Exosomes are synthesized and secreted by different cell types and contain proteins, lipids, metabolites and RNA species that reflect the physiological status of the cell of origin. As such, exosomes are increasingly being used as a novel reservoir for disease biomarker discovery. However, isolation of exosomes can be challenging due to their nonuniformity of shape and variable tissue of origin. Moreover, various analytical techniques used for protein detection and quantitation remain insensitive to the low amounts of protein isolated from exosomes. Despite these challenges, techniques to improve proteomic yield and increase protein dynamic range continue to improve at a rapid rate. In this review, we highlight the importance of exosome proteomics in neurodegenerative and neuropsychiatric disorders and the associated technical difficulties. Furthermore, current progress and technological advancements in exosome proteomics research are discussed with an emphasis on disease-associated protein biomarkers. Exosomes are a class of microvesicular bodies (MVB), 30–100 nm in size having a unique disc- or cup-shaped morphology . The secExosomes are composed of various clinically important biomolecules such as proteins, lipids, nucleic acids, and metabolites that likely reflect the physiological status of the cell. Thus, the transfer of exosome content has the potential to affect intercellular communication under various physiological and pathological conditions ,4. ExosoExosomes are involved in cell-to-cell communication at multiple levels. Different subtypes of cells in the CNS such as astrocytes, microglia, oligodendrocytes, neurons, and Schwann cells produce cell type-specific exosomes that may act as cargo delivery vehicles mediating communication between different types of cells . ExosomeAlthough the detailed mechanisms remain to be elucidated, the trafficking and proteolytic processing of the amyloid precursor protein (APP) have been implicated in Alzheimer’s disease (AD) . The rolExosomes have also been shown to have neuroprotective roles and to affect neurogenesis and cognitive impairment in amyloid-β animal models. One study used an AD mouse model generated by bilateral administration of Aβ-42 aggregates into the dentate gyrus . AdminisThe neuropathological accumulation of the microtubule-associated protein Tau, which leads to neurofibrillary tangles, is a widely explored phenomenon in AD pathology . SecreteMultiple studies have shown that α-synuclein and its aggregation in brain tissues play a significant role in the neuropathology of Parkinson’s disease (PD) . PreviouAmyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by the propagation of motor neuron death from one focal motor unit to other adjacent cells possibly through altered neuron–glial cell communication . SuperoxIn the past decade, exosome research has witnessed several advances in the application of new methodologies for the isolation, purification, and molecular characterization of exosomes from biological fluids such as plasma, serum, urine, saliva, CSF, conditioned cell culture media, and tissues. The common methods of exosome isolation, along with their advantages and disadvantages, have been discussed in some recent reviews ,43. TablCharacterization of purified exosomal fractions is generally based on various features such as size, shape, and protein or nucleic acid markers. Electron microscopy-based techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are the most commonly used techniques to characterize exosomes based on their size and morphology. These techniques provide higher accuracy and sensitivity (up to 0.1 nm to 3 nm) and are widely used for exosome characterization. The exosome concentration along with size and morphology can also be assessed by nanoparticle tracking  . This teRecently, asymmetric-flow field-flow fractionation (AF4) was successfully used in fractionating subpopulations of exosomes from conditioned media obtained from various cell lines based on their density and hydrodynamic properties by two perpendicular flows (forward laminar channel flow and variable crossflow) . This meImmunoblotting is another commonly used molecular technique for exosome characterization that is based on the presence of specific exosomal marker proteins. The combination of cytosol and membrane-bound reporter proteins could improve the specificity of analysis. Proteins associated with the ESCRT complex , tetraspanin family , which are involved in exosome biogenesis and protein sorting, were found to be specifically enriched in vesicle preparations compared to the cell lysate ,51. A reFluorescence microscopy-based methods have been designed to provide non-invasive imaging of exosomes in vitro and ex vivo. Specifically, exosomes have been labelled using various methods: (i) staining using free dye targeting the lipid bilayer or genetically engineered fluorescent protein; (ii) using suitable affinity probes such as immunoglobulins or aptamers; (iii) co-expressing fluorescent proteins with exosome-specific proteins . A recenIrrespective of the methods used in their isolation, exosomes to be used in functional analysis or biomarker discovery should be of the highest purity possible without contaminating protein aggregates and molecules, especially when the differential expression of specific exosomal proteins is to be used as an indication of disease status. Because of the inherent drawbacks of purification techniques, it is very important to have a systematic comparison of the effect of different isolation techniques. Consistent application of isolation methods is also required to enable comparison of various studies of exosomal biomarkers.With the caveats related to exosome isolation in mind, mass spectrometry-based proteomic methods are increasingly being used to complement and extend the more targeted approaches discussed above. Both exploratory and quantitative mass spectrometry analysis of exosomes isolated from different biological sources have revealed that they each contain specific sets of proteins rather than random cellular components. In addition to a conserved set of common proteins that are essential for vesicle biogenesis, structure, and trafficking, exosomes also contain proteins that are specific for the biological source and the particular experimental condition. Identification and characterization of these proteins provide crucial information regarding the molecular mechanisms that are involved in cargo sorting and trafficking, and clues about target cells . MoreoveMass spectrometry (MS)-based proteomics (LC–MS/MS) has the potential to provide comprehensive analysis of exosome protein content. The standard bottom-up approach is the most commonly used method for MS-based exosome proteome analysis. Briefly, after exosome purification, proteins extracted from an exosome lysate are proteolytically digested and subjected to peptide fractionation through one dimensional liquid chromatography (1D LC) or two-dimensional liquid chromatography (2D LC). An efficient peptide fractionation reduces the sample complexity and helps in obtaining deeper protein coverage. Pre-analytical steps in bottom-up proteomics play a crucial role in proteome coverage and quantification. These pre-analytical methods are mainly two types; (i) gel-based and (ii) gel-free. The most widely used exosome sample preparation method for MS analysis is by separating the exosome proteome in 1D SDS–PAGE. Subsequently the gel bands are proteolytically digested and subjected to LC–MS/MS analysis ,68,69,70Recently, gel-free methods such as in-solution digestion and filter-aided digestion (FASP) protocols have gained interest in exosome proteome analysis. In the first method, following the lysis of membranes, proteins are reduced, alkylated and proteolytically digested using standard protocols ,74. Contm/z) to protein concentration based on peptide signal intensity. Quantitative proteomics can be broadly classified into two types based on the mode of acquisition and sample preparation: (i) label-free and (ii) labelled approaches.Protein identifications with reproducible and reliable quantitative data are critically important when assessing the extent of differential regulation of proteins and their potential as candidate biomarkers for a clinical condition. Quantitative high-performance mass spectrometric techniques extrapolate the protein identification obtained on the basis of unique peptide mass/charge ratio  or spectral count, which is observed in the mass spectra. Both these measures approximately correlate with the abundance of protein in a sample. Label-free quantification has been used in multiple studies to quantify exosome proteomes ,78. The 13C or 15N in lysine or arginine during protein synthesis. Quantification is based on the ratio of the intensity of each labelled peptide to its unlabeled counterpart (endogenous peptide). SILAC has been used in exosome protein biomarker studies [Stable isotope labelling by amino acids in cell culture (SILAC) is a widely used strategy for metabolic labelling of proteins in cell culture. This technique is based on the principle of metabolically incorporating a stable isotope labelled  studies ,85. SILAExosomes are potentially a rich source of biomolecular cargo. Their ability to diffuse from the site of release and be retrieved from several biological fluids, where they may reflect pathological changes of cells present in relatively inaccessible tissues such as brain, makes them a promising target for biomarker discovery. As discussed above, multiple lines of evidence suggest that these vesicular bodies are implicated in age-associated neurodegenerative processes that may progress into cognitive impairment in later life. Thus, proteomic analysis of neural-derived exosomes (NDEs) isolated from CSF or serum/plasma could open a “window into the brain”, and might contribute to identification of biomarker candidates reflecting neuropathological mechanisms and disease progression ,87,88. TAnother comprehensive study using label free quantitative proteomics coupled with a machine learning method reported a panel of four exosome proteins that could identify AD patients with 88% accuracy . These bG93A) showed expression of SOD1G93A in both the exosomal surface and lumen [G93A mouse model and a non-transgenic mouse model showed that myelin–oligodendrocyte glycoprotein (MOG) was differentially regulated in both. The authors suggested that MOG might be a potential biomarker candidate to classify patients with early signs of ALS or neurologic dysfunction [Proteomic analysis of exosome-enriched fractions isolated from the CSF of sporadic ALS patients showed a significant differential regulation of various proteins . Comparend lumen . Quantitfunction .Another recent differential proteome analysis study of CSF exosomes from ALS patients showed that the proteasome-like Bleomycin hydroxylase, which has diverse protease activity, was significantly downregulated in ALS patients. Gene Ontology enrichment analysis demonstrated downregulation of the proteasomal core complex, endopeptidase complex, and carboxypeptidase activity that together support the hypothesis of alterations in protein homeostasis in ALS pathogenesis . FurtherIn a study linked to brain injury, proteomic profiling of EVs from the CSF of former National Football League (NFL) players identified the specific enrichment of proteins from neuronal cells, oligodendrocytes, microglia, and astrocytes . AlthougProteomic analyses of exosomes are also being used in studies related to other CNS disorders. Rett syndrome, a neurodevelopmental disorder linked to autism, is caused by mutations in methyl-CpG-binding protein 2 (MECP2) . FunctioExosomes transport cargo molecules in a bi-directional manner—from the periphery to the brain as well as from the brain to the periphery—and across the blood–brain barrier (BBB). This makes them an attractive source of biomarkers originating in the CNS that can be isolated from peripheral body fluids . The mecCompared to CSF biomarkers, blood biomarkers have the major advantage of sample availability and requiring less-invasive sample collection . RecentlA quantitative proteomics study of serum-derived exosomes from patients with PD at different stages identified various proteins to be differentially regulated compared to healthy controls . Serum ePotential biomarker candidates for chronic stress that can induce depression-like behaviors in rats were identified in serum-derived small extracellular vesicles (sEVs) . ProteomEmerging studies suggest that exosomes play different roles in pathological CNS conditions by modulating transcription, neurogenesis, synaptic plasticity, neural circuit development, and neuroinflammation see . Their f


This special issue on the effects of calorie restriction (CR) and intermittent fasting (IF) on health and diseases includes five scholarly reviews and four original articles that provide an insight into the molecular and cellular action mechanisms of epigenetically manipulated dietary paradigms.CR and IF are well publicized methods for health promotion and disease prevention. An interesting aspect shared by both is that their efficacy is demonstrable without modification of any dietary ingredient. CR, also often called as restriction in dietary intake, provides dietary allocation  to the experimental group .One unique feature of CR, first proposed as growth retardation in 1935 . AlthougRecent progress in Omics technologies has contributed to technical improvement that allows analysis at the level of the RNA, DNA, protein, and other cellular molecules and their intricate associations during aging. Therefore, it is now feasible to both resolve and coordinate complex biological processes during aging using recent methods, such as next-generation sequencing, proteomics, lipidomics, metabolomics, and epigenomics techniques. Systems biology and Omics integration can predict CR effects, cellular signaling pathways, and action mechanisms. These technical advances made in recent years provide a much-needed database on the regulation of inflammatory process. An expanded view of the inflammatory response in aging progression dubbed as “senoinflammation,” which describes both chronic inflammation and metabolic dysregulation, was suggested in our previous work . Based oIF is a dietary pattern alternating between fasting and non-fasting periods. Specifically, the IF diet in a particular mouse strain extended both mean and maximal lifespans . For exaThe growth hormone (GF)/insulin-like growth factor-1 (IGF-1) axis is an evolutionary well-conserved signaling axis, and the mechanisms underlying its effects on aging and longevity are modulated by CR in mammals . RecentlAnother cellular activity recently that was revealed as one of the major players in CR action is the involvement of autophagy. Autophagy is an important cellular management process that maintains an optimal cellular homeostatic condition under normal physiological and abnormal pathological states. Recent developments in CR research have proposed that the initiation of autophagy is associated with beneficial anti-aging response. CR-promoted autophagy plays a critical role under physiological states by sustaining proper homeostasis in the organism. In addition, autophagy induced by CR plays a protective role in different organs and tissues under many pathological and aging-associated conditions . While CFasting is known to account for physiological changes in the endocrine organs, particularly the pancreas. The study on every other day IF implementation for up to 3 months in a developing and matured healthy organism suggested that this regimen promotes β-cell dysfunction and muscle mass loss and increases fat reserves, especially in developing 30-day-old Wistar rats. More long-term studies are needed to explain the most effective IF regimen to minimize side effects . In receIn summary, we believe that this special issue provides useful information on the current research trends regarding the beneficial effects of CR and IF on longevity and health of humans. Further exploration of the effect of CR and IF on aging and age-related diseases will provide deeper insights into the interrelationships between health and diseases and enable beneficial interventions in aging mechanisms, thus aiding the development of new therapeutic approaches to improve health, diseases, and longevity.


Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and occurs mainly in patients with liver cirrhosis. The mammalian target of rapamycin (mTOR) signaling pathway is involved in many hallmarks of cancer including cell growth, metabolism re-programming, proliferation and inhibition of apoptosis. The mTOR pathway is upregulated in HCC tissue samples as compared with the surrounding liver cirrhotic tissue. In addition, the activation of mTOR is more intense in the tumor edge, thus reinforcing its role in HCC proliferation and spreading. The inhibition of the mTOR pathway by currently available pharmacological compounds  is able to hamper tumor progression both in vitro and in animal models. The use of mTOR inhibitors alone or in combination with other therapies is a very attractive approach, which has been extensively investigated in humans. However, results are contradictory and there is no solid evidence suggesting a true benefit in clinical practice. As a result, neither sirolimus nor everolimus are currently approved to treat HCC or to prevent tumor recurrence after curative surgery. In the present comprehensive review, we analyzed the most recent scientific evidence while providing some insights to understand the gap between experimental and clinical studies. Hepatocellular carcinoma (HCC) is the most frequent primary liver malignancy and it accounts for 85–90% of all hepatic cancers. HCC is the fifth most prevalent cancer and the second cause of cancer-related death worldwide. The natural history of liver cirrhosis is tightly linked with HCC: a chronic liver insult  among others), which is able to produce inflammation of the liver parenchyma, which in turn triggers the activation of hepatic stellate cells and portal myofibroblasts, thus resulting in progressive liver fibrosis and, finally, in liver cirrhosis . Both chMutations in oncogenes and tumor suppressor genes are the main pathogenic mechanisms underlying hepatocarcinogenesis as they are responsible for disrupting cell signaling pathways. In HCC, the major pathways involved in the oncogenic process are WNT/b-catenin, Hedgehog, hepatocyte growth factor/c-MET, vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK)/ERK (or Ras-Raf-MEK-ERK) and PI3K/AKT/mTOR . Among aIn the present manuscript we aimed to review the role of the mammalian target of rapamycin (mTOR) pathway in the pathogenesis and biological aggressiveness of HCC to better understand the potential clinical impact of its inhibition by currently available drugs. In addition, we have provided some insights regarding the limitations to reproduce the beneficial effects of mTOR inhibitors observed in experimental models into human trials.The mechanistic target of rapamycin (mTOR) is an evolutionary conserved serine/threonine kinase, which belongs to the phosphoinositide 3-kinase (PI3K)-related protein kinase family and integrates environmental information into cellular responses . The maimTOR is the keystone of at least two structurally different multiprotein complexes, known as mTOR complex 1 (mTORC1) and mTORC2 ,8,9. BotmTORC1 and mTORC2 respond to insulin and related growth factors through the interconnected MAPK/ERK and PI3K/AKT/mTOR signaling pathways ,14. ManyThe mTOR signaling pathway is frequently deregulated in cancer and metabolic diseases . Some meThe liver is a paramount organ for lipid metabolism . During Alcohol intake may disrupt normal hepatic lipid metabolism and may lead to fatty liver or steatosis by promoting hepatic lipogenesis while hampering fatty acids transport and oxidation. This metabolic reprogramming in alcoholic liver disease requires the activation of mTORC1 by downregulating DEP domain-containing mTOR-interacting protein  , and by Chronic infection by hepatitis B (HBV) and C (HCV) viruses is still a common cause of liver cirrhosis, although their prevalence is expected to continue to decrease in the upcoming years due to advances in antiviral therapy. The role of the mTOR pathway in chronic viral hepatitis concerns the regulation of cellular homeostasis. HBV and HCV use the mTOR pathway to enhance autophagy  to benefNASH is the liver manifestation of metabolic syndrome and it is characterized by hepatic steatosis, ballooning, inflammation and progressive fibrosis. NASH occurs as a result of an imbalance of energy intake/consumption or when there is an intrinsic problem with the lipid storing process. Within the hepatocyte, the accumulation of fatty acids leads to the production of lipid-synthesis intermediates, which causes steatosis through the mTOR pathway deregulation . Thus, NCholestatic liver diseases result from a repeated exposure to unconjugated primary biliary acids, which may activate mTOR through the IκB kinase β (IKKβ)/Tuberous sclerosis 1 (TSC1)/mTOR pathway as demonstrated in some precancerous conditions such as Barrett’s esophagus . In the Liver cancer development in different mouse models with genetic or dietary alterations is frequently preceded by hepatic steatosis and is associated with a deregulation of the mTOR pathway ,54,55,56As described above, the mTOR pathway is systematically deregulated among the most prevalent etiologies of liver cirrhosis and it may be found overexpressed in patients with HCC, where it confers a more aggressive biological behavior. Therefore, a pharmacological and molecular inhibition of the mTOR signaling pathway has been proposed as a therapeutic strategy for HCC . Next, wUDCA is a cytoprotective secondary bile acid which forms the backbone of the therapeutic arsenal against cholestatic liver diseases, mainly primary biliary cholangitis. UDCA therapy improves liver biochemistry parameters and delays the progression of the disease to liver cirrhosis . In a huStreptomyces hygroscopicus [Rapamycin , an antibiotic first isolated in 1972 by Sehgal and Cols from the bacterium scopicus , is a poscopicus . Rapamycscopicus , which iscopicus ,76,77,78scopicus .Rapamycin has shown potent antiproliferative and immunosuppressive properties against a large variety of tumor cells in vitro and suppresses growth of cancer cells in vivo ,81,82,83mTOR kinase inhibitors (TOR-KIs) are second-generation mTOR inhibitors, which emerged to solve the above referred limitations of rapalogs. TOR-KIs appear to be more potent than rapalogs because they inhibit the activity and associated functions of both mTOR complexes . Several of these compounds are currently being tested in preclinical experiments and early clinical trials ,87,88. HSorafenib is a multitarget kinase (multi-kinase) inhibitor that, among its multiple action mechanisms, interferes with the AKT/mTOR pathway by targeting VEGFR, PDFGR, c-Kit, c-RAF and B-RAF . SorafenThe oncogenic signaling pathways of HCC are dynamic and able to adapt to new situations by creating compensatory mechanisms, which provide tumor cells with the ability to evade individual drugs. Accordingly, new approaches based on the combination of different inhibitors targeting complementary critical points of these interconnected pathways have been tested. Yu et al.  recentlySorafenib produces heterogeneous cellular responses depending on the genomic context or mutational profile of the tumor. When sorafenib is used in combination with specific-targeted inhibitors driven from a close analysis of the mutational cell background, the observed effect was synergic regarding the inhibition of HCC proliferation. This fact could partially explain the suboptimal results of sorafenib, alone or in combination, in clinical trials where a heterogeneous and uncharacterized group of patients was included . A complRapalogs and TOR-KIs are associated with feedback loops that promote the (re)activation of these signaling pathways, thus limiting their therapeutic use. The simultaneous inhibition of both, mTORC1 and MAPK, or mTORC1/mTORC2 and RTKs, has been shown to enhance the effectiveness of each compound separately in preclinical models (in vitro and in vivo) ,89. The Recently, two-step senescence-focused therapies have been proposed as anti-cancer therapy . In thisAltogether, these results highlight two critical points to select the best therapeutic strategy in HCC. First, to define the components and relationships of the signaling pathways involved in tumor formation, and second, to know the genomic mutations affecting each patient. This knowledge would allow the tailoring of antiproliferative agents according to each patient and tumor characteristics, thus resulting in more effective and safer therapeutic approaches.The anti-proliferative properties of mTOR inhibitors in preclinical models would be of high value in patients with HCC as they would allow to prevent tumor recurrence after potentially curative therapies , or to delay tumor progression in patients with a more advanced disease. Unfortunately, clinical studies are scarce and with reduced methodological quality. Most of the studies are single-centre retrospective observations and those with a prospective and/or randomized design have failed to prove any benefit in terms of recurrence free survival and/or overall survival.In patients with unresectable or metastatic HCC, the mTOR inhibitor everolimus has been studied in combination with sorafenib. A phase I randomized trial was designed to explore the maximal dose of everolimus tolerated in combination with the standard dose of sorafenib . A total of 30 patients with compensated liver cirrhosis Child Pugh class A were included. The maximal tolerated dose of everolimus was only 2.5 mg per day. The combination of everolimus and sorafenib did not result in improved overall survival. The authors concluded that a phase II trial was not justified since a biologically active anti-proliferative dose of everolimus cannot be achieved in patients with liver cirrhosis, even in those with a preserved liver function . Despiten = 525) and with prolonged follow-up, have failed to demonstrate any benefit in reducing tumor recurrence rates after liver transplantation [In earlier stages of HCC, when the tumor is confined to the liver, liver transplantation may be the best therapeutic option as it allows for curing both, the tumor and the underlying liver cirrhosis . Howeverantation . This gaantation . Those pantation , are sysantation . The useantation .In light of the available evidence, mTOR inhibitors  have not been approved to treat or to prevent HCC and they do not form part of the routine clinical armamentarium. In clinical practice, everolimus is used as an immunosuppressive agent in liver transplant patients for a more effective preservation of kidney function. Many institutions have implemented everolimus-based immunosuppression in patients with HCC undergoing liver transplantation in order to prevent tumor recurrence, even in the absence of solid scientific supporting evidence. There is a need for more potent mTOR inhibitors and with an improved safety profile. The role of second-generation TOR-KIs in HCC is still to be explored. Since HCC occurs mainly in the context of chronic liver disease, randomized controlled trials may be conducted in patients with liver cirrhosis to study efficacy and safety.The mTOR signaling pathway is involved in cell metabolism and proliferation, which are key hallmarks of cancer and allow for HCC development, progression and spreading. The mTOR pathway inhibition exerts a pronounced antiproliferative effect in vitro and in animal models of HCC. However, clinical studies, especially those prospective and/or randomized, have failed to demonstrate any capacity of mTOR inhibitors to hamper tumor progression or to prevent tumor recurrence after potentially curative therapies such as liver transplantation. The gap between experimental and clinical observations may be partially explained by the dose-dependent toxicity of mTOR inhibitors in patients with chronic liver disease, which frequently motivates a significant dose reduction or drug withdrawal. There is a need to develop more potent mTOR inhibitors and with an improved safety profile in order to obtain a true antiproliferative  effect in patients with HCC. A detailed molecular profiling of HCC will probably become a powerful tool to select patients with HCC to receive mTOR inhibitors as part of a true personalized medicine.


Gliomas are a dismal disease associated with poor survival and high morbidity. Current standard treatments have reached a therapeutic plateau even after combining maximal safe resection, radiation, and chemotherapy. In this setting, stem cells (SCs) have risen as a promising therapeutic armamentarium, given their intrinsic tumor homing as well as their natural or bioengineered antitumor properties. The interplay between stem cells and other therapeutic approaches such as nanoparticles holds the potential to synergize the advantages from the combined therapeutic strategies. Nanoparticles represent a broad spectrum of synthetic and natural biomaterials that have been proven effective in expanding diagnostic and therapeutic efforts, either used alone or in combination with immune, genetic, or cellular therapies. Stem cells have been bioengineered using these biomaterials to enhance their natural properties as well as to act as their vehicle when anticancer nanoparticles need to be delivered into the tumor microenvironment in a very precise manner. Here, we describe the recent developments of this new paradigm in the treatment of malignant gliomas. Gliomas are a dismal entity, associated with poor survival and high morbidity. The current standard of care has reached a therapeutic plateau even after combining maximal safe resection and chemoradiation . In thisGliomas are the most common and devastating primary brain tumors, representing approximately 75% of these. According to the World Health Organization (WHO), gliomas are classified in four histological grades (I–IV), being the glioblastomas the corresponding WHO grade IV tumor. Glioblastoma (GBM) is the most common and aggressive among all gliomas, accounting for 57.3% of the tumors in this group, with around 12,500 new cases diagnosed every year only in United States . GlioblaThe current gold standard for the treatment of gliomas includes surgery and chemoradiation. Maximal safe resection is advised in all cases regardless of the WHO grade, given that the overall survival is positively correlated with the extent of resection (EOR) . HoweverA subset of gliomas cells have been pinpointed as the culprit of this recurrence. The glioma cancer stem cells (CSCs) are a subgroup of malignant cells with the potential of self-renewal, forming tumors that resemble the original pathology, as well as high resistance to current chemotherapeutics and radiation . These cin vitro and can be bioengineered to deliver a wide range of antitumor payloads such as proteins, oncolytic viruses, prodrugs, small interfering RNA (siRNA), and nanoparticles , mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs) are the most common multipotent stem cells used with this purpose (pluripotent cells such as the induced-pluripotent stem cells (iPSC) or embryonic pluripotent stem cells have also been described in cell therapy against glioma  were first described by Friedenstein more than 50 years ago . Furthermore, both groups were able to bioengineer the MSCs to deliver antitumor cargoes. To date, several reports on the use of MSC as key elements for glioma stem cell therapy have been published with exceptional promising results .On the other hand, not all are in agreement, as there has been a risen controversy in which some authors have described that MSCs could eventually support glioma tumor growth. Different types of MSCs such as BM-MSC, A-MSC, and UC-MSC have been associated with these proglioma effects through increased proliferation, cancer cells migration, angiogenesis, transition to epithelial–mesenchymal phenotype, and decreased glioma apoptosis ; howeverIn order to improve different aspects of stem cell therapies against glioma, different approaches have been studied. The use of nanoparticles for stem cell bioengineering is one of these potential approaches and will be discussed in the following section.Nanoparticles (NPs) are defined as nanomaterials sized between 1 and 100 nm in at least one of their external dimensions, which confer them a high surface/volume ratio . Due to In Nano-oncology, nanoparticles represent an important diagnostic and therapeutic tool, as they can be designed to interact with most biological system with great precision and specificity. This is possible due to their particular physicochemical characteristics and the possibility of making them able to target a specific tissue, specific cell types, or a specific cellular compartment  . The potligand targeting works by coating the nanoparticles’ surface with one or more ligands such as transferrin, epidermal growth factor (EGF), folic acid, arginyl-glycyl-aspartic tripeptide (RGD) peptide, hyaluronic acid, antibodies, and others (ligand targeting in glioma research).In order to achieve a targeted distribution at a cellular or even intracellular level, NPs can be functionalized via active targeting. Active targeting is achieved by different methods; a method called d others . These ltranscriptional targeting, which can occur at a transcriptional or posttranscriptional level  (PGA), poly(lactic acid) (PLA), poly(caprolactone) (PCL), and poly(lactic-co-glycolic acid) or (PLGA); polyalkyl alcohols such as polyvinyl alcohol or PVA; and polyethers such as poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG)  (PPG) . Currentl) (PPG) . Up to dl) (PPG) . This inl) (PPG) , as welll) (PPG) .magnetic nanoparticles, which are usually based in a core of iron oxide mNP with a large magnetic momentum under an external magnetic field, which allow its use as MRI contrast enhancer and thermotherapy agents (Plasmonic nanoparticles refer to mNPs such as gold (Au) or silver (Ag) NPs presenting with surface plasmon resonance (SPR), meaning that NP free electrons can be excited by electromagnetic fields (UV or infrared light) and resonate, creating the possibility to sense these changes (biosensors), produce heat , or create technologies such as surface-enhanced Raman spectroscopy (SERS) (Quantum dots (Qdots) are another group of inorganic NPs, usually smaller than 50 nm; these semiconductor NPs efficiently produce bioluminescence once excited by UV light, which has led them to be used in single cell and in vivo imaging , inertness, high stability, and easy functionalization, which give them different advantages when compared to organic NPs. Due to their cellular internalization ability and low immunogenic response, these nanoparticles were initially used as drug and gene delivery systems . Differey agents . Plasmony (SERS) . Quantum imaging .These nanoparticles are predominantly composed by carbon, and their discovery revolutionized diverse scientific fields . Carbon-Single nanoparticles are made of a single element such as gold, silver, copper, among others, and due to their homogeneity and electrochromic properties, they are widely used in electro-optical applications, energy conversion, and storage . Diversecore–shell (CS), when a central core (NP) is surrounded by one or more layers of different material [shell(s)], or as yolk–shell (YS). when the a movable core is located in a hollow cavity surrounded by a shell , genes, peptides, and others that can now be released in a controlled manner. Janus nanoparticles are a different type of nanomaterials; they possess a tunable asymmetric structure; their surface has two or more regions with different properties, which confer them unique properties as selective reactivity or directional interactions. The field of application is broad and innovative including its use as sensors, self-propelled carriers, or coatings  by encapsulating radiosensitizer hydrophobic drugs [aniopep-2-poly-(metronidazoles)n and doxorubicin (DOX)] in hydrophilic polymers (PEG2000); these NPs were functionalized to target gliomas cells and release its content only under hypoxic conditions, increasing radiosensitization as shown stration ; Table 3ligand targeting, or improvements on the enhanced permeability and retention (EPR) effect are required.The restricted permeability of the BBB has been one of the biggest challenges in the mission of effectively treating brain tumors. In nanomedicine, not all nanoparticles can efficiently cross this biological barrier despite their size and physicochemical characteristics; consequently, previously discussed strategies, such as ligand targeting is an active targeting method and required NPs to be designed with this purpose in mind, EPR effect refers to a passive targeting mechanism common to all NPs. EPR effect relies on pathophysiological characteristics of tumor vs. healthy vessels as well as NP size, which is larger than individual conventional chemotherapeutics . Due to their relatively larger size, NPs are not able to penetrate normal blood vessels but can easily cross diseased vessels such as those presented in brain tumors, leading to a selected distribution into cancer tissues. NP with diameters of at least 5–10 nm present reduced kidney excretion , prolonged blood half-life, and better accumulation in the tissue of interest. For instance, the plasma half-life of doxorubicin increases from 5–10 min to 2–3 days when this is encapsulated into liposomes. In order to achieve better results from the application of organic nanoparticles such as liposomes, micelles, etc., some polymers such as PEG can be used to decrease NP aggregation and opsonization by plasma proteins, thus adding to the improved blood half-life  and fibroblast growth factor (FGF) . Thus, LOverall, all the previously described strategies should be carefully weighted when trying to optimize the use of nanoparticles as nanocarriers. In this setting, when applied to an orthotopic glioblastoma model, the use of functionalized biodegradable polymeric nanoparticles coated or loaded with anticancer drug has been able to confer longer survivals in preclinical models . Among aOverall, these results point the use of nanocarriers as a promising enhancer of effective therapies for the treatment of patients with glioma .Nanomachines or nanobots are molecular self-propelled nanodevices considered as smart delivery systems that respond to specific triggers . DNA nanin vivo tracking after stem cell delivery. On the other hand, apart from nanoparticle surface modifications performed in an attempt to improve pharmacokinetics and pharmacodynamics parameters, stem cells appear as a reasonable option to overcome the suboptimal penetration, distribution, and retention associated to some nanomaterials when used as therapeutic nanocarriers. The use of stem cells in this context definitely add another option for a more targeted nanoparticle delivery. Thus, the benefit obtained from this combined approach using nanoparticles and stem cells is bidirectional.Nanomedicine has extend the reach to several cancer treatment approaches such as radiotherapy, chemotherapy, immunotherapy, and others. In the case of stem cell therapies, improvements in several aspects are clearly needed. In an attempt to consolidate the translational potential of this approach, nanoparticles have been used to enhance safety and efficacy, stem cell tumor homing, and Stem cells are known by their ability to serve as vehicles of antitumor cargoes. For this purpose, viral gene vectors have been traditionally used to transduce stem cells with a high degree of gene delivery efficiency resulting in constant payload production . Although newer generations of viral vectors present better safety profiles, these vectors have been associated with immunotoxicity as a response to viral proteins production or potential viral replication. They also would carry the hypothetical risk of uncontrolled viral genome integration and insertional mutagenesis, latent virus activation, and inflammatory responses leading to demyelination or neurodegeneration . In thisOur group reported on the use of biodegradable polymeric nanoparticles based on poly(beta-amino ester)s (PBAEs) to enable effective BMP4 gene delivery on human A-MSC, allowing for higher transfection rates than those of commercially available reagents. Transfected MSC retained their multipotency and their tumor-homing capacity and were functional, leading to extended survival in a rat orthotopic GBM model . Huang eIn the treatment of several malignancies, different nanoplatforms acting themselves as anticancer agents  or as caFurthermore, even if researchers could secure that nanoparticles reach the glioma tumor bulk, there exist other potential drawbacks that are imperative to highlight; they are related to the presence and location of glioma cancer stem cells (1) Nanoparticles are neither able to track infiltrative glioma cells leaving the tumor bulk to colonize distal healthy brain parenchyma nor (2) to reach the necrotic glioma core where blood flow is impaired. These areas do not present an EPR effect, which would facilitate nanoparticles to distribute across other areas of the tumor. Allowing nanoparticles to access the hypoxic central core would be crucial, as the treatment-resistant subpopulation of glioma cancer cells would predominantly locate in that area . Even afIn this scenario, coupling stem cell therapy to nanotherapeutics offers the possibility to solve the previously stated dilemma regarding the inadequate distribution of therapeutic nanoparticles to the hypoxic glioma core and distant infiltrative tumor foci. Thus, stem cells could extend the reach for nanoparticles to penetrate these areas. This approach implies nanoparticles to be conjugated to stem cell surfaces or internalized before migrating toward malignant gliomas. Furthermore, the internalization of nanoparticles inside stem cells would allow them to be up to fivefold larger than the usual nanoparticles used in cancer therapy, without entailing problems in crossing the BBB or a higher risk to be engulfed by macrophages or lymphocytes . This inneural stem cells were able to improve intracranial nanoparticle retention and tumor-selective distribution in an in vivo model by coupling huge nanoparticles to NSC surface  were loaded into BM-MSC. Osteogenesis, adipogenesis , and tumor homing were not affected by nanoparticle inclusion. The approach was associated with improved survival in a rat orthotopic glioma model when the modified MSCs were injected in the contralateral hemisphere , platelet-derived growth factor (PDGF), VEGF, tumor growth factor β1 (TGF-β1), interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and stromal cell-derived factor 1 alpha (SDF-1α) as well as stem cells surface proteins such as CD44, CXC chemokine receptor 4 (CXCR-4), integrin α4, and TGF-β receptors have been associated with increased MSC homing in gliomas (Several  gliomas .in vitro Boyden chamber experiments (in vitro and in vivo glioma models was found, and the SDF-1/CXCR4 signaling axis was associated to this phenomenon (The impact on stem cell behavior after being loaded with nanoparticles for different purposes has not been the principal focus of research. However, there already exist reports describing the increase in migration toward cancer cells after loading hMSC with iron oxide nanoparticles. This would be related to the overexpression of EGFR observed after nanoparticle inclusion and the characteristic elevated production of EGF by colon cancer cells used in the eriments . Interesenomenon .Stem-cell-based therapies rely on the ability of the grafted cells to target the organ of interest. In case of malignant gliomas, it is crucial to ensure stem cell tumor homing. In the preclinical setting, conventional methods for tracking migration and final fade of stem cells are traditionally based on bioluminescence imaging; however, poor spatial distribution and lack of translational applicability made necessary to establish a reasonably translational method that can be easily applied in a clinical setup.Cellular MRI-based tracking technologies have risen as gold standard for non-invasive, real-time monitoring of transplanted stem cells . This apThe conjugation of stem cells and SPIONs has allowed for tracking MSC migration and homing into glioma tumors in a rodent glioma model without compromising such migratory capacity . NSCs haEven when this approach was first evaluated in the clinical setting around 2006  and has Challenge and pitfalls associated with this relatively novel approach is proper of any disruptive technology. The ethical concerns associated with the use of particular stem cells, while seemingly addressed with modern techniques, need to be further discussed before extensive use can be assumed . ClinicaThe introduction of the nanotechnology in stem cell therapies has shown to be beneficial and hopefully will keep turning stem cell therapies into a less worrisome and more controlled therapeutic strategy. To date, we have explored NP for stem cell bioengineering and cell tracking; however, we believe that their malleability allows for further uses such as the ones previously described, alone or in combination, and even for stem cell functionalization .Finally, the combination of these therapies should not be limited to only nanoparticles and stem cells; this combined approach will need to explore if further value can be obtained by coupling with additional fields such as radiotherapy, thermotherapy, targeted systemic therapies, focused ultrasound, and other novel diagnostic techniques such as ultrahigh magnetic strength imaging and novel radiotracers in order to maximize its benefits.HR-G and KA-E screened titles for relevance and abstracted the data from the eligible full text articles. SK, AQ-H, and DT critically revised the manuscript with input from the entire team. HR-G and KA-E created the figures and tables. HR-G, KA-E, and DT worked on study conception and design. All authors analyzed and interpreted the data, drafted the manuscript, and read and approved the final draft.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.


By using the CCEP connectivity pattern intraoperatively, mapping and real-time monitoring of the dorsal language pathway is available. This intraoperative CCEP method also allows for mapping of the frontal aslant tract, another language pathway, and detection of connectivity between the primary and supplementary motor areas in the frontal lobe network. Intraoperative CCEP mapping has also demonstrated connectivity between the frontal and temporal lobes, likely via the ventral language pathway. Establishing intraoperative electrophysiological monitoring is clinically useful for preserving brain function, even under general anesthesia. This CCEP technique demonstrates potential clinical applications for mapping and monitoring large-scale cortical networks.To preserve postoperative brain function, it is important for neurosurgeons to fully understand the brain's structure, vasculature, and function. Intraoperative high-frequency electrical stimulation during awake craniotomy is the gold standard for mapping the function of the cortices and white matter; however, this method can only map the “focal” functions and cannot monitor large-scale cortical networks in real-time. Recently, an  To preserve brain function postoperatively, it is necessary to identify and have a thorough understanding of the neural connectivity between cortical eloquent areas. This is important to help neurosurgeons in maximally preserving brain function when surgically treating brain lesions.Intraoperative high-frequency direct electrical stimulation (DES) is a popular method to detect eloquent areas during brain resection. During awake craniotomy, DES introduces a biphasic constant-current stimulation (50–60 Hz) whilst optimizing intraoperative tasks  -based diffusion tractography. Diffusion tractography allows the visualization of in vivo large white matter pathways  in mapping functional brain networks and evaluate its clinical utility in preserving brain function intraoperatively.The CCEP technique was originally introduced as an extra-operative procedure preceding epilepsy surgery  and a consecutive slow negative potential (N2: peak latency 50–300 ms)  cortico-cortical propagation is directly conveyed through white matter fibers; and (2) cortico-subcortico-cortical propagation is indirectly conducted via subdural or depth electrodes  and somatosensory evoked potentials (SEPs) can map the motor cortices and output pathway , and the sensory cortices and ascending pathway, respectively, under general anesthesia  Subdural electrodes were covered on the ventrolateral frontal and lateral temporoparietal cortices, which were localized based on anatomical criteria or by using preoperative neuroimaging studies .(2) Single-pulse ES was applied to the ventrolateral frontal cortices. Based on the CCEP distribution (the largest CCEP connectivity) in the lateral temporoparietal cortices, we determined the stimulus site (the putative AL) and recording site (the putative PL).PLAL→ (stimulating the AL and recording CCEPs from the PL) monitoring was performed under general anesthesia, and in the awake condition by measuring the N1 amplitude at the largest CCEP response site.(3) To evaluate the integrity of the dorsal language pathway, online sequential CCEPALPL→ was recorded.(4) When available, DES was also applied only to the AL to confirm the language function, due to the time limitation in the intraoperative setting. To identify the reciprocal connection between the AL and PL, single-pulse ES was also applied to the electrode in the lateral temporoparietal area, and CCEPPLAL→ responses are observed to have a larger distribution than CCEPALPL→ responses  and the medial motor cortex (including the SMA) are detected using CCEPs  to the candidate electrodes through a pair of adjacent electrodes, and record the MEP of the contralateral extensor carpi radialis or first dorsal interosseous to detect the M1.Single-pulse ES is applied to the detected M1, and CCEPs are recorded from the medial frontal cortices, to probe the cortico-cortical connections between the M1 and the SMA proper. The SMA proper is defined by distinguished CCEP N1 responses in the medial frontal area. A 5-train ES is delivered to the estimated SMA proper, to confirm the SMA proper by recording MEPs from the upper and lower extremities.In a previous study, patients with preservation of the identified SMA proper did not display any new neurological deficits during or after surgery (Shibata et al., Intraoperative CCEP has some limitations. First, the data were obtained from patients with tumors or epilepsy. Their backgrounds, with regard to drugs, including antiepileptic drugs, location and pathology of the tumors, the grid coverage areas, and stimulus intensities varied across studies, and could have affected the CCEP results. Second, the subdural electrodes used have limitations in terms of spatial resolution and exploration of the cortical sulci (Ookawa et al., Many institutions, including our own, have recently reported the use of CCEPs for intraoperative mapping to preserve brain function, such as language and motor function. Although alternative preoperative neuroimaging studies, i.e., functional MRI or diffusion tractography, are still required, intraoperative CCEPs may allow efficient mapping and monitoring of functional networks. This single-pulse ES method may represent a new intraoperative monitoring technique to preserve language or motor function under general anesthesia only, analogous to MEPs or SEPs. We hope that further studies will establish intraoperative CCEP methods under general anesthesia.YY: data collection, literature review, and manuscript writing. RM, TKi, and TKu: data collection and manuscript review. KY and SM: supervision. All authors contributed to the article and approved the submitted version.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.


Bio-imaging is a tedious task when it concerns exploring cell functions, developmental mechanisms, and other vital processes in vivo. Single-cell resolution is challenging due to different issues such as sample size, the scattering of intact and opaque tissue, pigmentation in untreated animals, the movement of living organs, and maintaining the sample under physiological conditions. These factors might lead researchers to implement microscopy techniques with a suitable animal model to mimic the nature of the living cells.Zebrafish acquired its prestigious reputation in the biomedical research field due to its transparency under advanced microscopes. Therefore, various microscopy techniques, including Multi-Photon, Light-Sheet Microscopy, and Second Harmonic Generation, simplify the discovery of different types of internal functions in zebrafish. In this review, we briefly discuss three recent microscopy techniques that are being utilized because they are non-invasive in investigating developmental events in zebrafish embryo and larvae. Imaging a live organism is not easily accessible because matching a suitable animal model with a proper microscopy technique is challenging. In this context, different advanced microscopy approaches on different animal models were used either to explore the etiology of diseases or to explore the feasibility of discovering new drugs. However, some of these techniques suffer severe limitations.The most advantageous features of zebrafish are its transparency, its amenability to optical imaging, in addition to the ease of generation of transgenic lines. These features lead zebrafish to be an excellent animal model for addressing questions for in vivo studies in vertebrates. Different applications were implemented for this purpose, especially advanced microscopy techniques such as Fluorescence Correlation Spectroscopy (FCS), Multi-Photon Microscopy (MPM), and Light-Sheet Microscopy (LSM) –3. ReachHere, we briefly discuss the engagement of zebrafish in three models of advanced microscopy techniques: Two-Photon Microscopy (TPM), Second Harmonic Generation, (SHG), and Light-Sheet Microscopy (LSM). Different reasons were behind selecting these techniques include non-invasiveness, the high resolution in imaging intact biological samples with deep penetration, and success in imaging live zebrafish embryos and larvae without affecting their viability or behavior., and scientists performed successful labeling for apoptotic cells by genetically encoded fluorescent reporter protein. As an exceptional animal model, the authors described the patterns of apoptosis and the neuronal cell death at a single-cell resolution [Transparency of zebrafish is a dominant feature; therefore, many recent studies implemented zebrafish with advanced microscopy to explore significant events related to vertebrate development, functional genomics, organ functions, behavior, toxicology, and drug discovery –6. Live solution .Later, apoptosis was investigated in live zebrafish larvae through implementing a combination of Fluorescence Lifetime Imaging (FLIM) with Optical Projection Tomography (OPT). In this work, the application of FLIM OPT was a pioneer in monitoring apoptosis over time in vivo with genetically expressed Förster Resonant Energy Transfer (FRET) biosensors .2+ and cAMP at low resolution by expressing dual FRET sensors. The FRET was rapid, robust, and compatible with a varied range of sensors for functional microscopy in vivo. Therefore, it was proposed that FRET would facilitate the imaging of complex cellular systems in whole live organisms [+ (Pd2+) in living zebrafish larvae by FRET, with a significant advantage in monitoring Pd2+ due to that RN3 possessed excellent biocompatibility and low cell cytotoxicity [Zhao and colleagues utilized probes in live zebrafish embryos with a high spatial resolution to verify a functional imaging method through multiple (FRET). They showed a possibility to measure the levels of Carganisms . A new rtoxicity .The new high-resolution and non-invasive imaging techniques on transparent zebrafish embryos enabled scientists to visualize morphogenetic activities during the early development of zebrafish and understand the cell membrane and its organization in a whole animal model , 12. ManCalcium imaging in the zebrafish in vivo included a two-photon microscope, as reported by Renninger and Orger 2013. Genetically encoded calcium indicators have successfully unlocked the constrains in recording neural population activity in zebrafish. Two-photon Microscopy enabled imaging the whole brain with single-cell resolution, generating interesting brain-wide functional maps in zebrafish .By using deep Two-Photon Microscopy in zebrafish, microdomains in polarized epithelial cells were visualized by Laurdan dye, where the membrane order was quantified in the gut, kidney, and liver ductal epithelia during different developmental stages, from 3 to 11 dpf (days post-fertilization). It was found that the membrane order of polarized epithelial cells of the three types of tissues mentioned above is significantly changing during development with high membrane orders acquired at 6 dpf stage Fig. . This obDespite many advantages of Two-Photon Microscopy, there are also some drawbacks to such a technique. For example, photobleaching and photodamage are the most common artifacts at the focal volume, where photochemical interactions occur. Due to the high light exposure on the sample, it may induce unfavorable heat; this may lead to direct damage and considerable morphological changes to the sample, especially in pigmented tissue . But thiA higher harmonic generation technique, which includes the Second and Third Harmonic Generation, is noninvasive imaging utilized in zebrafish with a precise penetration, in the millimeter range, and sub-micron three-dimensional resolution Fig. .Fig. 3aIn this technique, complex in vivo developmental processes in zebrafish embryos were successfully detected without any pretreatment within less than 1-mm-thick. Even with high illumination and long-term observations, no visible damage was detected, and no adverse effect was found on embryos until the larval stage. The three-dimensional resolution enabled the researchers to acquire images of cellular processes occurring inside embryos and larvae. This effective technique provided a glance at the dynamics of cytological construction during embryogenesis, which added valuable findings to the research of developmental biology , 25. ForSecond Harmonic Generation (SHG) was employed to study the organization of collagen during the fin wound healing in zebrafish. The outcome of this work proposed the importance of collagen fibers during fin re-growth . In a siThe transparency, genetic liability, and availability of fluorescently tagged immune cells of zebrafish with using the SHG imaging provided new perceptions to tissue regeneration in wound healing in a live animal.Despite the effectiveness of SHG microscopy in imaging biological tissue, it still suffers from some limitations. One of these limitations is the restricted penetration depths (100–300 μm with laser excitation in the 800–1000 nm range). Consequently, this might make SHG microscopy inappropriate for some biological applications, mainly when the region of interest is deeply located within the tissue or organ; thus, it is unreachable by SHG. The biggest issue in SHG microscopy is that it can image only a few structural proteins or harmonophores such as collagen types I & III, actomyosin complexes, cholesterol crystals (ChC), centrosomes and mitotic spindles. Unfortunately, to date, scientists could not find a method that can discriminate between fibrillar collagen types, which could enhance wound repair and regeneration biology . HoweverBy exploiting the optical features of zebrafish, Light-Sheet Microscopy (LSM) became a preferred technique in imaging embryonic development. LSM owns various strengths such as deep optical sectioning, high frame rates, faint excitation intensities, minimal phototoxicity, moderate mounting techniques, rapid imaging with an efficient signal to noise ratio, and imaging specimens from different angles for a prolonged time , 31 Fig. In 2008Genetically encoded calcium indicator GCaMP5G was applied by Light-Sheet Microscopy to study the in vivo imaging of more than 80% of neurons in zebrafish larval brains. In a study, two populations of neurons with correlated activity patterns were successfully identified . Also, Lz stack and a large field of view to display the main morphogenetic relocations followed the changeover from optic vesicle to the optical cup stage [LSFM requires taking a large up stage . Wild tyup stage . Hence zup stage .Due to limitations in the optical range and scanning speed in confocal or Two-Photon Microscopy, it was challenging to image the whole zebrafish embryonic brains. Light-Sheet Fluorescence Microscopy (LSFM) was an efficient approach in imaging the entire head of live transgenic zebrafish embryos. By creating an inclusive 3D maps, researchers were able to image cranial neurons and blood vessels during zebrafish embryogenesis by LSFM. Blood cells circulating through the entire head, vagal, and cardiac vasculature at high resolution in a 3D were visualized, this came up with establishing a complete 4D atlas of embryogenesis and neural activity in zebrafish .Although LSFM is the only technique allowing volumetric imaging without exposing the sample to light outside the focal plane, LSFM frequently needs modifications in the experimental design and has illumination issues . When hiThe principle, advantages, disadvantages, and the applications of the Two-Photon, Second Harmonic Generation, and Light-Sheet Microscopy are tabulated in Table .Table 1Despite arguments and discrepancies about the visibility of using zebrafish as a research model, different zebrafish studies have been performed without changing its viability or behavior. Therefore, it is proven that zebrafish is a promising model in the microscopy field with a variety of applications due to its robust nature, its optical amenability, and genetic traceability.Two-Photon Microscopy is a promising approach in studying different topics in live zebrafish. Photobleaching within the focal volume is the main weak point of Two-Photon Microscopy when a proper laser intensity is used; the photodamage to the zebrafish organs during image acquisition is nearly absent.Two-Photon also showed a virtuous capability in zebrafish embryo imaging; hence, it is considered a powerful tool for addressing current issues in systems biology and high-content experimental investigation of embryonic development.The Second Harmonic Generation (SHG) has many advantages. It is suitable to image intact and living tissues without labeling. Besides, its near-infrared excitation is nonlinear, which enables optical sectioning with minimal autofluorescence background and minimal photochemical damage of biological samples. SHG involves no absorption because it is a non-resonant process. The constraint of weak signal intensity of harmonic generation has been fixed by increasing the repetition rate of the excitation light., mainly collagen and the muscle sarcomeres of zebrafish during its early development. It became more efficient in zebrafish when it used with genetically encoded fluorescent tags. There is a potential advantage of combing SHG with THG as this could identify gene expression levels in the embryonic nervous system in zebrafish in addition to recording and imaging the performance of the cell during the developmental stages of the zebrafish embryo  enables rapid imaging, high signal-to-noise ratios, and minimum rates of photobleaching and toxicity, three-dimensional imaging of live samples. These capabilities allowed scientists to quantify cellular dynamics in entire embryos throughout their development. LSFM owns an advanced ability, such as 4D in vivo imaging to reveal volumetric cellular structures in tissues during development. The LSFM has been combined with Structured Illumination (SI) to eliminate scattered and out-of-focus light. This powerful combination enhanced the resolution and contrast in the vertical and axial directions during imaging neural activity at the zebrafish larval stage.In summary, each of these three advanced microscopy techniques owns its advantages and disadvantages when it was implemented in vivo studies. In the future, we recommend for further improvement by combining two different modalities for addressing more effective visualization. A successful model was an integration of one and Two-Photon scanned oblique plane illumination microscopy that provided light-sheet scanning based rapid volumetric imaging ability at subcellular resolution. High-resolution nonlinear optical microscopy with zebrafish as an animal model can revoke many challenges that limit detecting changes at the tissue structure during pathoetiologies.


One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived xenografts to study prostate cancer. We describe emerging approaches using primary prostate epithelial cells and prostate organoids and their genetic manipulation for disease modelling. Furthermore, the use of human prostate-derived induced pluripotent stem cells (iPSCs) is highlighted as a promising complimentary approach. Finally, we discuss the manipulation of iPSCs to generate ‘avatars’ for drug disease testing. Specifically, we describe how a conceptual advance through the creation of living biobanks of “genetically engineered cancers” that contain patient-specific driver mutations hold promise for personalised medicine. The development of experimental models that accurately recapitulate cancer is crucial for the study of cancer biology and development of therapeutic treatments. This is a daunting challenge given the complexity and heterogeneity seen in many cancers, including prostate cancer, which results in variation in the curative effects from person to person ,2. ProstProstate cancer is a biologically heterogeneous disease and its complex nature provides a significant challenge for its clinical management. The nature of prostate cancer heterogeneity is characterised by interpatient, intertumoural , intratumoural, genomic and epigenetic heterogeneity, which raises considerable challenges when developing therapies . NeverthInduced pluripotent stem cell (iPSC)-based disease modelling has proven to be a powerful tool in biomedical research and personalised regenerative medicine by improving the understanding of the disease pathophysiology of various human inherited disorders at the cellular level. The emergence of three-dimensional multi-layered organoids has attracted widespread interest and has presented a unique opportunity for high throughput drug discovery which, combined with genome editing, has become an attractive model for cancer research. This review focuses on current prostate cancer preclinical models and how recent developments and the potential manipulation of human induced pluripotent stem cells (iPSCs) could hold promise for prostate disease modelling and personalised medicine.A large proportion of basic cancer research today is still undertaken in cancer cell lines . Whilst Primary benign human prostate cells can be cultured in vitro with relative ease from fresh patient biopsies. Isolating primary human epithelium (which grows from either acini structures or single cell digestions) and stroma  has long been a mainstay of translational research scientists ,39. HoweProstate cancer-derived xenografts (PDXs) involve implantations of cells or tissue from a patient into immuno-deficient mice. The efficiency is improved with co-engraftment with mesenchymal tissue  and into a vascular niche  ,44. PDXsOrganoids are in vitro 3D structures which authentically recapitulate the in vivo architecture, molecular make-up and function of the tissue of origin . OrganoiFailure to translate findings from preclinical models into the patient setting has been one of the factors contributing to the low success rate of anti-cancer drugs making it from the bench to the clinic, and highlights the need for more accurate experimental models . The usePDOs have served as a platform for cancer drug screening with studies demonstrating correlation between in vitro drug sensitivities and patient tumour molecular profiles. Gao et al. studied drug response in seven new human prostate cancer organoid lines derived from metastatic and circulating tumour cells expressing disease-specific mutations such as TMPRSS2-ERG fusion, PTEN loss, TP53 loss, SPOP mutations, FOXA1 mutations and CHD1 loss . CorrelaOrganoid cultures can be expanded long term relatively fast and cryopreserved enabling generation of organoid biobanks, such as the Hubrecht Organoid Technology (HUB) ‘‘living’’ biobank which generates organoids from a vast number of tumour specimens including prostate cancer aiding access to the scientific community . This isHowever, as advances in models for prostate biology have been hindered by the many challenges associated with primary prostate culture, use of iPSCs presents a promising complimentary approach. Yet, generation of prostate iPSCs has lagged behind that reported from other tissues. Therefore, we describe the significant advances made from using iPSCs to generate models in other systems to demonstrate how the prostate field could take lead from them.In 2006, Takahashi and Yamanaka demonstrated that stem cells with the same characteristics as embryonic stem cells (ESCs) could be generated from adult somatic cells by the simultaneous introduction of just four transcription factors , known as Yamanaka factors . iPSCs hDespite the huge advances in identifying cell-level phenotypes across a range of diseases, 2D models are restricted to cell culture-based systems and are mostly relevant to the cellular level, failing to represent complex diseases and tumour biology. Due to the significant interest in generating iPSCs from cancer cells to help elucidate the molecular mechanisms of cancer development, various cancer cell lines have been reprogrammed, including prostate cancer , melanomiPSCs have the potential to differentiate into multiple cell lineages and provide a promising source of specialised cells. The differentiation process to the target cell type is a pivotal step in the development of a model. Having gained insights from normal development, many strategies have been explored to guide the direct differentiation of iPSCs mainly by introducing cocktails of growth factors and/or small molecules at defined times and concentrations. In turn, iPSCs have provided models recapitulating single-cell and more complex multi-cell type tissues. For example, generation of human adipocytes  from iPSCs, exhibiting mature morphological and functional properties characteristic of in vivo fat tissue, has shed light on adipogenesis and provided a platform for the development of obesity- and diabetes- related therapies ,86. TGFβMore recently, iPSC-derived cancer organoids have been described. Using standard iPSC differentiation techniques coupled with recent advances in bioengineering, xenotransplantation and genome editing, iPSCs present new opportunities for the study of human cancer . These mThe groundbreaking work described by Yoshiki Sasai and colleagues, in a seminal paper , demonstFurther work from Yoshiki Sasai described the self-organisation of pluripotent stem cells to form optic cup structures, via temporal application of small molecules , displayIntestine organoids can also be derived from iPSCs in a 3D culture system by the use of extracellular support matrix and culture medium supplemented with pro-intestine growth factors . These oiPSC-derived liver organoids have been generated by co-culture of iPSCs, mesenchymal and endothelial cells. Liver buds were formed by self-organisation and cell-to-cell contact combined with paracrine signalling, resulting in induction of hepatic genes and expression of bile salt export pumps . More reiPSCs can also be differentiated into kidney organoids using a mesoderm induction step followed by 3D culture to promote self-organisation leading to organoid formation. These kidney organoids contained segmented nephrons connected to collecting ducts, surrounded by renal interstitial cells. Proximal tubules within the organoids were able to carry out endocytosis, as evidence of functional maturity . Kidney Lung organoids have also been successfully generated and used to study lung development and disease ,115. LunIn a seminal paper, it was shown that primary benign human basal prostate epithelium can initiate prostate cancer in immune-deficient mice and that the derived tumours realistically recreate histology of in situ human prostate cancer . This haRecently, a high throughput model of generating human prostate organoids from iPSCs has also been described, involving co-culturing iPSCs with rodent urogenital sinus mesenchyme (UGM). This simple differentiation protocol results in glandular structures in vitro that faithfully mimic prostate tissue histology and express key prostate markers such as AR, prostate specific homeobox protein NKX3.1 and secretory prostate specific antigen (PSA) . This apGenome editing technology has emerged as an extremely powerful tool that can greatly advance organoid-based research for the development of better targeted therapies . CRISPR-Effective treatment development for prostate cancer is hampered by the lack of patient-specific in vitro models that accurately recapitulate this disease. The prostate-derived iPSC generation of human prostate tissue both in vivo and in vitro is a new complimentary approach to established primary culture and PDX models . Togethe


The structure and organization of cellular membranes have received intense interest, particularly in investigations of the raft hypothesis. The vast majority of these investigations have focused on the plasma membrane of mammalian cells, yielding significant progress in understanding membrane heterogeneity in terms of lipid composition, molecular structure, dynamic regulation, and functional relevance. In contrast, investigations on lipid organization in other membrane systems have been comparatively scarce, despite the likely relevance of membrane domains in these contexts. In this review, we summarize recent observations on lipid organization in organellar membranes, including endoplasmic reticulum, Golgi, endo-lysosomes, lipid droplets, and secreted membranes like lung surfactant, milk fat globule membranes, and viral membranes. Across these non-plasma membrane systems, it seems that the biophysical principles underlying lipid self-organization contribute to lateral domains. The exquisite complexity and capability of biology are enabled largely by physical and/or biochemical compartmentalization. A predominant mechanism for biological compartmentalization is provided by lipid membranes, which partition intracellular space into membrane-bound organelles. Further, these membranes themselves can be compartmentalized into functional, lateral subdomains . The mosThe raft phenomenon has been studied extensively, with much of that attention focused on functional domains of the PM. The reasons for this inordinate attention to PM rafts are both historical and practical. The raft concept was originally proposed to explain the unique sterol- and sphingolipid-enriched composition of the apical PM in epithelial cells . In thisDespite these valid reasons for attention on mammalian PM domains, it is possible, if not likely, that similar principles of self-organization can drive membrane domains in other cell membranes. Lipids in other cell membranes have properties that tend to cause favorable interactions between various subclasses, leading to lateral heterogeneity. Examples of such properties include (a) acyl chain saturation, with relatively saturated lipids tending to pack together into ordered solid or liquid domains; (b) cholesterol abundance, with cholesterol interacting preferentially with relatively saturated lipids; (c) headgroup charge, with polyvalent cations or proteins capable of ionic cross-linking ; (d) speAccumulating evidence indeed strongly suggests, and sometimes directly shows, the presence of lipid domains in various organelles. In this review, we summarize observations of lipid domains found in non-PM membranes, including subcellular organelles  and non-cellular structures [milk fat and lung surfactant (LS)] . Where aThe ER is the largest cellular membrane system, consisting of a contiguous network of membrane tubes and sheets that comprises the cell’s center for lipid metabolism . AlthougMembrane protein transport from the ER to the Golgi requires binding to the Sec24 subunit of the coat protein complex II vesicle coat. However, glycosylphosphatidylinositol (GPI)-anchored proteins cannot directly bind to Sec24 because they lack a cytosolic domain. Instead, the P24-P23 protein complex, which can bind to Sec24D, acts as a cargo receptor to mediate the export of GPI-anchored proteins like CD59 or the folate receptor . BecauseMitochondria-associate membranes (MAMs) are ER subdomains that play crucial roles in inter-organellar crosstalk between ER and mitochondria required for the homeostasis of both organelles. It has been suggested that these MAMs also have unique lipid compositions that resemble lipid rafts . Indeed,The presence of lipid microdomains at inter-organelle contacts was also recently strikingly supported by observations of large intracellular vesicles induced by hypotonic swelling of adherent cells. This procedure results in the formation of large vesicles derived from various organelles, including ER, mitochondria, endosomes, and lysosomes  . ER-deriSaccharomyces cerevisiae. The function of these diffusion barriers is to retain misfolded or otherwise aberrant proteins in the mother cell, thus protecting the daughter cell from noxious ER stress. These domains are likely composed of sphingolipids, as these are the major saturated lipids in yeast, and inhibition of sphingolipid synthesis pathways  impaired diffusion barrier formation. Because inhibition of ergosterol did not abolish the diffusion barrier, the domains are probably So, rather than Lo. Importantly, such domains can serve as selective barriers to membrane inheritance: the long chains of sphingolipids, especially in an ordered state, present a relatively thick lipid bilayer, which tends to exclude membrane proteins with short transmembrane domains (TMDs) due to hydrophobic mismatch  phase. The So phase is similar to the Lo phase in its high degree of lipid order but distinguished by the negligibly low translational diffusivity of membrane components . This stmismatch . This memismatch  and resuConsistent with ordered nanodomains in yeast ER, large-scale lipid domains have been observed in mammalian ER under conditions of perturbed metabolism and lipid synthesis . FeedingThe physical state of the ER  must be carefully regulated to fulfill its versatile functions. The ER membranes must be fluid and compliant to accommodate nascent membrane proteins with diverse physical properties  but may Unlike the ER, which is a continuous network with a thin and readily deformable membrane to allow facile insertion of nascent membrane proteins, the Golgi apparatus is a polarized organelle composed of stacked cisternae functionally compartmentalized into cis-, medial-, and trans-Golgi . The majSuch Golgi raft domains have been heavily implicated in protein sorting to the PM in non-polarized cells  and, morRaft-based sorting in membrane traffic relies on proteins’ preferential partitioning to ordered membrane domains. This partitioning relies on biophysical differences between membrane domains: raft domains are relatively thick and tightly packed, whereas more disordered domains are thinner and less packed. Thus, thicker raft bilayers favor the incorporation of proteins with long TMDs . ConsistEndosomes and lysosomes organize the sorting, recycling, and degradation of PM membrane components internalized via endocytosis. Endocytosis involves numerous mechanistically parallel pathways, several of which have been associated with raft domains, including caveolae , clathriPerhaps because of the unique lipid composition of the lysosome, some of the clearest microscopic evidence for raft domains in living cells comes from studies of the vacuole in yeast, which is analogous to the mammalian lysosome. While in the logarithmic phase, the yeast vacuole appears microscopically homogenous. In striking contrast, vacuoles of yeast grown to stationary phase show micron-sized coexisting liquid domains with distinct ergosterol abundances . The depLipid droplets are often misconceived as structureless and relatively inert fat particles, when in fact, these organelles are vital for cellular homeostasis via energy storage and detoxification of free fatty acids. Although not directly related to ordered membrane domains, LDs present some fascinating biophysical features worth brief consideration. Droplets are typically spherical intracellular organelles 0.1–100 μm in diameter. Their major structural feature is a large core of neutral lipids  surrounded by a phospholipid monolayer derived from the ER . LDs areEnveloped viruses, including important pathogens like human immunodeficiency virus (HIV), influenza, Ebola, and coronaviruses, are encapsulated by a lipid membrane originating from the membranes of host cells during virus budding. In many of these cases, the viruses bud from the PM with an envelope relatively rich in cholesterol and sphingolipids, suggesting the possibility of raft involvement in membrane assembly . Indeed,The involvement of raft domains in HIV assembly and budding was recently supported by an exciting set of microscopic studies relying on validated lipid and protein probes for membrane domains . The nasThe precise roles of membrane domains in viral function have not been completely resolved; however, several possibilities have been suggested by experimental evidence. First, similar to protein sorting in eukaryote membrane traffic, rafts may function as sorting platforms for selective incorporation/exclusion of viral proteins. Consistent with this notion, many viral envelope proteins are posttranslationally palmitoylated, including those of influenza , HIV Bh, the sevLung surfactant is a thin film of lipids and proteins at the surface of alveoli secreted by type II alveolar epithelial cells. The main function of LS is to reduce the surface tension at the air–alveoli interface and thereby protect alveoli from collapse during the rapid area compression–expansion involved in the breathing cycle . SurfactMammalian milk is rich in lipids packaged in the form of fat globules surrounded by a milk fat globule membrane (MFGM). The MFGM is an unusual tri-layer, whose inner monolayer is comprised of polar phospholipids and proteins surrounding a core of neutral triglycerides. This typical LD-like configuration is then surrounded by an outer bilayer, presumably acquired during extrusion of the LD-like fat globule through the apical surface of mammary epithelial cells . The speThe presence of lipid domains on non-PM membrane systems is fully consistent with, and a logical extension of, the lipid raft hypothesis. The evidence above combines to strongly support the relevance of lipid domains in various biological membranes, highlighting a general principle of membrane compartmentalization regulating various biological processes. The underlying biophysical insights generated from the intensive interest in PM lipid rafts are equally applicable to non-PM membrane systems. The experimental challenges of studying intracellular membranes have been a formidable barrier to a detailed understanding of their structure and organization: such membranes are not accessible to external labels or manipulations; organelles are often small and have a variety of complex shapes ; and various membranes are tightly packed inside the cells making unambiguous identification difficult. However, several methods have been recently developed that allow observations of large-scale domains, such as enlarging organelles through hypotonic swelling or fatty acid feeding . In paraAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.