissues of the tumor. The uniform increase of interstitial fluid pressure and the dysfunctional lymphatic vasculature caused by the high permeability of tumor blood vessels exacerbate the pressure on tumor blood vessels, resulting in insufficient perfusion inside the tumor, which is not conducive to drug diffusion. On this basis, the dense ECM induced by poor tissue perfusion further impedes molecular diffusion, limits drug penetration and ultimately reduces the efficacy of antitumor drugs .Recent studies in multiple animal models have confirmed that increased ECM stiffness reduces cell apoptosis induced by chemotherapy and hinders the efficacy of treatment, as stromal stiffness interferes with the distribution of chemotherapeutic agents and may induce insensitivity to chemotherapy , 88. AftHowever, the opposite pattern was observed in another breast cancer cell line MCF-7. When the same concentrations of cisplatin and paclitaxel were added on 5.3, 46.7 and 2710 kPa gel matrix, the viability of MCF-7 cells decreased with the increasing matrix stiffness, suggesting that MCF-7 cells have lower sensitivity to antitumor drugs on soft substrate . SimilarThe vascular permeability of tumor tissue may also be one of the underlying mechanisms by which the matrix stiffness affects sensitivity of chemotherapy . In tumoThe stiffness and elasticity of liver tissue caused by cirrhosis are one of the risk factors of liver cancer, and sorafenib is the standard therapy for advanced hepatocellular carcinoma . Huh7 ceLiver metastases (LM) are the leading cause of death in nearly 50–75% of colorectal cancer (CRC) patients. A significant increase in stromal stiffness in fresh and cryo-preserved LM tissue in colorectal cancer were observed compared to primary tumor (pTU), 1.5 kPa and 0.3 kPa, respectively. Activation of metastasis-associated fibroblast (MAF) in LM with higher expression of COL-1, a-SMA, and p-MLC2 significantly contributed to matrix stiffening through ECM remodeling compared to pTU. Meanwhile, a hypertension disease signature in MAFs of LM were observed, and qPCR analyses on freshly isolated MAFs versus liver-derived fibroblasts revealed a significant increase in expression of all the key components of the renin-angiotensin system (RAS). Patients with anti-RAS treatment, losartan or captopril, significantly reduce the activity of MAFs and matrix stiffness of LM in CRC through inhibiting the YAP/TAZ signaling, which in turn increases the efficacy of anti-angiogenic therapy . The combination treatments of Bev and anti-RAS drug prolonged the overall survival of CRC patients who underwent resection of LM compared with non-RAS drug + Bev group , which further illustrate the possibility of matrix stiffness as a new target for tumors. However, the RAS inhibitors do not change the stiffness of non-metastatic liver tissue, which means the mechano-based therapy might not be beneficial until tumor cells infiltrate the liver . As a reCancer stem cells (CSC) are a small subpopulation of cells with stem like characteristics in solid tumors, which can maintain an undifferentiated state and self-renewal ability, becoming one of the main sources for drug insensitivity and resistance –103. SoxRadiotherapy as an adjuvant therapy induces tumor cell death or slows tumor growth by stimulating the production of free radicals and reactive oxygen species and disrupting the DNA double helix . AlthougOpposite, some studies showed that the matrix stiffness has no effect on the sensitivity of tumor cells to radiotherapy. Lacombe et al. investigated the response of prostate cancer cell line PC3 to 2 Gy radiation on conventional cell culture substrates (~ Gpa) and decellularize spinach leaves (21.8 ± 3.3 kPa) by assessing the short-term DNA damages in tumor cells. Although, matrix stiffness regulated the proliferation of tumor cells via Yap/TAZ pathway, the DNA damages were effectively repaired after 6 h of irradiation in different stiffness conditions and there was no significant difference in the radiosensitivity of PC3 cells on both substrates after 24 h X-ray irradiation . Given tImmune checkpoint inhibitors and adoptive T cell therapy are the two main T-cell-based immunotherapy in tumor, but an elevated percentage of patients with solid tumors fail to respond to these therapies inexplicably . RecentlOverall, the increased matrix stiffness by excessive accumulation and linearization of collagens inhibits the function of immune cells and limits T cell-based therapy, leading to the immune escape of tumor cells as the tumor progresses and poor efficacy of immunotherapy. Accordingly, one important challenge in the field is to develop strategies targeting tumor fibrosis in order to reverse immune exclusion and to improve T cell-based immunotherapy .The effect of matrix stiffness in the tumor microenvironment on tumorigenesis and progression has received increasing attention recently. It affects not only cell functions including cell morphology, proliferation, and invasion, but also the sensitivity to chemotherapy, radiotherapy and immunotherapy in cancer cells and animal models. However, how matrix stiffness influences tumor carcinogenesis, progression and therapeutic efficacy is one of the many unanswered questions yet to be addressed. Some potentially conflicting results suggests the effect of matrix stiffness on tumor growth and treatment depends on the tumor type and is not applicable to all tumor types. Thus, it must be determined whether matrix stiffness is a causative factor that drives tumorigenesis or contributes tumor resistance to chemotherapy or radiotherapy in different tumor types or the same tumor with different subtypes. With a better understanding of matrix stiffness, the potential for matrix stiffness to be an effective target for the treatment of cancer patients remains very high.


Helium bubbles are known to form in nuclear reactor structural components when displacement damage occurs in conjunction with helium exposure and/or transmutation. If left unchecked, bubble production can cause swelling, blistering, and embrittlement, all of which substantially degrade materials and—moreover—diminish mechanical properties. On the mission to produce more robust materials, nanocrystalline (NC) metals show great potential and are postulated to exhibit superior radiation resistance due to their high defect and particle sink densities; however, much is still unknown about the mechanisms of defect evolution in these systems under extreme conditions. Here, the performances of NC nickel (Ni) and iron (Fe) are investigated under helium bombardment via transmission electron microscopy (TEM). Bubble density statistics are measured as a function of grain size in specimens implanted under similar conditions. While the overall trends revealed an increase in bubble density up to saturation in both samples, bubble density in Fe was over 300% greater than in Ni. To interrogate the kinetics of helium diffusion and trapping, a rate theory model is developed that substantiates that helium is more readily captured within grains in helium-vacancy complexes in NC Fe, whereas helium is more prone to traversing the grain matrices and migrating to GBs in NC Ni. Our results suggest that (1) grain boundaries can affect bubble swelling in grain matrices significantly and can have a dominant effect over crystal structure, and (2) an NC-Ni-based material can yield superior resistance to irradiation-induced bubble growth compared to an NC-Fe-based material and exhibits high potential for use in extreme environments where swelling due to He bubble formation is of significant concern. Structural components exposed to extreme environments in nuclear reactors are subject to degradation due to irradiation damage and elevated temperatures . High-enNanocrystalline (NC) materials have intricate microstructures composed of grains less than 100 nm in size and consequently have a large volume fraction of grain boundaries (GBs) compared to their coarse-grain analogs. Molecular dynamics simulations and experiments have demonstrated that GBs can absorb point defects and defect clusters, act as sinks for irradiation-induced defects, and contribute to defect reduction by effectively annihilating defects . Due to On the charge to produce more advanced nuclear materials, one method is to design and develop bulk-nanostructured alloys with enhanced swelling and creep resistance as well as satisfactory corrosion tolerance. Experimental evidence of the correlation of radiation tolerance with grain size is crucial in investigating the performance of candidate nanomaterials for future nuclear applications and for evolving mesoscale predictive models to deconvolute the significance of underlying mechanisms. From coarse-grain studies, it is generally accepted that austenitic stainless steels possess good creep and fatigue resistance at elevated temperatures compared to ferritic/martensitic steels . AdverseIn this work, thin NC Ni (FCC) and NC Fe (BCC) film samples were chosen as model metals to examine (1) how grain boundary density affects bubble formation and bubble damage in the grain matrices, and (2) compare this effect on one FCC and one BCC model material. Our hypothesis is that the increased grain boundary density will lead to low bubble formation and bubble damage in the grain matrices, but the effect of grain boundary density can also be phase-dependent (FCC vs. BCC). in situ transmission electron microscopy (TEM) coupled with helium bombardment was used to implant helium, and automated crystal orientation mapping (ACOM) was used to characterize the post-irradiation microstructure, defect morphology, and grain orientation. A comparison of grain size versus bubble densities in the NC regime revealed that bubble density increased with grain size in both the Ni and Fe; however, the bubble densities were more than 300% greater in Fe compared to Ni. A kinetic rate theory (RT) model was employed to explain differences in helium–void and helium–interface interaction proclivities. The results indicated that in NC systems, helium atoms are more biased towards GBs in Ni compared to Fe, whereas helium atoms and vacancies are more inclined to bond and form bubbles in the grain matrix in Fe compared to Ni. Thus, contrary to standard belief, our results suggest that an NC-Ni-based material can yield a superior radiation tolerance compared to an NC-Fe-based material, exhibiting a high potential for use in extreme environments where swelling due to helium bubble formation is of significant concern.6 to promote grain growth, while actively monitoring to obtain a stabilized equilibrium nanocrystalline microstructure. Both films maintained a columnar microstructure, while the Ni film was composed of grains ranging from 10 nm to 100 nm in size, and the Fe film was composed of grains ranging in size from 10 nm to 150 nm.Thin NC Ni and Fe films (approximately 100 nm-thick) were deposited via physical vapor deposition onto 6 at the in situ Ion Irradiation TEM at Sandia National Laboratories [22 m−2 for both Fe and Ni. The damage distribution of the ions was simulated using Stopping Range of Ions in Matter (SRIM 2013) Monte Carlo code , respectively. The samples were irradiated in situ using a Colutron implanter attached to a JEOL 2100 LaBratories . Each sacode see ; the meacode see . The fincode see  is 1.5 ×6 TEM. Defect and grain characterization measurements were conducted using the protocol outlined in [Post-irradiation, bubble characterization was performed using under-focused bright field Fresnel imaging, a proven method which provides an accurate and prompt means of identifying and measuring bubbles, using a JEOL 2100 LaBlined in ; ACOM waAfter implantation, in both the Ni and Fe films, small cavities were found throughout the grain interior matrices and the GBs; these cavities were presumed to be helium–vacancy bubbles based on previous studies . Figure 2 whose density increases with grain size, regime 2 contains grains between 3000 nm2 and 7500 nm2 with scattered densities, and regime 3 encompasses grains >7500 nm2 with densities approaching a saturation value and eventually decreasing as grain size continues to increase into the ultrafine grain domain. A similar breakdown is found in the Ni plot but with all regimes shifted to smaller grain size ranges: regime 1 contains grains <1000 nm2, regime 2 contains grains between 1000 and 4000 nm2, and regime 3 with grains >4000 nm2. In regime 3 in the Fe plot, the bubble density approaches saturation value near 0.055 bubbles per nm2 at the verge of the NC/ultra-fine grain transition (around 100 nm diameter), whereas in the Ni plot, bubble density saturates between 0.015 and 0.020 bubbles per nm2 in grains larger than 4000 nm2 (around 63 nm diameter). Bubble density saturation suggests a steady state between defect absorption to interfaces such as GBs and helium–vacancy complex formation and coalescence.In an earlier manuscript , El-AtwaCrystal structure and grain size have proven to be influential factors in defect cluster formation. Large-scale molecular dynamics simulations of the irradiation of NC Ni and Fe show both similarities and differences in defect production. In both materials, the NC grain size promoted interstitial-free grain interiors but with different vacancy cluster structures forming during displacement cascades . It has ) planes ,31. Note) planes , have be) planes .Under bombardment conditions resulting in the production of Frenkel pairs with primary knock-on atom (PKA) energies below the SFT-formation threshold, faulted Frank loops of a vacancy nature are expected to form and be visible in the form of “black dot” clusters in the TEM in Ni at the temperature and irradiation parameters in the present study ; nonethesionally . When thHelium bubbles in crystals occur when vacancy clusters are stabilized internally with helium atoms, leading to the nucleation of bubbles that are thermally stable and trap helium in the bulk. The capacity of bubble growth in an irradiation environment is governed by several factors including helium availability, vacancy production, prominence of defect sinks, and irradiation temperature. The helium implantation profiles shown in The vacancy migration energies of Fe and Ni  set the temperature for the onset of stage III kinetics (recovery) during isochronal irradiation studies . ConsequIn a variable temperature implantation study, El-Atwani et al. measured bubble density as a function of grain size in two NC Fe samples irradiated to the same damage dose at two different temperatures . NC Fe wTo the first order, the differences in bubble density in Fe and Ni can be rationalized in terms of the partition of helium under irradiation in both materials. As indicated above, helium atom migration is nearly athermal (temperature-independent), so one can understand helium accumulation as a process in which atoms are immediately apportioned among the existing defect sinks. The high GB density existing in both materials due to their NC structure provides a strong driving force for helium absorption. However, vacancy clusters in Fe are well predisposed to capture helium atoms to stabilize themselves and give rise to bubble nuclei, while small faulted loops in Ni display a much lower helium capture propensity. This propensity can be quantified to the first order via the helium–vacancy binding energy, which, as shown in The results of the current study clearly show that the density and size of bubbles formed are quite different in the two materials and are dependent heavily on microstructure, i.e., the presence of GBs acting as effective sinks. Evidence of crystal structure influencing irradiation damage defect production is apparent in the defect yield in BCC metals being reported as much lower than in FCC metals ,46,47,48The microstructural influence of GB density may have more implications in assorted metals and compounds as well as alloying elements—such as Ni, Si, P, Ti, and C—that have varying effects on defect evolution behavior in different alloys . Since hIn the same vein, the capacity of GBs to limit bubble growth in acute extreme conditions is questionable and requires more thorough investigation. In a sequential self-ion irradiation, helium implantation, and annealing study in NC Ni, Muntifering et al.  observedmVn) evolution have found that the stable configuration of complex clusters is impartial to cluster size and discrete numbers of helium atoms but dependent on the helium atom to vacancy ratio, m:n [mVn clusters as a function of m, the number of helium atoms [Consideration of the gas atom pressure inside bubbles is known to have relevant implications for material performance; how complex composition affects bubble diffusion and agglomeration are of particular concern. Nevertheless, due to experimental restraints, the stoichiometric ratios of the bubbles resulting from the implantations in the current work were unspecified. Model investigations of helium–vacancy complex  Ni and (BCC) Fe films. Bubble density and size were measured and compared with respect to grain size. A decreasing trend in bubble density as grain size decreases is distinct for both samples. Contrary to findings in coarse-grain studies, the bubble density in the grain matrices in Fe was 300% greater than in Ni, suggesting that the NC Ni may possess an enhanced resistance to changes in volume due to helium bubble growth. A kinetic RT model was employed to differentiate between helium–defect and helium–interface interaction proclivities; the model results indicate in that in NC systems (grain size < 100 nm), helium is partitioned more equitably between bubbles and GBs in Fe, whereas it preferentially accumulates at GBs in Ni, resulting in a microstructure where bubbles are more prevalent in NC Fe than in Ni. Moreover, many factors such as complex composition, irradiation parameters, material chemistry, and GB sink efficiency all affect the kinetics and play a significant role in defect evolution and morphology in irradiated NC materials and require further exploration.


One of the major complications of diabetes mellitus is diabetic cardiomyopathy. One of the mechanisms that initiates the irreversible deterioration of cardiac function in diabetic cardiomyopathy is mitochondrial dysfunction. Functionally impaired mitochondria result in greater levels of oxidative stress and lipotoxicity, both of which exacerbate mitochondrial damage. Mitochondrial health is constantly monitored by mitochondrial quality control mechanisms. Mitophagy selectively degrades damaged mitochondria, thereby maintaining the healthy pool of mitochondria and preserving myocardial function. Mitophagy in diabetic cardiomyopathy is mediated by multiple mechanisms in a time-dependent manner. Potential targets for the treatment of diabetic cardiomyopathy include increased oxidative stress, mitochondrial dynamics, and mitochondrial clearance. Thus, stimulation of mitophagy represents a promising strategy for the alleviation of diabetic cardiomyopathy. Major triggers of diabetes include a sedentary lifestyle, obesity, and dyslipidemia (2). More than half of patients with diabetes develop cardiomyopathy, characterized by the presence of hypertrophy, diastolic dysfunction, and arrhythmia, which is consistently accompanied by mitochondrial dysfunction at the cardiomyocyte level (3). Mitochondria occupy approximately 30% of the cardiomyocyte volume and supply large amounts of ATP to support the energy demands of contraction. The presence of dysfunctional mitochondria in cardiomyocytes leads to increased oxidative stress and triggers a vicious cycle of mitochondrial damage and production of reactive oxygen species (ROS) (4). Mitochondrial health is maintained by various regulatory mechanisms, including mitochondrial fusion and fission, mitophagy, and biogenesis (5). Mitophagy is a major mechanism of degradation by which damaged mitochondria are selectively sequestrated in double-membraned vesicles called autophagosomes and subsequently eliminated by lysosomes (6). Our goal in this review is to summarize the molecular mechanisms of mitophagy and discuss rational therapeutic considerations for improving the quality of mitochondria and alleviating cardiac complications in patients with diabetes.The incidence of diabetes has increased dramatically over the past three decades (1). Diabetic cardiomyopathy was first described in 1972 and was later accepted as a distinct clinical entity in the 1974 Framingham Heart Study (8). It is defined as a cardiac dysfunction in the presence of diabetes mellitus that is not caused by ischemic, valvular, or hypertensive heart disease. Other types of cardiomyopathies, including hypertrophic, restrictive, and dilated cardiomyopathies, are also excluded as potential causes of the dysfunction (9). In the early stage, diabetes causes diastolic dysfunction and heart failure with preserved ejection fraction, but it may later lead to systolic dysfunction, called heart failure with reduced ejection fraction, in the chronic phase (10). Other structural and functional changes observed in the heart during diabetes include ventricular hypertrophy, cardiac steatosis, and cardiac arrhythmia (3).Although type 1 and type 2 diabetes have different underlying mechanisms, both lead to similar detrimental cardiovascular outcomes. Cardiovascular complications in diabetes are associated with high morbidity and mortality rates and are caused by accelerated atherosclerosis, coronary artery disease, myocardial infarction, and heart failure (12). However, diabetic hearts lack metabolic flexibility, depending on fatty acid oxidation more critically than healthy normal hearts because of the impaired uptake and utilization of glucose due to insulin deficiency or resistance (14). In healthy cardiomyocytes, the low levels of electron leakage from the electron transport chain at the time of oxidative phosphorylation can be cleared by antioxidants, such as glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase (SOD). However, in diabetic hearts, greater amounts of ROS are produced as a result of increased levels of fatty acid oxidation, exceeding the cells’ capacity to neutralize oxidants. Mitochondrial proteins, including those in the electron transport chain, are damaged by oxidative stress and, in turn, produce more ROS (15). In addition, the NADPH oxidase, Nox4, is an important source of ROS in hyperglycemia, and its upregulation may be involved in early structural changes seen in diabetic cardiomyopathy caused by type 1 diabetes (16). Increased calcium influx into mitochondria from the endoplasmic reticulum has detrimental effects on both mitochondrial and cardiac functions in diabetes (17). Damaged and futile mitochondria undergo fragmentation, separation, and elimination. However, as we will discuss later, they can accumulate if the clearance mechanism is insufficient, leading to the production of more ROS, which, in turn, exacerbate mitochondrial dysfunction (19). Mitochondrial dysfunction also leads to the attenuation of fatty acid oxidation. An imbalance among fatty acid intake, lipolysis, and fatty acid oxidation leads to the accumulation of cytotoxic lipids, including ceramides and diacylglycerols, a condition termed lipotoxicity (20). Mitochondrial dysfunction, energetic insufficiency, and lipotoxicity all contribute to impaired cardiac function in diabetic cardiomyopathy (22). Diabetic cardiomyopathy is also accompanied by inflammation and cellular senescence, but their involvement is not discussed in this article. (The readers are referred to an excellent review of the involvement of inflammation and cellular senescence in the development of diabetic cardiomyopathy (23)).The adult heart is a high energy demand organ and continuously produces a large amount of ATP, using fatty acid as the major source of fuel (60% of the ATP produced) (5).Damaged mitochondria not only are dysfunctional but can also be toxic since they exacerbate ROS production, release cell death-promoting factors, and induce mutations in mitochondrial DNA. Thus, it is critical for the cell to monitor the quality of mitochondria and expeditiously eliminate those that are dysfunctional. Mitochondrial quantity, quality, and morphology are under constant regulation by several different mechanisms. These include mitochondrial dynamics , mitophagy, and mitochondrial biogenesis  (5).(24). In mitochondrial fusion, depolarized mitochondria merge with others, allowing each mitochondrion to compensate for its own defects by mixing with new constituents to complement its own mtDNA. It is generally believed that fusion is a useful mechanism that increases cellular viability (5). In contrast, mitochondrial fission involves asymmetric segregation (25) of damaged mitochondria that occurs at sites of contact between mitochondria and the endoplasmic reticulum via recruitment of Drp1. In general, mitochondrial fission serves to prepare the cell for subsequent mitophagy (26). Fusion and fission are delicately balanced with each other, and tipping the scale in favor of either one can become detrimental to the cell (27). Mitochondrial fusion is regulated by Mfn1 and Mfn2 on the outer mitochondrial membrane and Opa1 on the inner mitochondrial membrane. Regulators of mitochondrial fission include Drp1, FIS1, Mff, and MiD49/51. Drp1 translocates from the cytosol to mitochondrial fission sites with the help of other fission proteins (28). There are also special mitochondria-endoplasmic reticulum contact sites, termed mitochondria-associated membranes. These are involved in mitochondrial fission and the regulation of mitochondrial calcium balance and oxidative stress (29). Mfn2 and FUNDC1 are present at these contact sites and are involved in the regulation of the Ca2+ homeostasis of mitochondria and endoplasmic reticulum. Inhibition of Mfn2 decreases significant mitochondrial calcium overload and oxidative stress and improves both cardiac systolic and diastolic dysfunction under hyperglycemic conditions. FUNDC1 on these contact sites also has an important function, and its deletion causes impaired mitochondrial fission with larger mitochondria dominating in the cell. Although this could be perceived as a protective mechanism for the heart, loss of FUNDC1 results in mitochondria with decreased membrane potential, leading to diastolic and systolic dysfunction with fibrosis (30).Mitochondria are dynamic organelles that frequently change shape, size, and quantity. Damaged mitochondria lose their membrane potential, which is the driving force for ATP production, and this occurs even more rapidly in diabetic hearts (31). However, downregulation of Drp1 in cardiomyocytes below baseline leads to dysfunctional mitochondria with decreased mitochondrial membrane potential, increased opening of the mitochondrial permeability transition pore, and compromised ATP production (32). In addition, increased mitochondrial fission during hyperglycemia protects mitochondrial quality since inhibiting fission increases cell death, whereas Drp1 overexpression prevents high glucose-induced cardiotoxicity (33). These studies all suggest that the delicate balance of fusion and fission relative to one another is an essential part of maintaining mitochondrial quality in diabetic hearts (34).The role of mitochondrial fusion and fission in the heart appears complex and context dependent. For example, inhibition of Drp1 with Mdvi-1 increases fused mitochondria and improves cardiac function during doxorubicin treatment (35). Autophagy occurs via macroautophagy, microautophagy, and chaperone-mediated autophagy. In macroautophagy (hereafter autophagy), cellular constituents are delivered to lysosomes in double-membraned autophagic vesicles that are formed via the well-coordinated actions of autophagy-related (Atg) genes. In contrast, in microautophagy, lysosomes directly engulf dysfunctional and damaged organelles, and chaperone-mediated autophagy involves lysosomal sequestration of cellular constituents with the help of chaperones that tag specific KFERQ sequences of target proteins (36). General autophagy occurs via multiple steps. AMPK, the energy sensor of the cell, detects decreased energy/ATP levels in the cell and initiates autophagy by phosphorylating Ulk1. Autophagy then begins with the formation of a layer of membrane from the endoplasmic reticulum membrane and continues via the formation of multiple complexes that activate one another, including Beclin1/Vps34/Vps15, involved in nucleation, and LC3-Atg12/5, involved in elongation. Finally, the double-membraned autophagic vesicle maturates and closes with the incorporation of LC3-II. Autophagosomes then fuse with lysosomes for final degradation of the engulfed cellular components (37).The term autophagy refers to the mechanisms by which a cell “eats itself,” degrading cellular materials to maintain cellular homeostasis (12). Autophagy either can occur non-selectively or can target particular organelles, including mitochondria and peroxisomes. When autophagy selectively targets mitochondria, it is called mitophagy (38). Autophagy is activated or deactivated in accordance with the type of stress (39), and the direction of the change in autophagy can have different consequences in different pathologies, ranging from cellular protection to cellular death (40). At the time of an initial insult to the organism, as in consumption of a high-fat diet (HFD) by mice, autophagic flux generally increases (41). However, the activation of autophagy is often transient, and the subsequent inactivation may prevent the organism from being able to cope with the pathologic stress, thereby leading to the development or deterioration of the disease process (42).The high levels of autophagy seen during the first few hours of life, when nutrient availability is relatively low, demonstrate a highly adaptive aspect of this mechanism (45). One of the most well-studied mechanisms of mitophagy occurs via the PINK1-Parkin pathway. Mutations in PINK1 and Parkin are involved in the pathogenesis of early-onset Parkinson’s disease (47). PINK1 is a serine-threonine kinase that exists in two forms and resides in two subcellular compartments. Mitochondrial PINK1 is the longer form, accumulating specifically on the membrane of damaged and depolarized mitochondria. Presenilin-associated rhomboid-like protein, a protease, cleaves the long form of PINK1, thereby turning it into the shorter cytosolic form and preventing its accumulation on healthy, functioning mitochondria with a high (polarized) membrane potential (48). PINK1 stabilization and phosphorylation of the ubiquitin ligase, Parkin, allow the activation of Parkin at the outer mitochondrial membrane. Although PINK1 accumulation triggers the acceleration of Parkin recruitment to mitochondria, a previous study found that the actions of PINK1 may not be essential, and that mitophagy may ensue in the presence of Parkin alone (49). After Parkin is recruited to mitochondria, it tags damaged mitochondrial proteins via ubiquitination. Ubiquitinated proteins are then bound by adapter proteins possessing both a ubiquitin binding domain and LC3 interacting regions (LIRs), including p62, optineurin, and NDP52. After separation of the damaged portion from the healthy area through either Drp1-dependent or -independent mechanisms, autophagosomes carrying mitochondria merge with lysosomes for degradation. In another form of mitophagy, proteins harboring LIRs, including FUNDC1, BNIP3, and NIX/BNIP3L, expressed on the damaged mitochondria are recognized by LC3 (15). Growing lines of evidence suggest that degradation of damaged mitochondria is also mediated through a mechanism, termed alternative mitophagy, that is independent of the conventional machinery, including Atgs and LC3. Although the functional significance of the alternative mechanism of mitophagy remains unclear, it appears to act as a compensatory mechanism when conventional mechanisms of mitophagy are downregulated, as in chronically pathologic conditions in vivo.Mitophagy is a mitochondria-specific form of autophagy that selectively degrades dysfunctional/damaged or unnecessary mitochondria to prevent their harmful effects or accumulation in the cell (50).Degraded mitochondria are replaced with new mitochondria through mitochondrial biogenesis. Mitophagy and mitochondrial biogenesis coordinately regulate mitochondrial content to maintain energy metabolism. For example, Parkin-induced degradation of Paris may stimulate mitochondrial biogenesis by alleviating Paris-induced suppression of PGC-1α, a transcription coactivator and the master regulator of mitochondrial biogenesis (22). During the early phase of HFD consumption, the majority of autophagosomes in the heart are colocalized with mitochondria, and mitophagy is activated through a Parkin-dependent mechanism. This activation of mitophagy is attenuated in cardiac-specific Atg7 knockout (Atg7 cKO) mice, which are autophagy-deficient, suggesting that mitophagy during the early phase of HFD consumption utilizes the mechanism commonly used in general autophagy (22). Atg7 cKO mice exhibit more severe mitochondrial dysfunction and cardiomyopathy than wild type mice, suggesting that the heart activates autophagy to stimulate mitophagy, which plays an important role in eliminating damaged mitochondria and maintaining cardiac function during the acute phase of HFD consumption. In another study, the development of cardiomyopathy after 4 months of HFD consumption was similarly attenuated in PINK1 knockout mice (51). It should be noted that, although the mouse heart exhibits signs of diastolic dysfunction 1 month after HFD consumption, other morphological features of cardiomyopathy, including hypertrophy, steatosis, inflammation, and fibrosis, are not obvious until after 3 months of HFD consumption. This activation of mitophagy during the acute phase of HFD consumption is an important defense mechanism against cardiac remodeling and cardiac dysfunction; fully established features of cardiomyopathy are manifested during the acute phase of HFD consumption only when the activation of mitophagy is inhibited (22). This suggests that early intervention to maintain mitochondrial quality is essential to delaying the progression of cardiomyopathy.Although the activation of autophagy and mitophagy has been demonstrated in diabetic hearts, the extent of mitophagy activation and the underlying molecular mechanisms appear to be time dependent. In a mouse model of obesity-induced cardiomyopathy, autophagy is activated in the heart in response to HFD  consumption, peaking at 6 weeks  (22). Du(22). Inactivation of autophagy is commonly observed in many experimental models of diabetic cardiomyopathy. Although the mechanisms mediating the rapid inactivation of autophagy remain unclear, it has been shown that signaling mechanisms inhibiting autophagy, including one mediated by the serine-threonine kinase, Mst1, are activated (36). Further investigation is needed to elucidate the specific mechanisms by which autophagy is inactivated during the chronic phase of diabetic cardiomyopathy. Targeting these inhibitory mechanisms may also delay the progression of diabetic cardiomyopathy.Despite its critical importance, the activation of general autophagy and mitophagy is transient, and the level of autophagy and mitophagy declines to baseline by 20 weeks of HFD consumption alternative mitophagy, continues to be observed even after general autophagy is inactivated (52). Since systolic cardiac dysfunction is not yet observed at this time point, alternative mitophagy presumably acts as a compensatory mechanism. Delayed activation of alternative mitophagy after inactivation of conventional mitophagy is also observed in the heart during acute pressure overload, suggesting that alternative mitophagy acts as a backup mechanism to delay the development of mitochondrial dysfunction in response to stress. Mitophagy activated during the chronic phase of HFD consumption is mediated by an Ulk1- and Rab9-dependent but Atg7- and Parkin-independent mechanism, consistent with the aforementioned alternative mitophagy. Suppression of alternative mitophagy in Rab9 S179A knock-in or cardiac-specific Ulk1 knockout mice exacerbated the development of mitochondrial dysfunction and cardiomyopathy (52). Thus, regardless of the underlying mechanisms involved, mitophagy activation appears critical for the maintenance of mitochondrial function in both the acute and chronic phases of obesity and diabetic cardiomyopathy. In fact, TAT-Beclin1 (TB1), a cell-permeable short peptide that potently induces autophagy and mitophagy by mobilizing endogenous Beclin1 from its intracellular storage site, can reactivate or enhance mitophagy under a wide variety of conditions, thereby alleviating obesity-induced cardiomyopathy (22). Compared to the wealth of knowledge about the molecular mechanism of the conventional form of mitophagy, such as the involvement of PINK1 and Parkin, the molecular mechanism of alternative mitophagy remains poorly understood. Alternative mitophagy is often observed after conventional mitophagy is activated. The accumulation of dysfunctional mitochondria as a result of inactivation of conventional mitophagy alerts the cell by stimulating signaling mechanisms, including TFE3, which in turn upregulates transcriptional mechanisms (52). Further investigation of the mechanisms modulating the transcriptome that orchestrates the initiation of alternative mitophagy may provide useful information for improving mitochondrial quality control during the chronic phase of diabetic cardiomyopathy.Interestingly, although general autophagy is no longer active after 20 weeks of HFD consumption, mitophagy, primarily mediated through (53). Following diagnosis of diabetic cardiomyopathy, cardiac changes are rapidly progressive, and there is a high risk of progression to heart failure. Since mitochondrial dysfunction is the most common driver of diabetic cardiomyopathy, excess oxidative stress, lipotoxicity, mitochondrial dynamics, mitophagy, and mitochondrial calcium homeostasis all constitute potential therapeutic targets. A similar concept has been proposed for the treatment of neurodegenerative disease, where improving mitochondrial quality control mechanisms alleviates the disease progression (54). Some studies have used mitochondria-targeting agents to decrease the production of ROS and improve impaired mitochondrial function, which ultimately prevents undesirable cardiac outcomes even in other cardiac pathologies. Cardiac structural and functional changes and even vascular damage can be prevented by administration of the mitochondria-targeting antioxidant, MitoQ (56). Another study demonstrated that treatment with MnSOD, an antioxidant, preserved mitochondria and cardiac structure in diabetic OVE26 mice, which are associated with diminished levels of MnSOD (57). However, despite the promising results of antioxidant therapy in animal studies, its use has not been proven to be beneficial in humans (58). Manipulation of mitophagy represents another possible approach to prevent mitochondrial dysfunction. Several agents target the different steps of mitophagy. Intraperitoneal injection of TB1 into mice increases LC3-II primarily in the mitochondrial fraction and reverses functional changes associated with diabetic cardiomyopathy. TB1 mobilizes endogenous Beclin1 from its intracellular anchorage site and stimulates mitophagy, the level of which is insufficient for the maintenance of mitochondrial function during the chronic phase of diabetic cardiomyopathy (22). High expression of the transcriptional and epigenetic regulator, Bromodomain-containing protein 4 (BRD4), during HFD consumption can be inhibited by JQ1, a chemical inhibitor of BRD4, resulting in increased PINK1-Parkin-dependent mitophagy and alleviation of the structural and functional changes associated with diabetic cardiomyopathy (51). Melatonin, a natural regulator of the sleep-wake cycle (59), accumulates in mitochondria and increases PINK1-Parkin-mediated mitophagy in diabetic hearts (60). Metformin also upregulates autophagy through its effects on AMPK and Beclin1 and preserves cardiac function in diabetic cardiomyopathy (61). Mst1, a serine-threonine kinase and a component of the Hippo pathway, inhibits Parkin-mediated mitophagy and causes the accumulation of dysfunctional mitochondria. Thus, a small molecule inhibitor of Mst1 may rescue depressed autophagy and improve both diastolic and systolic functions (63). SGLT-2 inhibitors, highly effective antidiabetic agents, prevent cardiovascular complications of diabetes mellitus type 2 (64). The dual inhibitor sotagliflozin and SGLT-2 inhibitor empagliflozin decrease cardiac remodeling (66). Empagliflozin improves diabetes-related microvascular injury through inhibition of mitochondrial fragmentation in cardiac microvascular endothelial cells, which results in decreased oxidative stress. It leads to improved cardiac nutrient and oxygen supply and increased resistance of cardiac microvascular cells to hyperglycemic insult (66). Of the many beneficial effects of SGLT-2 inhibitors, their effect on mitochondria appears particularly important. SGLT-2 inhibitors generally suppress mitochondrial fission by regulating the relative amounts of mitochondrial fusion-fission proteins, although some may exert their effects through stimulation of AMPK, with the same outcome (67). Ipragliflozin increases the level of mitochondrial fusion proteins and protects mitochondrial shape and structure in kidney proximal tubular epithelial cells (68). H2S, an endogenous gasotransmitter, decreases mitochondrial fragmentation by regulating fusion-fission proteins, but it can also improve mitophagy during hyperglycemia and hyperlipidemia (69). Small molecules identified through screening for the ability to induce mitochondrial fragmentation and autophagosome formation ameliorate proteostasis defects associated with neurodegenerative diseases in Caenorhabditis elegans in a Pink1-dependent manner (70). Thus, similar approaches may allow identification of modalities that would effectively protect mitochondria in the presence of diabetic cardiomyopathy.Currently, there is no effective treatment regimen to reverse or control the progression of diabetic cardiomyopathy. Strict glycemic control with prevention of other related complications is the most accepted initial approach. Nutrition is a cornerstone for successful management of the disease. Ketone ester supplementation has been shown to improve cardiac function in diabetic mice Diabetic cardiomyopathy is insidiously progressive. Unfortunately, there is currently no specific treatment to reverse or slow the pathogenesis of diabetic cardiomyopathy. As discussed in this review, however, inducing or enhancing the mitochondrial quality control mechanisms represents a reasonable approach to treat and potentially reverse the progression of diabetic cardiomyopathy. Nevertheless, many questions remain. First, how mitochondrial quality is affected and how autophagy/mitophagy is regulated at various stages of diabetic cardiomyopathy need to be clarified in the human heart. It would be useful to identify convenient biomarkers. Second, the endogenous mechanism by which general autophagy is inactivated after transient activation during the acute phase of cardiomyopathy needs to be clarified. This would provide hints to prolonging the endogenous mechanism of autophagy into the chronic phase of diabetic cardiomyopathy. Third, since alternative mitophagy is the predominant form of mitophagy during the chronic phase of diabetes, it can be a primary target in patients with chronic diabetes for longer term. Thus, the molecular mechanisms of alternative mitophagy, namely, how damaged mitochondria are identified and how autophagosomes are formed, need to be clarified. The endogenous (upstream) signaling mechanisms that stimulate alternative mitophagy also require further clarification. Finally, although induction of autophagy and mitophagy is generally protective, the optimal timing and extent of autophagy and mitophagy induction need to be explored carefully since inappropriate timing and/or intensity could exacerbate cell death and heart failure. By addressing these questions, it may be possible to identify a specific intervention to best preserve mitochondria in patients with diabetes and delay the development of diabetic cardiomyopathy.NoneThis work was supported in part by the U.S. Public Health Service grant numbers HL67724, HL91469, HL102738, HL112330, HL138720, HL144626, HL150881, and AG23039 (J.S.), the American Heart Association Merit Award 20 Merit 35120374 (J.S.), and the Fondation Leducq Transatlantic Network of Excellence 15CVD04 (J.S.).M.K., D.Z., and J.S. wrote the manuscript. The authors reviewed and approved the manuscript for publication.Junichi Sadoshima is one of the Advisory Board members of JMA Journal and on the journal’s Editorial Staff. He was not involved in the editorial evaluation or decision to accept this article for publication at all.


Noncanonical functions of the autophagy machinery in pathways including LC3-associated phagocytosis and LC3-associated endocytosis have garnered increasing interest in both normal physiology and pathobiology. New discoveries over the past decade of noncanonical uses of the autophagy machinery in these distinct molecular mechanisms have led to robust investigation into the roles of single-membrane LC3 lipidation. Noncanonical autophagy pathways have now been implicated in the regulation of multiple processes ranging from debris clearance, cellular signaling, and immune regulation and inflammation. Accumulating evidence is demonstrating roles in a variety of disease states including host-pathogen responses, autoimmunity, cancer, and neurological and neurodegenerative pathologies. Here, we broadly summarize the differences in the mechanistic regulation between autophagy and LAP and LANDO and highlight some of the key roles of LAP and LANDO in innate immune function, inflammation, and disease pathology. LAP and LANDO diverge from autophagy and regulate inflammation. Macroautophagy, autophagy or canonical autophagy henceforth, is an evolutionarily conserved process for either the nonspecific engulfment of cytoplasmic material or specific targeted engulfment of damaged organelles as well as misfolded or aggregated proteins into double-membrane autophagosomes  (16–21).In addition to differences in the autophagy machinery as will be discussed, the source of cargo is a major defining factor delineating autophagy from LAP and LANDO. As mentioned, autophagic cargo is derived from intracellular sources, whether that be intracellular debris, damaged organelles, or intracellular pathogens . In starThe physiological roles for the conjugation of LC3 to single membranes have best been characterized in the context of LAP. As will be expounded upon herein, LAP is activated in response to phagocytic cargoes including dead cells following recognition by efferocytic receptors such as T cell immunoglobulin and mucin domain containing 4 (TIM4) in macrophages  complex 1 (mTORC1) and its inhibition, which results in the recruitment and activation of the autophagy machinery (+ vesicles to lysosomes (+ autophagosome including PI(3)P.Following LC3 decoration and autophagosomal sealing, LC3 interacts with microtubules via a variety of motor and motor adaptor proteins including PLEKHM1, PLEKHM2/SKIP, FYCO1, and JIP1 and kinesins and dyneins, leading to trafficking of the sealed autophagosome to lysosomes for fusion P phosphatase that dephosphorylates PI(3)P to PIP is sufficient in abrogating the maturation of the phagosome without altering phagosome formation P. During LAP and LANDO, VPS34 produces PI(3)P in the outer leaflet of the phagophore/endosome following cargo encapsulation and vesicle sealing (Membrane PI(3)P can serve as a platform for the recruitment of a variety of effector proteins, many of which use PX and FYVE domains to interact with PI(3)P. The main PI(3)P effector proteins found in the autophagy pathway are the WIPI family of proteins, which alternatively bind PI(3)P using the WD β-propeller PROPIN lipid binding domain   of Rubicon alter its role in regulating LAP activation and may possibly aid in delineating LAP from LANDO. The intracellular bacteria In addition to acetylation, Rubicon has multiple putative phosphorylation sites including serine-44 and serine-92 in the N-terminal region, which are phosphorylated by hormonally up-regulated Neu-associated kinase (HUNK) . Given tTo date, the best characterized functions of LAP and LANDO in governing innate immune function are in the context of inflammatory signaling and, to a lesser extent, in the processing and presentation of pathogenic antigen. LAP and LANDO modulate anti- and proinflammatory cytokine production in response to immunological stimuli including dead cells, a process termed efferocytosis, extracellular pathogens including bacteria and fungi, as well as aberrant extracellular protein aggregates including the AD peptide Aβ. As expected, this diverse set of stimuli known to engage either LAP or LANDO stems both from physiological scenarios, such as the clearance of dead cells following routine turnover, and under pathological settings, including neurodegenerative diseases and cancer.The phagocytic clearance of apoptotic corpses, a process termed efferocytosis, is of paramount importance to maintaining tissue homeostasis . BindingLike the contribution of LAP in suppressing autoimmunity, LAP functions within tumor-associated macrophages (TAMs) to facilitate dead cell clearance and mitigate inflammation and is, however, beneficial for the tumor microenvironment. LAP deficiency in TAMs promotes M1-like proinflammatory polarization and compromises tumor growth  such as TLRs is known to activate LAP  , which are responsible for elimination of microorganisms from the cytosol  (Streptococcus pneumoniae infection that engages LAP (S. pneumoniae model and may contribute to age-related susceptibility (Over the past 30 years since the characterization of the autophagy machinery by Ohsumi and colleagues (Looking forward, the establishment of new experimental techniques including single-cell sequencing and spatial transcriptomics will be invaluable for decrypting the intricate control and cross-talk between canonical autophagy and noncanonical functions of the autophagy machinery in pathways such as LAP and LANDO, the impact of aging on the regulation of these pathways, and their important and often obligatory roles in organismal biology.