Reports of its successful implementation were numerous and convincing, especially for infectious diseases, but the therapeutic mechanism of such an intervention was unclear, with hypotheses ranging from modulation of immune responses to purely psychosomatic effects  revived the definition of autotherapy, applying it specifically to those approaches that stimulate inherent tissue responses, manipulate stem cell niches, and modulate endogenous tissue microenvironments to enhance tissue healing and regeneration . As it relates to tissue engineering and regenerative medicine, this could be achieved by the transient modulatory effects of cell therapy, administration of stem-cell derived factors such as extracellular vesicles (EVs), secreted ligands or extracellular matrices (ECMs), or from synthetic small molecules, genetic engineering tools, biomaterials or scaffolds. The entire field of autotherapy is extremely broad, but in this collection, we focus on approaches that foster a pro-regenerative environment through stimulation of stem cells and/or modulation of the immune system.Modern autotherapeutic concepts can be crudely divided into i) strategies that modulate the stem cell niche  ii) those that foster a pro-regenerative environment  and (iii) direct lineage reprogramming of stem cells or trans-differentiation of differentiated cells  stressing the importance of local control in the propagation of a pro-regenerative environment. In particular, Dr Lumelsky emphasizes the contribution of the stem cell niche in this process, stressing that its individual components, stem cells, stromal cells, extracellular matrices and soluble mediators play distinct but equally important roles in maintaining regenerative homeostasis. In addition, the contribution of the immune system is discussed, with emphasis on the need for precise modulation of inflammatory and post-inflammatory processes to support tissue regeneration rather than fibrosis. Within this context, the potential of synthetic biomaterials to control cell adhesion characteristics, biomechanical forces, and soluble factor delivery is discussed. In keeping with the emphasis on biomaterials and their potential for immune modulation, Karkanitza et al. reviews our current understanding of immune reactions to biomaterials, and considers how this knowledge might be employed to drive regeneration and reduce the probability of adverse reactions.We begin with Dr Lumelsky’s opinion article  during bone formation. Using canine MSCs in mouse recipients, they describe for the first time that MSCs and sub-therapeutic levels of bone morphogenic proteins synergize to drive bone repair. In these studies, the implanted MSCs were essential for rapid repair, but did not persist at the site of injury suggesting that MSCs, when triggered with very low levels of BMP, stimulate the host’s inherent bone repair mechanisms rather than directly differentiate into osteoblasts. This study represents an excellent example of cell-mediated autotherapy and contributes to the rapidly growing number of studies supporting autotherapy as the predominant mechanism of MSC-mediated healing. Han et al. offer an alternative approach for therapeutic enhancement of adipose derived MSCs through ex vivo exposure to decellularized adipose tissue ECM. In this work, the authors demonstrate that culturing of MSCs on decellularized adipose tissue ECM enhanced secretion of regenerative and immune-modulatory factors. This process could, in the future, improve the autotherapeutic potential of adult stem cells.One of the major challenges in the translation of experimental therapies to clinical implementation is the selection of appropriate animal models for preclinical testing. In most cases, and especially for musculoskeletal applications, a large animal model is required, mimicking as closely as possible, the load and weight bearing characteristics of the human body. Although largely overlooked, the canine system is an excellent model for skeletal regeneration, with similar anatomy to humans, well-developed surgical techniques, and the capacity to perform gait analysis. Liebman et al. present an intriguing strategy for the stabilization of labile biological ligands hepatocyte growth factor and fibroblast growth factor 2 by attachment to immunoglobulins. In a porcine model of myocardial ischemia and perfusion, the immunoglobulin-sequestered factors were stable and promoted repair of cardiac tissue. Sankar et al. describe the broad autotherapeutic applications of RAD16, a synthetic peptide with hemostatic properties that also serves as an ECM analog for cell attachment and migration. To address the issue of radiation-induced salivary gland damage during treatment of head and neck cancer, Gilman et al. employed the non-steroidal anti-inflammatory drug indomethacin to inhibit prostaglandin E2-mediated inflammatory processes and promote regeneration of the gland. In a related study, Nam et al. employ hydrogels to deliver Laminin-1 peptides for enhanced autotherapy of salivary glands through stimulation of the salivary stem cell niche and by modulation of macrophages to adopt a pro-regenerative phenotype. Xu et al. demonstrate that simple, naturally occurring substances can be employed as autotherapeutic tools. In their study, a simple preparation of platelet rich plasma stimulated regenerative characteristics of human dental pulp cells, providing some credence to the reports of autotherapy in the early 1900’s.While stem cells have the potential to deliver multiple bioactive factors with the means to adapt to the dynamic healing environment, some autotherapeutic effects can be achieved by simple and direct administration of defined factors. Fetz et al. and Hymel et al. represent excellent examples of the utilization of synthetic materials to regulate local release of therapeutic factors into the regenerative microenvironment. Fetz et al. employed electrospun polydioxanone to slowly release chloroquine which in turn caused neutrophils to downregulate inflammatory factors and adopt a more resolving, putatively regenerative phenotype. Using a similar approach, Hymel et al. utilized nanofibers to release bioactive lipid into the myogenic microenvironment to enhance myofiber growth and promote regenerative immunological processes.We hope this diverse collection of studies showcases many aspects of the autotherapy concept and recent efforts in developing innovative approaches to enhance our inherent capacity for tissue healing and regeneration.


Effective cancer therapeutics for brain tumors must be able to cross the blood-brain barrier (BBB) to reach the tumor in adequate quantities and overcome the resistance conferred by the local tumor microenvironment. Clinically approved chemotherapeutic agents have been investigated for brain neoplasms, but despite their effectiveness in peripheral cancers, failed to show therapeutic success in brain tumors. This is largely due to their poor bioavailability and specificity towards brain tumors. A targeted delivery system might improve the efficacy of the candidate compounds by increasing the retention time in the tumor tissue, and minimizing the numerous side effects associated with the non-specific distribution of the chemotherapy agent. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence (NIRF) compounds that have recently emerged as promising agents for drug delivery. Initially explored for their use in imaging and monitoring neoplasms, their tumor-targeting properties have recently been investigated for their use as drug carrier systems. This review will explore the recent developments in the tumour-targeting properties of a specific group of NIRF cyanine dyes and the preclinical evidence for their potential as drug-delivery systems in the treatment of primary and metastatic brain tumors. Keywords used to search for the literature used in this review pertaining to the field of heptamethine cyanine dyes.Heptamethine cyanine dyes, MHI-148, IR-783, IR-780, DZ-1, IR-786, drug-dye conjugate, heptamethine carbocyanine dyes, *near infrared dye, cyanine conjugate.Truncations were used in the terms as shown to ensure thorough capture of research pertaining to the field. Literature pertaining to the field of HMCDs in the use of photodynamic therapy were generally excluded, unless the dyes were conjugated to a small molecule inhibitor, as the application of HMCDs in photodynamic therapy has been thoroughly reviewed.The term ‘brain tumors’ refers to a mixed group of neoplasms growing inside the central nervous system (CNS) . PrimaryIn adults, the global age-standardized rate of primary brain tumors is approximately 6.8 per 100,000, with the highest rates seen in Europe . Of the The only established treatment for high-grade astrocytomas consists of maximal surgical resection of the tumor, followed by concurrent radiation and chemotherapy with temozolomide (TMZ), prolonging the median survival by only four months . SeveralIn children, brain tumors represent the second most common cancer, comprising approximately 24% of all pediatric malignancies . MedulloMetastatic tumors are the most frequent type of intracranial tumor in adults, with reported incidence rates between 2-14 persons per 100,000 population . ConsequChemotherapeutic treatments for primary and metastatic brain tumors are limited by the reduced BBB penetration of the existing anticancer agents . The BBBThe BBB is comprised of a functionally complex, tightly regulated neurovascular unit that includes endothelial cells, pericytes and astrocytic end feet . TogetheA number of strategies have been investigated to overcome the limitations of the BBB in the treatment of brain cancers: the first is to bypass the BBB, the second is to increase its permeability, and the third is to use carrier molecules that are permeable to the BBB –22.Intraventricular infusion and intracerebral implants are the main strategies used to bypass the BBB , 23–25. Tumor Treating Fields (TTFields), developed by Novocure, is a non-invasive, loco-regional anti-mitotic treatment which uses alternating electric fields in the intermediate frequency range (200 kHz) to disrupt cancer cell division with minimal systemic toxicity . TTFieldTo overcome the limitations of the BBB, several approaches to physically and chemically disrupt the barrier have been developed. The main strategies include osmotic disruption through intracarotid infusions of mannitol, modification of tight junctions by the administration of bradykinin analogues, and focused ultrasound techniques , 24, 25.Nanoparticles have been explored to protect drug entities in the systemic circulation and facilitate the uniform delivery of drugs across previously impenetrable barriers . SeveralTraditional and modern chemotherapy and radiotherapy can have undesirable effects on the CNS, which can result in the discontinuation of the treatment or dose-adjustments that prevent the achievement of a desirable therapeutic outcome [reviewed by ]. TraditRadiation necrosis is a delayed complication of radiation therapy for brain tumors and a dose-limiting factor for stereotactic radiotherapy, a standard treatment for brain metastases , 43. GenAcute and chronic complications of the CNS following chemotherapy treatment are common. Acute complications include headaches, seizures and acute encephalopathy, which can have detrimental effects on patient outcome, limiting the duration of treatment . ChronicExisting research recognizes the preferential tumor uptake of a class of NIRF HMCDs. This has been demonstrated in a variety of cancer cell lines, tumor xenografts, spontaneous mouse tumors in transgenic animals and human tumor samples , second, they possess an alkyl chain that attaches to the terminal tetramethyl indoline ring (orange), and they contain an ionizable functional group, such as carboxylic or sulfonic acid (purple) Figure 1Ex vivo NIRF imaging demonstrated that fluorescence signal intensities were six-fold higher in the tumor when compared to the normal surrounding kidney tissues. These studies highlight the potential clinical translation of these NIRF dyes as tumor-targeting anti-neoplastic agents.Recently, a group of HMCDs that preferentially target and accumulate in tumors have been described   , 87. Altin vitro studies support an important role of Src and PDGFR in human glioblastoma. However, in vitro and in vivo studies have revealed that dasatinib is a substrate for efflux transporters that are expressed on the BBB , which promotes OATP expression . OATPs ae manner . OATPs he manner .in vitro paradigm that compared the fluorescent intensity of cells in serum-free culture media. Typically, cells treated with a pan-OATP inhibitor demonstrated a reduction in fluorescent intensity, implying that the uptake of the NIRF agents were, in part, via the OATPs. In contrast, cells treated with hypoxia-mimetic agents, such as dimethyloxalylglycine and cobalt chloride, stabilized HIF1α and increased the expression of OATPs, thus resulting in an increase in the fluorescent intensity. Several groups have reported that upon uptake, HMCDs are localized to the mitochondria of tumor-cells  in comparison to the pKa of other low molecular weight aminothiols present in the plasma   . This mend thiol . Presentnd thiol . Drug innd thiol ]. Hence,34 directly, or possibly associate with one of the binding sites before being relayed to the free thiol. There is evidence for the relay of non-covalently bound HMCDs to a covalent bond   with C1  . Using ir organs . Moreove24 hours . This suC3  is a mitochondria-bound enzyme that degrades monoamine neurotransmitters and dietary monoamines . MAOA inC3 . Evidenthe tumor . The samma cells . They fuma cells . Synergima cells .C4)  inhibitors have shown promise in the treatment of many cancers, with several clinical trials underway to expand their indications . PARP inhibitors (PARPi) can sensitize glioblastoma cells to radiation, making them a useful adjuvant therapy to use with alkylating agents such as TMZ , which iC4) . We demoIncreasing the BBB penetration and specificity of traditional chemotherapy agents are essential in achieving a desirable outcome for patients and reducing neurotoxicity. Recent advances in oncology research have identified aberrant signaling pathways in tumors, which has led to the development of successful TKIs in the treatment of various cancers . DespiteTKIs are recognized as powerful candidates for the treatment of GBM, as aberrant tyrosine kinase signaling is central to the pathogenesis. There are two main tyrosine kinase signaling pathways implicated in the tumorigenesis of GBM; the Ras/MEK/ERK pathway, and the PI3K/AKT/mTOR pathways – both of which are being investigated as potential therapeutic targets . These cC5). The authors highlight a modest 4.7-fold improvement in cell viabilities, despite a 15 and 30-fold reduction in the IC50 for Src and Lyn kinases, respectively. Moreover, they demonstrate tumor-specific accumulation of the conjugate for up to 72 hours, with less signals detected in other organs such as the liver and the lungs   . This deree-fold . At the ree-fold . Given tg cancer . OverallIn vivo and in vitro investigations have contributed substantially to our understanding of tumor biology and pathogenesis, providing clear indications for the repurposing of existing anticancer agents. However, the scarcity in recent clinical trials’ success has demonstrated that expanding the indications of existing small molecule anticancer agents for brain tumors and metastases can be challenging. As detailed in this review, the BBB remains a fundamental obstacle for delivering anticancer agents into the brain. Besides, achieving a high degree of tumor specificity and retention, without inflicting additional neurological injury, remains a formidable challenge in obtaining therapeutic effectiveness. For this, HMCDs may provide a unique platform to facilitate the targeted delivery of anticancer agents that have been previously excluded from the brain.Despite the abundance of preclinical trials conducted to identify novel effective therapies for brain tumors and metastases, translation into actual clinical benefits have been limited. HMCDs are an advantageous candidate for development as a drug-delivery system by virtue of its favourible tumor-specificity and biocompatibility. The primary thing to consider when designing HMCD-drug conjugates is the biological innertness of the carrier system. Literature suggests that the N-alkyl chain length alters the uptake and photophysical properties of HMCDs, but the understanding of the pharmacodynamic and toxicity profiles of different HMCD analogues is lacking . A thoroAs discussed, the mechanisms of uptake of HMCDs across the BBB and into the tumor cells are unclear. NIRF studies of the biodistribution of HMCDs provides compelling evidence for the tumor-specific accumulation of these dyes and drug conjugates, yet the understanding of the biology of this statement is lacking. The reliance of OATPs for the uptake of HMCDs is generally characterised through pharmacological inhibition of these transporters with a non-specific OATP inhibitor, bromosulfophthalein. However, a more detailed understanding of the expression profile and reliance of these transporters is required. OATPs are ubiquitously expressed throughout the body, hence the biodistribution of HMCDs is an important factor to consider in the development of these compounds.Particular emphasis should also be laid on the stability of HMCD-drug conjugates. Several groups report an increase in the potency of HMCD-drug conjugates in comparison to the drug alone; however, the mechanism of this is unclear , 71, 76.in vitro, and mouse xenograft models are typically used to establish the biodistribution of the HMCD-conjugates in different organs. There is a lack of literature assessing the pharmacodynamic profiles of HMCDs and HMCD-drug conjugates. The in vitro accumulation and increased potency of HMCD-conjugates, coupled with the tumor-specific biodistribution is compelling, but few groups have translated this to an increase in overall- or progression-free survival in vivo. A thorough analysis of the survival benefits of HMCD-drug conjugates relative to the drug alone in an appropriate in vivo model is necessary for the clinical translation of this technology.Other limitations exist in the methodology commonly used to evaluate the activity of HMCD in research. In preclinical studies, the efficacy of HMCD-drug conjugates is generally characterised Indeed, a deeper insight into deciphering the biological mechanisms of tumor cell uptake and retention of HMCDs are required to enable rational design for their use as drug-carriers. Concomitantly, animal models need to be used more extensively to evaluate their pharmacokinetics and survival benefits. Ultimately, HMCDs provide a novel strategy to reinvigorate existing chemotherapies by exploiting tumor-intrinsic properties to deliver chemotherapies effectively to brain tumors and minimize toxicity.EC: Researched and wrote the entire manuscript. PC: Aided in the writing of the manuscript, especially the medicinal chemistry sections. WD: Supervised the medicinal chemistry sections of the manuscript. JJ: Conceived and supervised the writing of the medicinal chemistry sections of the manuscript. MD: Supervised the biomedical sections of the manuscript. TP: Conceived, wrote, and supervised the entire manuscript. All authors contributed to the article and approved the submitted version.Neurological Foundation of New Zealand; Douglas Charitable Trust; Auckland Cancer Society Research Centre; Hugh Green Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microplastics (MPs) are ubiquitous in environmental media, and their harmful effects on MPs on the ecosystem have attracted more and more attention. Once released into the environment, MPs can trigger oxidative degradation through ultraviolet (UV) to cause photoaging. Photoaging significantly affects the properties of MPs, which leads to changing their environmental behaviors and increasing environmental risks. In this review, the generation of MPs under UV irradiation and the influence of environmental factors on the photoaging of MPs were discussed. Photoaging of MPs is an important process affecting the migration, transformation and interaction of pollutants in water and soil. In order to fully predict the fate and environmental interaction of MPs, more researches are needed in the future to explore the photoaging behavior of different types of MPs under natural environmental conditions. As one of the most important materials in the 21st century, plastics have an impact that cannot be underestimated on our society and daily life . Since tAccording to statistics, the most commonly used plastics are polystyrene (PS), poly (PET), polyurethane (PUR), poly(vinyl chloride) (PVC), polyethylene (PE) and polypropylene (PP), which accounted for about 81% of European plastic demand in 2016 ,13,14,15With the investigation of the distribution of MPs in the environment and the exploration of their biological toxicity, the pollution of MPs in environmental media has become one of the issues that researchers are highly concerned about. The distribution of MPs has spread to every corner of the earth; humans have found traces of it in the coastal estuaries, oceans, equatorial waters and north and south poles ,26,27,283 and that in estuary sediments is 20–7900 pieces/kg [Statistics showed that the abundance of MPs in the coastal waters of China is 0.13–545 pieces/mieces/kg . In the ieces/kg . At the ieces/kg ,30.MPs are considered potential sources of chemical contaminants due to the release of intermediates during the aging process of MPs. Plastics contain various chemical additives, including stabilizers, plasticizers, fillers and antioxidants, which are released into the environment during the process of plastic fragments forming MPs ,25,31. AMPs undergo aging in the environment, and the aging process mainly includes mechanical breakage, photo-degradation, thermal degradation and biodegradation. The combined influence of various environmental factors on the MPs’ aging process is complex, leading to unpredictable changes in the fate of MPs in the environment. UV light is considered to be the most critical factor in the aging process of polymers. The chemical oxidation induced by light is enhanced, showing a synergistic aging effect. Polymer photo-oxidation reaction generally includes four processes: chain initiation, chain growth, branch formation and chain termination. The photo-degradation of polymer is when unsaturated structure or additives absorb UV light to form free radicals and then interact with oxygen to generate more free radicals, which accelerates its aging. Under the combined action of UV light and oxygen, the aging speed of the polymer would be further accelerated and eventually lead to the collapse of the polymer chain . Hence, Understanding the impact of UV on the fragmentation process and mechanism of plastics is of great significance for predicting the production of MPs in the environmental medium. Current research proved that the chemical oxidation of MPs could be enhanced by light, showing a synergistic aging effect. Polymer photo-oxidation reactions generally include chain initiation, chain growth, chain formation and chain termination. Under the combined action of UV irradiation and oxygen, the aging rate of the polymer will be further accelerated. The photo-degradation of polymer is initially due to the unsaturated structure or additives in the polymer absorbing UV light, forming free radicals, which then interact with oxygen to produce more free radicals, thus accelerating its aging and eventually causing the polymer chain to collapse .UV irradiation can change the surface roughness, morphology and chemical properties of plastics, accelerate the weathering process of plastics seriously and eventually lead to the formation of MPs . Song etThe surface morphology of microfibers changed after UV light and cracked to produce smaller particles in the marine environment . UV irraDuring the UV degradation process, the oxidation of plastic particles, the generation of surface cracks and the formation of fragments are common photoaging forms ,48,49,50Under UV irradiation, new carbonyl groups, vinyl groups, hydroxy peroxides and obvious local micro-cracks are generated on the plastic surface, which reduces the performance of the plastic and increases its brittleness ,53. The The COVID-19 pandemic has promoted explosive growth in the use of masks. According to research, the chain structure and chemical composition of PP in masks change during the UV aging process, and a mask could release more than 1.5 million particles into the water environment . The che−, Br−, NO3−, CO32−, SO42− and natural organic matter, existing in the environment would affect the photoaging reaction of MPs in the environment.The various photochemically reactive components, such as ClPrevious researches showed that sulfides that are ubiquitous in the environment could accelerate the oxidation of MPs during UV irradiation, which induced physical damage (embrittlement and cracking) and chemical conversion (increased O/C ratio and formation of C–S) of MPs. The spontaneous oxidation of sulfides leads to the formation of hydroxyl free radical (•OH), which is an oxidizing agent that attacks the carbon atoms in the polymer chain, causing the surface oxidation and chain scission of polymers . TherefoThe presence of inorganic substances in the environment could also affect the photoaging behavior of MPs. For example, iron oxide inhibited the photoaging of plastic by reducing its light absorption efficiency. Iron red pigment reduced the photoaging rate of PP-MPs significantly, which mainly depended on the reaction of Fe(III) with organic acids and reactive oxygen species derived from MPs under irradiation conditions ,63,64. H− could also effectively sequestered •OH radicals, which may weaken the promotional effect of •OH on the photoaging of MPs [−. This is due to the reaction of Cl2•− generated in seawater under irradiation conditions with HO2• to prevent the formation of O2•−, thereby inhibiting the photoaging process of PP-MPs [On account of the variability in water composition, the photoaging processes and mechanisms of MPs in different water environments are complex. Salinity ions affect the photo-degradation rate of MPs in aquatic ecosystems by influencing UV penetration depth and free radical reaction, which also leads to the otherness of the photoaging mechanism of MPs in the freshwater system and seawater system. The increase in salt concentration led to the high refractive index of water and salt crystals that formed on the surface of MPs; thereby, the inhibition of the MPs photo-degradation rate was caused by the reduction in the light absorption efficiency ,66. At tg of MPs . The resg of MPs . The phof PP-MPs . Neverthf PP-MPs ,70.Photoaging significantly affects the physical and chemical properties of MPs, and further influences their transportation and fate in the environment. Therefore, how the aging of MP under UV radiation further exacerbated its negative impact on the environment was summarized in the review. The aging process caused by UV light could alter the surface charge density and even the Hamaker constant of plastics, thus affecting their aggregation behavior in the environment . PreviouPrevious studies testified that the interaction between MPs and organic pollutants is influenced by hydrophobic forces ,74,75,76The photoaging process could change the surface properties of MPs and affect the adsorption capacity of hydrophilic and hydrophobic organic pollutants ,78,79. TThe photoaged MPs can also enhance the adsorption capacity for hydrophilic organics. Among them, PS and PVC after photoaging by UV irradiation showed higher adsorption of ciprofloxacin (CIP) than unaged MPs, and the main mechanism of adsorption between photoaged MPs and CIP was the physical interaction (electrostatic interaction and intermolecular hydrogen bond interaction) ,60. The 2+ and Zn2+ increased, and the adsorption capacity and adsorption strength of PP-MPs and tire wear particles for Pb2+ and Cd2+ rose. The results of the above researches proved that the correlation between the aging degree of MPs and the adsorption capacity of heavy metals was existed [MPs are good carriers of heavy metals in the environment, and photoaging can also affect the adsorption performance of MPs to heavy metals. The adsorption capacity of MPs for heavy metals after photoaging is higher than that of original MPs, which was speculated to be related to the increase in specific surface area of aging MPs and the generation of oxygen-containing functional groups ,86. With existed ,84,86,873DOM*) generated by DOM under irradiation conditions that promoted the photoaging reaction of PVC [Additives and life-extension compounds mainly function through the following five ways: (1) light shielding, (2) ultraviolet absorption, (3) excited state quenching, (4) peroxide decomposition and (5) free radical scavenging . Photochn of PVC .UV light irradiation accelerated the leaching of dissolved organic matter in MPs (MP-DOM), and the trihalomethane (THM) produced by the chlorination reaction of MP-DOM was harmful to the environment . MP-DOM Toxicological studies on MPs proved that MPs have certain toxic effects on organisms. Nevertheless, MPs exposed to ultraviolet radiation for a long time have a stronger toxic effect on organisms. Compared with raw PS-MPs, long-term exposure to photoaged PS-MPs solution at low concentration (1 μg/L) resulted in stronger neurotoxicity to nematodes, which is due to the neurotoxicity of aged PS-MPs by affecting the neurotransmission of dopamine, glutamate and serotonin . The pho2O2 treatment increased its toxicity to bacteria [Epinephelus moara) [The organic matter  leached from PS after UV and UV/Hbacteria . The agebacteria . Compares moara) . These fTo better understand the ecological and environmental risks of photoaging of MPs, more attention should be paid to the following aspects in future studies:It is encouraged to collect MPs samples from the environment for surface chemical analysis, revealing the changes in the adsorption behavior of MPs with different degrees of photoaging toward different types of organics. The impact of NPs produced by MPs in the photoaging process on the groundwater environment and soil ecosystem should also be paid more attention to in the future.The current researches on the photoaging process of MPs are mostly carried out in artificial laboratory simulation or under the natural control conditions of a separate system, which is different from the actual natural environment under the combined action of multiple environmental factors. Hence, the researches on the photoaging process of MPs in natural environments need to be strengthened urgently. It is recommended to conduct long-term simulation experiments of MPs under outdoor environmental conditions. In addition, long-term experiments are needed to reveal further the biophysical and chemical interactions between photoaged MPs and biological communities in the environment.The photoaging mechanism of MPs is understood to a certain extent, but it is more at the level of qualitative description. There is still a lack of quantitative model verification for the aging relationship between MPs and various environmental factors. While the aging mechanism of MPs is being further explored, a parameterized model is urgently needed to be established in order to quantitatively analyze the contribution rate of various environmental factors to the photoaging of MPs.


COVID-19 has proved to be a social disease due to its widespread diffusion in the general population, the serious harm it causes on affected patients and its impact on the economy and social life of burdened countries. While socialization is a risk factor for the spread of the SARS-C0V-2, health protection measures such as isolation and lockdown further aggravated the “social” burden of COVID-19. Diseases with social impact require a management approach based on social medicine, integrating health, social and economic responses.There is evidence that a low socio-economic status (SES) is strongly associated with higher rates of both incidence and mortality attributable to COVID-19 [SES also affects the living environment, the eating habits, the occupational status and the access to health care services, ultimately influencing health . Most deGiven the current scenario caused by the COVID-19 pandemic, interventions to tackle socio-economic disparities should be considered as priorities like the search for effective curative and preventative treatments . Indeed, existing inequities worsened during the pandemic and aspects like access to food, education, and psycho-social support must be carefully weighed in a holistic approach.The application of social medicine in the current COVID-19 pandemic requires the recall of a long standing medical tradition. The history of recent epidemics, plagueing Italy in the early twentieth century, has shown that the ravages of war, movement of armies, migration of entire populations, poverty, overcrowding and poor hygienic housing conditions contributed to increase the spread of deadly communicable diseases. Social medicine, established in Italy in 1922 to contain the massive spread of tuberculosis and malaria, assumed a key role to promote the first European policies of capillary health education across Europe, contributing to prevent and control relevant infectious threats .social medicine extends the interest and alters the emphasis of the older public health, just as social pathology extends the interest and alters the emphasis of earlier epidemiological study” [As clarified by John A. Ryle, “l study” . Specifi“the whole of the economic, nutritional, occupational, educational, and psychological opportunity or experience of the individual or the community” [When confronted with public health, which is primarily intended to focus on the environment , social medicine differs by encompassing mmunity” . BasicalPeople are simultaneously biological and social organisms, and thus human health and disease are affected by social factors as well as by biological factors. Included in the basic idea and concept of social medicine is that the interdisciplinary program between medicine and social science would provide the former with knowledge and skills needed to analyze the social causes of health and illness in the same way as the alliance between medicine and laboratory sciences had provided new insights into the biological, chemical and physical bases of disease .Social medicine explicitly investigates social determinants of health and disease, rather than treating such determinants as mere background to biomedical phenomena.The proposition of social medicine deserves emphasis, and especially so today – its intellectual breadth, its political and economic depth, its essential humanism.Contemporary social medicine is critical to understand and prevent diseases, improving healthy life conditions in the general population and the efficiency of health systems.Today's health challenges require novel approaches involving the implementation of new technologies and advanced methodologies such as tele-medicine, tele-rehabilitation, tele-consultation and new digital infrastructures for modern data communication 5G technology, Big Data and their management through artificial intelligence algorithms will define an epochal change in health care delivery, processing huge amounts of health data in real time .These technologies will allow clinical research to identify new models of diagnostic, therapeutic and preventative interventions, optimizing health care expenditures yet supporting the most fragile population. Recent studies suggest that countries implementing an integrated health, social and economic public policy response will be able to overtake the current pandemic not only healthier, but also economically and socially stronger .


Mesoporous silica nanocarrier (MSN) preparations have a wide range of medical applications. Studying the biocompatibility of MSN is an important part of clinical transformation. Scientists have developed different types of mesoporous silica nanocarriers (MSNs) for different applications to realize the great potential of MSNs in the field of biomedicine, especially in tumor treatment. MSNs have achieved good results in diagnostic bioimaging, tissue engineering, cancer treatment, vaccine development, biomaterial application and diagnostics. MSNs can improve the therapeutic efficiency of drugs, introduce new drug delivery strategies, and provide advantages that traditional drugs lack. It is necessary not only to innovate MSNs but also to comprehensively understand their biological distribution. In this review, we summarize the various medical uses of MSN preparations and explore the factors that affect their distribution and biocompatibility in the body based on metabolism. Designing more reasonable therapeutic nanomedicine is an important task for the further development of the potential clinical applications of MSNs. The completion of the Human Genome Project (HGP) marks the entry of human beings into the postgenome era.. The “prThe mechanism of interaction between nanoparticles and various cells and tissues in the human body is the key to determining the medical applicability of nanotechnology. Compared with soluble molecules and drugs, the delivery of drugs using nanocarriers is more complicated because the behavior of nanocarriers is related not only to the molecular weight, solubility, and physical and chemical properties of the loaded drugs but also to the pore size distribution, and particle size of the nanocarrier. Coatings, targeting groups and other factors are also closely related. Therefore, for drug delivery using nanocarriers, it is necessary to detect, understand and optimize the release parameters, distribution, metabolism, excretion and biocompatibility of the drugs in the body for optimal clinical results. These are the current challenges in nanomedicine.In this review, we summarize the current reliable research data and describe the application of mesoporous silica as a nanocarrier in gene therapy, photodynamic therapy, molecular targeted drug delivery, vaccine delivery, and imaging, among others. MSN distribution and metabolism are discussed in detail. The importance of biodistribution and biocompatibility is explained, and key points of future research are discussed.MSNs have the advantages of an ordered mesoporous structure, an adjustable pore size, a high specific surface area, and easily modified active surface groups . MSNs arIn vivo experiments showed that the smallest tumor volume and greater anticancer efficacy in the mesoporous silica nanomedicine group; additionally, body weight monitoring and hematoxylin and eosin (H&E) staining of organ  tissue sections showed no histopathological abnormalities, indicating MSN-DOX@PDA-PEG has good biocompatibility and low in vivo toxicity.At present, chemotherapy is one of the most effective methods for the treatment of cancer, and chemotherapy, surgery and radiotherapy together form the three pillars of cancer treatment. In chemotherapy, drugs are used to kill cancer cells to achieve the goal of treatment. However, whether is the drugs are taken by mouth, injection, intraperitoneal injection, or other routes, they will enter the systemic circulation and cause uncontrollable side effects. MSNs have a high drug loading capacity and can be loaded with small-molecule drugs, especially hydrophobic drugs; at the same time, this approach can prevent drug degradation and physiological toxicity to healthy tissues caused by premature drug exposure. Thus, MSNs are of great significance for the delivery of hydrophobic anticancer drugs, drugs with narrow therapeutic windows, and chemotherapeutics with significant side effects. Drugs with narrow therapeutic windows is also called narrow therapeutic index (NTI). NTI drugs can be defined based on the steepness of the dose–response relationship and the degree of overlap between the effective and the toxic concentrations. MSNs are good tool about solving the problem of NTI, because they could improve the efficacy and reduce the side effects of drugs . Duo et In vivo fluorescence imaging was performed to evaluate its targeting performance. The results of in vitro fluorescence imaging showed aggregation of the targeted drug in the tumor. After 36 h, the content of heparin in the tumor was higher than that in the heart, spleen, kidney and other organs. When tested in tumor cells  and normal cells (3T3), the normal cells showed a viability of greater than 80%, demonstrating low toxicity and good activity even at relatively high concentrations, in turn indicating that this MSN has good biocompatibility. Shao et al (3O4 as the head and DOX-loaded MSN as the body (M-MSN-DOX); this multifunctional nanocomposite material showed excellent targeting, with greater uptake by HepG2 tumor cells. The administration of DOX and M-MSN-DOX via a tail vein injection for 16 days effectively inhibited the growth of orthotopic liver tumors and the deterioration of other analyzed liver tumor mouse model parameters, including body weight, blood chemistry and major organ and tissue toxicological indicators. No pathological damage was observed in mouse brain, heart, liver, spleen, lung or kidney tissue sections, indicating that this mesoporous silica nanomedicine not only has good biocompatibility but is also effective for improving the efficacy and reducing the side effects of the drug.Mesoporous silica nanomedicine can not only improve the therapeutic effect of the anticancer drug DOX but also reduce its side effects. Hu et al.  designedao et al  designedThe current important standard for cancer treatment  is basedIn vivo experiments showed that the tumor inhibition rate in the free DOX group was 50.7%, while that in the H-MSN-DOX/siRNA group reached 87%, demonstrating a significant inhibitory effect on drug resistance. At the same time, mice in the free DOX group lost weight, but those in the other groups, including the H-MSN-DOX/siRNA group, showed normal weight changes. Compared with the injection of free drug, administration of the H-MSN-DOX/siRNA delivery system greatly enhanced the therapeutic efficacy while significantly reducing the side effects of the delivered anticancer drug. MSNs loaded with different drugs may overcome the problem of drug resistance by strategically improving the therapeutic efficacy of the drugs. Meng et al , inhibitor of apoptosis protein (IAP) and FLIP protein expression, leading to reduced drug sensitivity. These two mechanisms are sometimes active at the same time. In view of the unique properties of mesoporous silica, different drug delivery strategies have been designed to overcome drug resistance. The energy-dependent endocytosis of MSNs promotes cell absorption, improves drug efficacy and bypasses drug efflux pumps to overcome drug resistance. Sun et al.  designedng et al  designedng et al  designedThe combination of medications can improve the therapeutic effect of the drugs; additionally, different drugs can be selected for loading according to different treatment strategies and individual differences. This approach can reduce the side effects of anticancer drugs and avoid the development of drug resistance.Gene therapy has broad prospects, but naked nucleic acids are not easily internalized by cells; they have poor biological stability and a short half-life and are subject to intracellular degradation prior to entering the nucleus. The systemic delivery of carrier particles can overcome these problems. At present, gene delivery systems can be divided into two categories: viral vectors and nonviral vectors. Although viral vectors have advantages in delivering genes, they have potential safety hazards. Nonviral vectors have been a research hotspot in recent years, including polymers, recombinant proteins, cationic compounds and inorganic nanoparticles. However, cationic materials are usually associated with high toxicity, recombinant proteins show low cost performance, and liposomes can enable effective gene transfection but are unstable. Compared with other nanoparticles, inorganic nanoparticles are simple to prepare and easy to functionalize and have good biocompatibility and excellent physical and chemical stability. MSNs have favorable characteristics for gene delivery, including a narrow pore size distribution and the ability to can effectively protect the cargo up to the release point, and the surface chemical structure facilitates the optimization of adsorption and release characteristics. Thus, MSNs are considered promising carriers for gene therapy.The current methods to improve the performance of mesoporous silica-loaded genes are as follows: 1) positive charge functionalization, such as by amination  and catiin vivo through electrostatic interaction. Compared with free siRNA, the CP-MSNPs reduced the intracellular degradation rate of siRNA by 47.4%, and the cell internalization rate reached 99.2% within 24 h. In vivo microscopy monitoring in a tumor mouse model showed that the maximum concentration at the tumor site could be reached in 20 min, targeting and effectively inhibiting the gene expression of glycolytic pyruvate kinase isoenzyme (PKM2). Wang et al  and photodynamic therapy (PDT) are important class of therapy approach by MSNs, have been demonstrated the priorities to elevate cancer therapeutic efficacies and diminish undesired side effects through different mechanisms in cancer treatment. Photothermal therapy (PTT)  is a minTo improve the therapeutic effect, synergistic effects between photothermal agents and antitumor drugs  have becCopper sulfide (CuS) nanoparticles have the characteristics of a low synthesis cost, a wide NIR absorption range, good biocompatibility and a good NIR photothermal conversion efficiency. An increasing number of nanodrugs based on the photothermal effect of CuS are being developed . Liu et in vitro chemo-photodynamic synergetic experiments indicated that the NPs had excellent reactive oxygen species generation and remarkable cancer cell killing efficiency under laser irradiation.Wang et al. reported The critical challenges in photodynamic and photothermal chemotherapies of cancer are the limited penetration depth of light, instability of photosensitizers, and non-degradable character of inorganic nanomaterials . ImproviTargeted drug delivery systems (TDDSs)  consist Active targeting is achieved through the use of specific targeting ligands, which can enhance specific uptake by cells and increase the intracellular drug concentration. Quan et al.  designedMSNs allow high loading of drugs, and protects them inside the channeled pores until they reach target and release. The MSNs with optimal drug allow potential possess high loading of drugs, sustained and controlled release, and targeted delivery actions. An increasing number of targeted nanodrugs have been modified through diverse preparations to achieve targeted delivery for different needs.Cancer immunotherapy can be achieved with cancer vaccines, , which aThe use of mesoporous silica in auxiliary imaging technology has made good progress  and can The MSN that is used in fluorescence imaging is optically transparent due to its nanoscale particle size  and will2+. The functionalized MSNs showed significant contrast enhancement on MRI both in vivo and in vitro. Similarly, Li et al.  to study their cytotoxicity and inflammatory potential. In vitro and in vivo experiments showed that the toxicity of mesoporous silica was lower than that of nonporous nanoparticles and that changing the surface charge of MSNs can further reduce their cytotoxicity. Further analysis showed that particle size, zeta potential and surface modification were important factors affecting MSN cytotoxicity. Yu et al.'s . The advantage of intravenous injection, subcutaneous injection and intratumoral administration is that these methods can overcome the “first-pass effect” of the liver; after subcutaneous injection, according to the size of the nanoparticles, large nanoparticles (tens of nanometers) enter the lymphatic circulation, and small nanoparticles (a few nanometers) enter the blood circulation through the capillaries. It has been shown that larger materials can last longer at the injection site, and intertumoral injection has been the most common method used in animal experiments; ; this mein vivo. Nanoparticles 80–390 nm in size were injected into the tail vein of mice and rats. The biodistribution of the nanoparticles was tracked using fluorescein isothiocyanate (FITC), and the results showed higher concentrations of particles in the spleen, liver, and lungs and lower concentrations of particles in the kidneys and heart. The size of nanoparticles also showed a negative correlation with the circulation time. MSNs 80 nm in size showed the longest cycle time, and PEGylation could significantly extend the cycle time. Larger nanoparticles showed faster degradation, which may have been caused by the RES.(The physical and chemical properties of nanomaterials affect not only the biological distribution but also the biodegradation rate of the nanomaterials in the body. The biodistribution of nanomaterials is related to many factors, including the size, surface properties and dissolution rate of the nanomaterials, as well as tissue- or organ-dependent factors, such as tissue permeability and barrier properties. After intravenous, oral and subcutaneous delivery, nanomaterials can be found in the liver, spleen, kidneys, bone marrow, central nervous system, and local and systemic lymph nodes, , but the the RES.. Xie et  the RES.. By chan the RES..The biodistribution of MSNPs is closely related to their physical and chemical properties. The size of the nanoparticle greatly affects its biodistribution, and the surface charge, surface modification, geometric shape and biodistribution are closely related to the biodistribution. While surface modifications have the greatest influence, different surface modifications have different degrees of influence. This suggests that when designing nanoparticles, the size and surface modifications should be selected according to the drug delivery strategy and treatment requirements.in vivo. At present, scientists have paid attention to the immunotoxicity and cytotoxicity of nanoparticles. However, research on MSN that can cross the blood-brain barrier is still insufficient, because it may damage nerve cells. And the research on the influence of MSNs on DNA is far from enough. In future experiments, more evidence is needed to clarify the biological toxicity of MSN, particularly the following four aspects: immunotoxicity, cytotoxicity, (Now, we understand the metabolism of MSNs, the factors that affect their metabolism, and their distribution in the body. However, understanding these facets is not sufficient. To realize the clinical transformation of MSNs, only investigating the acute toxicity and subacute toxicity of nanoparticles cannot fully reflect their biological toxicity . It is noxicity, , neurotooxicity, , and genoxicity, .Immunotoxicity: The body’s first line of defense includes monocytes and macrophages. Nanoparticles entering the human body interact with immune cells and can cause the production of proinflammatory factors, in turn leading to cell apoptosis, which is considered to be the mechanism of nanoparticle-induced immunotoxicity.. When thCytotoxicity: MSNs can induce oxidative stress and mediate apoptosis in a size- and dose-dependent manner through the mitochondrial pathway. ROS-mediated toxicity is considered to be an important mechanism of nanoparticle toxicity..Neurotoxicity: Very small nanoparticles can be transported across the blood-brain barrier and damage nerve cells. Studies have shown that direct contact with neuroblastoma cells induces adverse reactions, including markers of Alzheimer’s disease..Genotoxicity: MSNs can directly interact with DNA, consuming antioxidants, oxidizing DNA, disrupting the cell cycle and causing abnormal gene expression. These are considered to be the potential mechanisms of nanoparticle-induced genotoxicity..Currently, it is believed that the main mechanism of biological MSN toxicity is the induction of oxidative stress, which causes toxicity to cells, including immune and nerve cells, as well as DNA. Of course, the toxicity of MSNs is strongly related to the dose, particle size, shape, and surface properties of the MSNs. This is also a research direction that will continue to be explored in the future. For the development of effective mesoporous silica-based clinical drugs, it is necessary to study the potential mechanisms of toxicity.This review provides a detailed introduction to the applications of MSNs in the field of medicine and how they can improve the bioavailability of drugs, reduce antitumor drug resistance, and facilitate targeted therapy and bioimaging, among others. MSNs have achieved remarkable results in animal experiments. The US FDA has also approved the first silica material, C dots, for clinical research. Porous silica holds great promise. Scientists have conducted much research to develop MSNs and apply them as drug delivery systems or in therapeutic strategies. The biodistribution and long-term fate of these MSNs are critical to the applicability and efficacy of drugs, and gaining an understanding of these outcomes is essential to promote the clinical application of mesoporous silica.In the development of drug delivery strategies, scientists continue to make advances in synthesis methods, surface modifications, and targeting strategies. Composite materials and multifunctional materials that can achieve active targeting and bioimaging while delivering drugs are constantly emerging. In oncology, better therapeutic effects can be achieved through the combination loading of pharmaceuticals and genetic vectors, with the potential to result in a full response. Now, not only reactive oxygen and acid-base responses but also precision delivery under external conditions can be achieved via controlled release in response to photonic, ultrasonic, or magnetic stimuli. In future research, the development of multifunctional responsive materials through different targeting and response strategies and in combination with various imaging technologies will lead to the efficient treatment of target tissues with reduced side effects.Currently, research on the biodistribution and biosafety of mesoporous silica is very important for its application as a nanocarrier. Studying the biological toxicity of MSNs, particularly their immunotoxicity, cytotoxicity, neurotoxicity and genotoxicity, is important for achieving the clinical application of MSNs.


Extracellular matrix (ECM) is a kind of connective tissue in the cell microenvironment, which is of great significance to tissue development. ECM in muscle fiber niche consists of three layers: the epimysium, the perimysium, and the endomysium . These three layers of connective tissue structure can not only maintain the morphology of skeletal muscle, but also play an important role in the physiological functions of muscle cells, such as the transmission of mechanical force, the regeneration of muscle fiber, and the formation of neuromuscular junction. In this paper, detailed discussions are made for the structure and key components of ECM in skeletal muscle tissue, the role of ECM in skeletal muscle development, and the application of ECM in biomedical engineering. This review will provide the reader with a comprehensive overview of ECM, as well as a comprehensive understanding of the structure, physiological function, and application of ECM in skeletal muscle tissue. Skeletal muscle is an important organ of locomotion and metabolism in the body, which plays a very important role in maintaining the exercise balance, glucose metabolism , and eneECM is involved in skeletal muscle development from embryonic stage  to senesWith the further understanding of the mechanism of proliferation and differentiation of muscle cells, more and more attention has been paid to the important role of cell niche in development. ECM has also been widely used in the fields of developmental biology, regenerative medicine, and bioengineering due to its important role in regulating cell physiological functions and its unique biological characteristics. Although there have been numerous studies demonstrating the important role of ECM in skeletal muscle development, it is not very systematic. To provide a more comprehensive and systematic concept of the function of the extracellular matrix of skeletal muscle, we provide an overview over the current state of knowledge concerning the structure, composition, function, and application of ECM in skeletal muscle tissue.Multilayer ECM is a common feature of vertebrates. In 2011, Charvet et al. demonstrated the genesis of ECM during muscle fiber development using zebrafish as a module. They emphasize that the development of myocomma originates from the segmentation period formed by sparse and loosely arranged collagen fibers . During To study the structure of ECM visually, researchers have developed a number of tools. Recently, Mayorca-guiliani et al. developed a method to visualize the structure of ECM in detail, called in situ decellularization of tissue (ISDOT). They isolated natural 3D ECM scaffold from tissues with ECM structure and components, and then the structure of ECM could be determined by mapping the protein . In addiIn morphology, the ECM of skeletal muscle tissue can be divided into three independent and interconnected layers: the epimysium is a dense connective tissue that wraps the whole muscle; the perimysium originates from the epimysium and wraps the muscle bundles; the endomysium, also known as basal lamina, is a kind of sophisticated membrane around each muscle fiber . The epiThe basal lamina is a supramolecular ECM structure, including the inner layer (adjacent to the sarcolemma) and the outer mesh layer Fig.  26–28].–28.26–28Extracellular matrix is composed of three main proteins, namely, collagen, non-collagen and proteoglycan. Collagen is the largest component of ECM protein in skeletal muscle. In addition, there are receptors and regulators present in extracellular matrix, such as integrin  and matrCollagen is the most abundant component of ECM in skeletal muscle tissue. There are three types of cells that can produce and secrete collagen in mouse skeletal muscle, namely fibroblasts, fibro/adipogenic progenitor cells, and skeletal muscle progenitor cells (MPCs) . GilliesTypeI collagen can significantly inhibit myogenic differentiation. Myoblasts and myotubes can synthesize CTGF in the presence of TGFβ and lysophosphatidic acid, thereby inhibiting myoblast over-differentiation by promoting the expression of a variety of ECM components, such as typeI collagen and integrin . AlexakiType IV collagen is one of the main components of basal lamina that can promote the IGF1 mediated migration, differentiation, and fusion of myoblasts, thus promoting the regeneration of skeletal muscle . Col4α1 Type VI collagen play an important role in maintaining the physiological function of skeletal muscle. Type VI collagen expressed and secreted by fibroblasts, almost no expression in the muscle cells . However+ -paxillin (PXN) pathway could enhance Laminin organization and maintain the stability of basal lamina, thus contributing to improve the muscular dystrophy phenotype [Laminin is located in the basal lamina of muscle fibers , 65, whihenotype .The canonical expression of different subtypes of laminin protein chains is conducive to regeneration of damaged skeletal muscles. Laminin-1 can maintain the adhesion of muscle fibers on basal lamina, improve muscle performance of mdx mice, relieve degeneration and inflammation of skeletal muscles, shorten regeneration cycle, and promote proliferation and migration of myoblast cells –71. InjeFibronectin is localized in epimysium, perimysium, and endomysium. In addition, fibronectin protein also co-locates with tenascin-C at the tendon junction . FibroneFibronectin can promote the adhesion and differentiation of myoblasts but inhibit the migration and division , 80. FibDystrophin and dystroglycan are important links between cytoskeleton and extracellular matrix, which can maintain the integrity of cell membrane. The N-terminal of dystrophin protein binds to actin through two major actin binding sites , and each ABD consists of two calmodulin homologous domains . There aDystrophin forms dystrophin-glycoprotein complex (DGC) along with dystroglycan and other proteins, such as dystrobrevin and utrophin –97. DGC Proteoglycan is an important component in the ECM of skeletal muscle, including glycosaminoglycans, fibromodulin, and heparin sulfate glycosaminoglycan (HSPG) etc. Proteoglycan is involved in connecting the internal cytoskeleton and ECM, while mice with proteoglycan deficiency will exhibit muscle degeneration and muscular dystrophy .Glycosaminoglycans combine with fibrous proteins to improve myoblast proliferation and differentiation . FibromoECM is a highly nonlinear elastic material whereas muscle fibers are linear and elastic . ECM serStudies showed that the supportive and regulatory role of ECM is essential for the formation of muscle tube, and this effect occurs in the early stages of myogenic differentiation , 112. LiLikewise, the physical activity of muscle cells also affects the composition of the ECM. Kaasik et al. have shown that muscular unloading and reloading could influence the composition of the ECM. Unloading could down-regulate the expression level of typeI, III, and IV collagen, while reloading could strengthen the expression of collagen, MMP-2, and tissue inhibitor of metalloproteinase-2 (TIMP2) in the fast muscle fibers . In seruThese studies indicated that myogenic differentiation can regulate muscle microenvironment, which in turn regulates the cell behavior during skeletal muscle development. The ultimate purpose of the interaction between cells and their niche is to better “serve” the development of tissues. In this process, the cell is the functional actor and the extracellular matrix acts as a regulation factor.Skeletal muscle stem cells, also known as satellite cells, are activated when skeletal muscle development or damaged, and subsequently proliferate, differentiate, and fuse to form new muscle fibers. In the development of skeletal muscle, ECM provides a stable microenvironment for the migration, adhesion, proliferation, and differentiation of satellite cells. Overexpression of ECM proteins can lead to alteration in niche of satellite cells and weaken the differentiation ability of satellite cells, thus affecting the development of skeletal muscle , 59, 120However, different components of ECM have different effects on myoblast behavior. Studies showed that the promoting effect on proliferation and differentiation of satellite cells of complete ECM and laminin were better than collagen and fibronectin, while fibronectin and laminin can improve the adhesion and differentiation ability of satellite cells but inhibit the proliferation and migration of cells , 80. MorIntact ECM can support regeneration of muscle fibers in damaged skeletal muscles. Zhang et al. produced d-ECM from porcine skeletal muscle, liver and kidney, and modified with heparin hyaluronic acid hydrogel (ECM-HA-HP), studies have shown that satellite cells show stronger ability of proliferation, differentiation, and fusion on muscle ECM-HA-HP (mECM-HA-HP) substrate, which can be used for cell therapy of skeletal muscle dysfunction . Also noIn the early stage of muscle fiber injury, ECM hyperplasia results in increased skeletal muscle tissue stiffness, and this orderly deadhesion, and fibrosis is designed to protect skeletal muscle from further damage , 87, 125According to these studies, we can conclude that ECM, as an important component of muscle fiber niche, plays an important role in muscle fiber regeneration and skeletal muscle development. ECM component proteins are secreted by a variety of cells surrounding muscle fibers, such as fibroblasts, endothelial cells, and skeletal muscle connective tissue cells –131. TheECM components are essential for the development of neuromuscular junction (NMJ). Study showed that ECM proteins could promote the activity of acetylcholinesterase . In addiIn addition, various proteins in the ECM, such as collagen , integriThe integrity of the neuromuscular junction and the transduction of synaptic signals are the keys to the motor function of skeletal muscle, while the abnormal deposition of ECM protein will lead to the disorder of the connection between motor neurons and muscle fibers , 138. ItECM can exert transverse stress on fibers and have axial strain . If the After skeletal muscle injury, ECM can improve muscle function to a certain extent by regulating the force transmission at the injured site rather than relying on skeletal muscle regeneration . The stiIn addition, ECM is also the major contributor to the passive tension of skeletal muscle . StudiesMain conclusion is that excessive accumulation of extracellular matrix can significantly impair the mechanical properties of skeletal muscle, including active and passive tension. Therefore, the remodeling of extracellular matrix and the correct expression of each component are of great significance in the clinical treatment of muscle weakness.Although the characteristics, components, and function of the ECM vary in different tissues, it is common that any deficiency in ECM properties can cause pathophysiological responses, such as chondrodysplasia , Ehlers-ECM is necessary for tissue development, so it has a good application prospect. Decellularize ECM (dECM), which is derived from in vivo, is widely used in the field of bioengineering and regenerative medicine because of its excellent histocompatibility and biological properties. It can be used as a biological scaffold to promote the formation of functional tissues. Kao et al. prepared pig bladder matrix hydrogels using Sodium Dodecyl Sulfate Decellularization Method, and the results showed that the SDS Decellularization Method provides a more stable and safer access to the Decellularization bladder matrix due to reduced immunogenicity and can be used as a potential candidate scaffold for tissue remodeling . NikniazIn recent years, researchers have developed dECM active materials for clinical treatment using bioengineering techniques. Trevisan et al. constructed mouse decellularized diaphragm ECM, which can promote the activation, proliferation and differentiation of skeletal muscle progenitor cells to form a powerful three-dimensional skeletal muscle structure, providing a promising tool for clinical application of diaphragm regeneration in the future . Lee et ECM materials can support cell attachment and proliferation in vitro, and have good anti-inflammatory and immunosuppressive properties in vivo, which can improve the success rate of cell transplantation . In the ECM is a complex and sophisticated structure whose components are synthesized and secreted by many types of cells. In this paper, the characteristics and functions of ECM in skeletal muscle tissue are discussed in detail. The cytoskeleton forms a close connection with ECM through DGC, laminin, and proteoglycan, etc. ECM can not only maintain skeletal muscle morphology and contraction as a scaffold, but also regulate various physiological functions of skeletal muscle, such as signal transmission of motor neuron, glucose metabolism, and regeneration after injury. Furthermore, decellularized ECM as biomaterials is widely used in bioengineering and regenerative medicine because of its unique and superior biological characteristics. Therefore, the in-depth study of ECM is beneficial for researchers to further explore the mechanism of skeletal muscle development, and provide new insights for clinical treatment of skeletal muscle diseases and the development of biological materials.However, ECM does not function independently and also requires the involvement of multiple cytokines, such as Integrin, MMPs, and TGFβ. Integrin, as receptor of extracellular matrix proteins, coordinate with extracellular matrix to regulate the adhesion , prolife


Genetic and acquired defects of lower motor neurons, peripheral nerves, or skeletal muscle are responsible for several neuromuscular disorders.The neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve, and defects in the development and maintenance of the NMJ are responsible for the pathogenesis of several neuromuscular disorders, including congenital myasthenic syndromes, autoimmune myasthenia gravis, spinal muscular atrophy, and amyotrophic lateral sclerosis (ALS); moreover, defects in muscle and nerve communication occur physiologically during aging.The molecular players involved inthe formation and maintenance of theNMJ have been deeply studied; however, the role of skeletal muscle in NMJ dismantlement still needs to be fully elucidated. In this Special Issue, entitled “The Role of Skeletal Muscle in Neuromuscular Diseases: From Cellular and Molecular Players to Therapeutic Interventions”, we collected recent research advances and ongoing studies focused on the muscle denervation associated with aging and neuromuscular diseases. Two of the published papers are focused on the role of skeletal muscle in ALS disease, one on NMJ dismantlement in relation to muscle accumulation of oxidative damage, and the others are focused on the role of skeletal muscle in different diseases, such as muscular dystrophies.ALS is a complex and multifactorial disease in which several mechanisms contribute to the development of the pathology; among these, there are glutamate-induced excitotoxicity, protein aggregation, mitochondrial defects, and the alteration of metabolic homeostasis. All these pathogenic mechanisms are integrated into two different hypotheses: (i) the dying back hypothesis, by which signals released by muscle can retrogradely influence nerve and neuron survival, and (ii) the dying forward hypothesis, which describes a progressive neuron degeneration that first affects the neurons of the motor cortex, and then spreads through the nerve, ultimatelyinducing NMJ dismantlement.The articles published in this Special Issue related to the role of skeletal muscle in ALS are“The Skeletal Muscle Metabolism: Origin or Prognostic Factor for Amyotrophic Lateral Sclerosis (ALS) Development?”and “Circulating myomiRs in Muscle Denervation: From Surgical to ALS Pathological Condition” ,2. TheseThe first article mainly reviews the relationship between the dysfunction of skeletal muscle energy homeostasis and muscle denervation. The author claims that in ALS patients and mouse models, the onset and the development of the pathology are associated with mitochondrial dysfunction in skeletal muscle, which leads to the alteration of calcium homeostasis, and thus to the deregulation of the respiratory chain, inducing the imbalance of tissue energy homeostasis. Indeed, one of the main features of ALS in humans is the loss of body weight associated with insulin resistance and glucose intolerance; moreover, a high body mass index is considered a positive prognostic marker for ALS and is linked to a lower risk of developing ALS. The loss of metabolic flexibility precedes hypermetabolism in ALS mouse models andis responsible for the alteration of the balance between glucose and fatty acid flux. Moreover, hypermetabolism is already detectable during theasymptomatic phase of the disease in ALS mouse models and human patients, and persists throughout the course of the disease, suggesting that the maintenance of body metabolic balance represents an interesting therapeutical tool to counteract ALS pathology. In this context, the author reviews pharmacological strategies for targeting the energetic balance, including DCA, RAN, and TMZ, and assert that the restoration of glycolysis could represent an interesting route for the future treatment of ALS patients.Whole-body metabolic changes in ALS involve different tissues, including skeletal muscle fibers; indeed, during muscle denervation, a selective and progressive degeneration of the motor neurons that innervate glycolytic fibers occurs. Several pieces of evidence associate metabolic alterations in muscle fibers with muscle transcriptome changes, and in a recent work of Musarò and coworkers , the traMiR-206 plays a key role in muscle–nerve crosstalk since its level has been described to be modulated when muscle–nerve homeostasis is altered, as is the case in ALS. Moreover, several studies have demonstrated that miR-206 can be released in the bloodstream and its circulating levels can change according to muscle trophism. However, whether miR-206 exerts a protective or a detrimental function is still debated. In the article “Circulating myomiRs in Muscle Denervation: From Surgical to ALS Pathological Condition”, the levels of circulating miR-206 are investigated during muscle denervation conditions, in both pathological and surgical denervation. In particular, the authors define a hormetic role of circulating miR-206, since its levels increase in relation to the progression of the disease and are modulated during reinnervation and not in chronic denervated muscles.Aging is characterized by a gradual loss of principal physical abilities, leading to reduced mobility and a loss of independence. Although many factors contribute to the pathophysiological effects of aging, one of the major events appears to be related to the impaired integrity of the neuromuscular system, which connects the brain and skeletal muscles via motor neurons and neuromuscular junctions (NMJs). In particular, NMJs undergo dramatic morphological and functional modifications during aging that result in an inexorable decrease in skeletal muscle mass and strength, a condition known as sarcopenia. As reported by Dobrowolny et al., muscle–nerve communication can be affected by the age-related alteration of ROS levels, and increased oxidative stress has been correlated with the etiology of a variety of disorders, including ALS and other neurodegenerative diseases . In addiIn recent years, several studies have shown that epigenetic modifications such as miRNA-dependent gene expression, DNA methylation, and histone acetylation can regulate the signals between motoneurons and NMJ and can be associated with the development of several age-related diseases ,15. The In the article of Verdile et al., recent progress related to the role played by alternative splicing (AS) in the establishment of functional NMJs and its contribution to the onset of NMJ-related neuromuscular disorder are discussed. Indeed, although AS is a highly sophisticated process of gene expression, it is prone to errors, leading to mis-splicing events that can be involved in a variety of human diseases, including muscular dystrophies and neurodegenerative diseases. In particular, the authors report important findings which demonstrate that manipulating the splicing of dystrophin pre-mRNA to induce the skipping of specific exons can restore a correct reading frame, representing an important strategy for the treatment of Duchenne Muscular Dystrophy. Moreover, in the same paper, it is reported that in Myotonic Dystrophy Type 1 (DM1), aberrant splicing occurs in genes involved in the maintenance of intracellular calcium homeostasis, resulting in a dysregulation of the mechanism of excitation–contraction coupling, thus inducing muscle degeneration ,17,18.


The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma. The fusion of vesicles with other lipid bilayers is essential for intracellular trafficking and release of neurotransmitters and hormones ,2,3,4. RTraditionally lipids have been regarded as structural elements playing a relatively minor role in the molecular mechanisms of exocytosis whereas proteins such as SNAREs  are thought to be the central elements that generate the specificity and force needed for overcoming the repulsion of the negative charges within lipid bilayers that oppose fusion ,12,13,14In addition, some lipid components aggregate to form microdomains that facilitate recruitment of the proteins that catalyse exocytosis. For example, phosphatidyl inositol 4,5-biphosphate (PIP2) microdomains seem to act as beacons for coordinating F-actin bundles involved in recruiting SNARE proteins, and are needed for the translocation of secretory vesicles to their specific docking sites ,18,19,20Furthermore, lipids can be incorporated into secretory proteins such as SNAP-25 via post-translational modification consisting in the acylation of cysteine residues by palmitate, a saturated 16-carbon fatty acid and in that way affect the location and the function of this protein . It seemIn addition to SNAP-25, other proteins participating in secretion such as synaptobrevin-2 have been demonstrated to be palmitoylated. This protein is modified by palmitoylation during development since the modification is only found in adult rats and not in embryonic cells . FurtherAs can be deduced from the multiple roles played by lipids mentioned above, their function in exocytosis is far more complex than that deduced from being the basic structural elements forming membranes, and this is further supported by recent data on the direct modulation of the secretory machinery by signalling lipids.As mentioned above there are several roles that could be assumed by lipids in order to influence the secretory process ranging from structural determinants to specific “markers” locating the position of exocytotic active sites. Nevertheless more recently it has been highlighted that certain type of lipids acting as intracellular signals, the so called signalling lipids, could interact directly with the SNARE proteins constituting the secretory machinery. Signalling lipids such as sphingosine or arachidonic acid (AA) are released from structural phospholipids by the action of phospholipases ,33, and These signalling lipids interact with a variety of parts of the secretory machinery such as SNAREs and in that way they modulate exocytosis. In this sense AA interaction with the t-SNARE syntaxin 1 was the first reported evidence of a direct interaction of signalling lipids with SNAREs linked to the exocytotic fusion machinery in 2005 . In thisThe AA regulation of the secretory machinery was further emphasized when it was reported that the protein α-synuclein accumulating in the neuronal cytoplasm during the pathogenesis of Parkinson disease, was able to sequester AA and in this way impede the activation of exocytosis caused by this signalling lipid . TherefoRecently, by screening the ability of a variety of lipids in changing the formation of the SNARE complex in vitro it was proven that specifically sphingosine and some related lipids were able to induce synaptobrevin-2 binding to syntaxin-1 and SNAP-25 dimers formed in the target membrane  Figure . InteresAnother study suggested that sphingosine could additionally act through a different molecular mechanism involving the SNARE syntaxin-1 . In thisInterestingly, a physiological derivative of sphingosine, sphingosine-1P also has been found to alter the secretory response in neuroendocrine chromaffin cells ,41 by a Therefore, it is clear that the direct interaction of sphingosine or physiological derivatives such as sphingosine-1P with different elements of the secretory machinery may cause a complex modulation of the exocytotic response in neuronal and neuroendocrine cellular models, and that the complexity of such interactions deserves further study.To study the way sphingolipids affect the secretory process, it is important to appreciate that this is a multi-step process starting with the translocation of the vesicles to the plasma membrane using active transport involving cytoskeletal elements ,44, matuOur initial study describing the enhanced formation of SNARE complexes by sphingosine , also inLater on, two of the groups participating in this study and using distinct cellular systems studied more deeply the fusion exocytotic steps affected by sphingosine. First, and by using capacitance techniques to distinguish unitary fusion events in pituitary lactrophophs, Robert Zorec’s group demonstrated that sphingosine raises the frequency of fusion of both small vesicles and large dense granules . In addiWe reached a similar conclusion using amperometry to measure catecholamines from cultured bovine chromaffin cells . We usedIn a recent report, intracellular sphingosine-1P was also found to accelerate the rate of fusion pore expansion in chromaffin cells from mouse , thus inIt is important to note also that both sphingosine and sphingosine-1P might regulate other steps of the secretory process such us the initial entrance of calcium after cell stimulation ,41, synaNot only sphingosine and its natural derivatives have been found to alter exocytosis in a variety of cellular systems as mentioned above, but more recently a fungi-derived sphingosine analog, FTY-720, also known as Fingolimod, has been reported to influence the secretory pathway. Initially, FTY-720 was employed as an immunosuppressant agent, since its phosphorylated form binds to the sphingosine-1P receptors , and theCrucially, FTY-720, has been shown to induce the formation of the SNARE ternary complex in vitro, and, as a consequence, enhanced the exocytosis in neuroendocrine cells , thus miIn a recent paper, we reported that FTY-720 has a complex effect on secretion from neuroendocrine chromaffin cells because, in addition to modifying the amount of quantal release and the frequency of vesicle fusion in cells stimulated by depolarizations, we also observed that the recruitment of vesicles in response to multiple pulses decreases, indicating that late phases of vesicle transport are also altered . The resIn conclusion, it is possible that both sphingosine and FTY-720, in addition to altering the vesicle fusion properties in neuroendocrine and neuronal systems through the alteration of SNARE function, these small lipids could additionally alter the transport of vesicles through F-actin cytoskeleton, impairing vesicle access to secretory sites and that this could be related with the alteration of calcium dynamics by sphingolipids .Our more recent study revealed that FTY-720 not only mimics sphingosine properties in relation to the fusion of vesicles and the plasma membrane during exocytosis but also exhibits new properties in relation to the fusion and fission of other cellular structures such as mitochondria that had not been reported previously .Using confocal fluorescence microscopy of chromaffin cells, we detected substantial alterations in the shape and size of mitochondria that were accompanied by simultaneous alterations in the number and size of chromaffin granules. Fluorescent microscopy revealed that soon after adding 20 μmol/L of FTY-720 the fluorescently labelled vesicles decreased in size and, within 5 min of drug incubation, started to merge with mitochondria that had been labelled with a different dye. These merging organelles then increased in size and roundness at 15 min of initiation of the FTY-720 incubation. The changes were confirmed by electron microscopy that additionally showed a dramatic change in the fusion properties of chromaffin granules in the interior of the cellular cytoplasm  as seen Interestingly, the fusogenic alterations induced by FTY-720 were mediated by changes in the function of SNAREs since the expression of non-fusogenic SNAP-25 forms prevented the homotypic and heterotypic fusions . TherefoIn summary, the results obtained with FTY-720 highlight that sphingolipid derived drugs might be acting on multiple targets involved in a variety of cellular and pathological processes, and that we need to consider that molecular mechanisms based in the alteration of SNARE fusogenic activity associated to exocytosis, homotypic, and heterotypic organelle fusion and fission processes could underlie the pathological alterations observed in neurons and neuroendocrine cells after application of sphingosine and derivatives.In view of the important role of sphingolipids in neuronal and neuroendocrine signalling pathways , and theIn addition, a plethora of recent studies in a variety of animal models suggest that FTY-720 could have additional therapeutic applications ranging from the modulation of astrocyte and neuronal function, neuronal excitability, and even anti-cancer properties. Among the benefits, it has been shown that FTY-720 can stimulate neuronal gene expression, axonal growth and regeneration , and thiRegarding neurodegenerative disorders: It has been shown that FTY-720 enhances hippocampal synaptic plasticity and memory in Huntington’s disease models , attenuaThe effects of FTY-720 on mitochondria—dramatically altering the membrane potential with the consequence of generating a rapid apoptosis that induces cell death—might be associated with effects on the proliferation of cells from cancers of neuronal origin that have been reported. For instance, FTY-720 induced apoptosis in neuroblastoma cells  and glioThe anti-tumour effects of FTY-720 are observed in cancers from other, non-neuronal origins, for example in the treatment of leukaemia , with thWhat could be the molecular mechanisms involved in the FTY-720 effect preventing cancer cell proliferation and inducing cellular death? Some studies indicate that this compound mainly induces mitochondria-involved apoptosis see  in a varTaken together, it is clear that FTY-720, an example of a drug that mimics sphingosine structure, could be used in the therapy of neuronal and neuroendocrine syndromes, and as an anti-tumour agent, due to the action upon a variety of molecular targets that influence the physiology of multiple cellular systems.The role of lipids in exocytosis in neurons and neuroendocrine cells has been evolving dramatically in the last years to pass from being considered as merely structural components to a more active role in the direct regulation of the secretory molecular machinery ,88,89.In particular sphingosine, released from the structural membrane sphingolipids by the action of sphingomyelinases, and considered a signalling lipid, interacts directly with synaptobrevin-2, which promotes the formation of the SNARE complex, and increases membrane fusion by exocytosis in neuronal and neuroendocrine cells ,38. FurtThe direct regulation of the secretory machinery by sphingosine and sphingosine-1P was further emphasized when a structural analog FTY-720, approved for the oral treatments of certain forms of relapsed multiple sclerosis , was shoWhether or not these effects on exocytosis are linked to the plethora of possible therapeutic uses of FTY-720 in the normal and pathological states is a matter that might be worth further study. This drug influences the activity of neurons and astrocytes by stimulating neuronal gene expression, axonal growth, plasticity, and regeneration . In smalThe molecular mechanisms that might be associated with the potential use of FTY-720 to induce apoptosis in cancer cells from a variety of neuronal and non-neuronal origins are not fully understood. In relationship to neuronal function, effects limiting the proliferation of neuroblastoma , glioblaTaken together, it is now evident that sphingolipids, their derivatives and related drugs such as FTY-720 could act directly upon a variety of cellular targets that affect membrane fusion processes associated with neurotransmitter release by exocytosis and also that affect intracellular transport and organelle fusion and fission. These newly discovered molecular mechanisms may help to explain the ability of sphingolipids to influence the course of neuronal syndromes and even cancer in neuronal and other tissue models.In conclusion, studies of the molecular effects of sphingolipids on membrane fusion and related processes increase our understanding of the physiological regulation of neurosecretion and provide a promise to conceive new therapeutic approaches against neurological syndromes ,75,76,90


Stimulated and spontaneous interactions of electron wavepackets with optical near fields were explored with complementary techniques. In striking agreement with theory, scientists have demonstrated the dependence of spontaneous and stimulated quantum mechanical processes on the spatial distribution of optical modes. In 1923, Louis De Broglie proposed that matters are waves, a contradictory description of quantum mechanics; thus, physics had to wait until the 1940s so that both quantum phenomena could be unified within the framework of quantum electrodynamics. Interestingly, a semiclassical theory, i.e., the theory that constitutes the interaction between matter waves, such as electron wavepackets and classical light (described by Maxwell’s equations), gained importance whenever spontaneous emission is negligible. However, the latter phenomena cannot be neglected when a few-photon process is considered: a situation that occurs during the interaction of an electron wavepacket with nanostructures to probe, for example, cathodoluminescence (CL) or electron energy-loss spectroscopy (EELS) responses1.The first quantum hypothesis reluctantly proposed by Max Planck, in an effort to describe black-body radiation in 1900, revolutionized the world of physics. The subsequent interpretation of the photoemission effect by Albert Einstein using Planck’s radiation theory initiated the birth of 2 and performing holography3, highlight the unambiguous quantum nature of electrons as matter waves, thus verifying semiclassical theories. However, with the advent of ultrafast electron microscopy, which synchronizes laser excitations with pulsed electrons through the photoemission process, quantum electrodynamics experiments with an interplay between spontaneous and stimulated processes are becoming feasible. The field of photon-induced near-field electron microscopy (PINEM), invented by the late Nobel Laureate Ahmed Zewail4, involves stimulated processes instead of the spontaneous interactions of the EELS and CL spectroscopy techniques.In parallel to quantum mechanical developments, the electron microscopy field emerged after the invention of the first electron microscope by Ernst Ruska and Max Knoll. Taking advantage of extremely short wavelengths of matter waves, such as ultrafast electron wavepackets, an ultimate resolution of a few tens of picometers is theoretically achievable. Everyday, experiments with electron microscopes, including recording diffraction patterns5. In an intelligent experiment, EELS, CL, and PINEM spectra were acquired and carefully analyzed. To conduct the experiments, a gold nanostar structure was used that, thanks to its sharp tips, hosts only single-mode dipolar resonances at its tips. By controlling the polarization of the laser beams in the PINEM experiment, it is possible to selectively excite certain tips. Since sharp tips do not sustain higher-order multipole resonances, both electrons and incident photons couple to the same mode when the light polarization and the propagation direction of electrons and incident photons allow for this. By carefully analyzing the spatial dependences of the EELS, PINEM, and CL rates, Liebtrau et al. showed the same spatial dependences for both stimulated and spontaneous processes in good agreement with theory, signifying the importance of the modal representation in describing the coupling efficiency between an external excitation and the system. Interestingly, theoretical predictions demonstrate similar spectral dependencies for both PINEM and CL that depend on the radiative nature of the mode and differ from EELS spectra. Comparing the CL and PINEM processes , the electron wavepacket demonstrates a quantum walk on the energy ladder specified by photon energies , which leads to tightly bound evanescent tails of the plasmonic resonances so that the phase-matching condition could be met for only slow electron wavepacketsTechnological developments in electron microscopy thus push the field toward performing quantum optics experiments and placing well-controlled electron beams next to single-photon emitters, as unique tools for performing such experiments. As perfectly demonstrated in the work by Liebtrau et al., the marvelous ability of electron microscopes to control the position of the beam and focus it into deep subwavelength spots allows quantum optics experiments to be performed on demand. This ability of electron microscopes circumvents the need to employ complicated schemes for controlling the position of single-photon emitters, such as quantum dots with respect to nanostructures. Therefore, by introducing an external laser field and controlling its intensity, it becomes possible to switch between spontaneous and stimulated processes.9. New experiments that compare CL, EELS, and PINEM in graphene enabling multiphoton spontaneous processes could add a novel perspective in this regard. Moreover, although theoretical works predict an optimum energy for the electron that enables a strong coupling to the field, PINEM experiments with slow electrons within the range of intermediate electron energies reachable by point-projection microscopes, and scanning electron microscopes should be performed to test, for example, the validity of nonrecoil approximations10.CL and EELS hence both work within spontaneous interaction processes, where only single-photon exchanges are normally possible; though it was recently demonstrated that using slow electrons interacting with graphene, one could expect multiphoton processes as well