Parkinson’s disease (PD) is a neurodegenerative disorder with motor deficits due to nigrostriatal dopamine depletion and with the non-motor/premotor symptoms (NMS) such as anxiety, cognitive dysfunction, depression, hyposmia, and sleep disorders. NMS is presented in at least one-fifth of the patients with PD. With the histological information being investigated, stem cells are shown to provide neurotrophic supports and cellular replacement in the damaging brain areas under PD conditions. Pathological change of progressive PD includes degeneration and loss of dopaminergic neurons in the substantia nigra of the midbrain. The current stem cell beneficial effect addresses dopamine boost for the striatal neurons and gliovascular mechanisms as competing for validated PD drug targets. In addition, there are clinical interventions for improving the patient’s NMS and targeting their autonomic dysfunction, dementia, mood disorders, or sleep problems. In our and many others’ research using brain injury models, multipotent mesenchymal stromal cells demonstrate an additional and unique ability to alleviate depressive-like behaviors, independent of an accelerated motor recovery. Intranasal delivery of the stem cells is discussed for it is extensively tested in rodent animal models of neurological and psychiatric disorders. In this review, we attempt to discuss the repairing potentials of transplanted cells into parkinsonism pathological regions of motor deficits and focus on preventive and treatment effects. From new approaches in the PD biological therapy, it is believed that it can as well benefit patients against PD-NMS. In brain degenerative conditions on the middle-aged or the elderly ages, Parkinson’s disease (PD) occurs with progressively worse motor defects such as bradykinesia, myotonia, resting tremor, and rigidity and develops a wide range of non-motor/premotor symptoms (NMS).Parkinson’s associated symptoms mainly relate to the diseased and pathological changes in the brain . Their PNMS can appear earlier than PD motor dysfunction and fluctuate with the motor symptoms later. For example, among approximately half of Parkinson’s patients, there are anxiety  and/or depression, which are presented years before typical motor PD symptoms occur, while PD-associated mood and sleep disorders usually develop during the course of PD . Use of Stem cell trials with surgical procedures have been proposed as a viable treatment option in Parkinson’s patients . Mild coTherapeutic effects on motor improvement have been related to the protective maintaining of a relative balance for acetylcholine/dopamine in the brain regions. In addition to problem-solving of motor fluctuations using the oral levodopa medication or the surgical deep brain stimulation alone, increased trends of incidence/prevalence rates of PD-NMS are demonstrated . We reviFor PD neuropsychiatric symptoms of anxiety and depression, selective serotonin reuptake inhibitors (SSRIs)/antidepressants  have included citalopram (Celexa), escitalopram (Lexapro), fluoxetine (Prozac), fluvoxamine , paroxetine , sertraline (Zoloft), and vilazodone (Viibryd). In PD combating depression , SSRIs may aggravate tremor in 5–20% of Parkinson’s patients. Amitriptyline that is considered clinically useful but with adverse reactions of anticholinergic side effects drives particular attention in cardiac arrhythmia or cognitive decline according to the guideline. In Parkinson’s patients with hypertension and/or diabetes, it is necessary as well to prevent and treat cerebrovascular disease-related cognitive impairment (Table There are anti-PD drug-associated psychiatric symptoms (inducing PD psychosis) in patients with the anticholinergic drugs, amantadine, MAO-BI , dopamine agonist, and levodopa. For an adjustment of unsatisfactory anti-PD drugs, low-dose quetiapine treatment is proposed with the mechanisms of histamine H1 and alpha-1 adrenergic receptors that may have negative metabolic effects and significant adverse effects. Dementia/cognitive impairment can be presented with the influence of anti-PD psychotic drugs. For protecting the cognitive function, use of trihexyphenidyl and other anticholinergic drugs should be avoided. Many other common situations also include an impulse control disorder usually identified in Parkinson’s patients with dopamine agonist, which can become significantly worse with the higher dose of levodopa . MeanwhiIn patients with concurrent insomnia/sleep-wake disorder, the use of levodopa controlled-release, long-acting dopamine agonist  or sleep hygiene consultation may be related to an excessive daytime sleepiness/sleep attack. With clarification for the cause of excessive daytime sleepiness, the patient develops drowsiness after each dose of dopamine agonist . AdditioParkinson’s patients develop rapid-eye-movement (REM) sleep behavior disorder (RBD) by a specific loss of normal muscle atonia in REM sleep. There is currently a lack of RCT studies on PD-NMS RBD treatment. In the many clinically supportive researches, presentation of RBD indicates a neurodegeneration progression, but it needs to be excluded for RBD induced by some medications . Due to Fatigue identification and clarification with PD have been performed. Our group recently test an acute exercise-induced fatigue scale (AEIFS) as an evaluation for the fatigue chronically developed . The patIt may have been evidenced in promoting the renewal of cardiomyocytes, well-controlled blood sugar, repairing wounds or restorative neurological/behavioral functions, etc. for BMSC (commonly blood sources) and UC-MSC  . AdditioAn ideal strategy in brain diseases would be taking advantage at full capacity of an efficient use for cell therapy, of an application to the source selection considerations on neural cells /vascular cells, and of a biological and physiological tone in the posttransplantation host microenvironment . For preIn a general example for a mesenchymal stromal cell, given stronger plasticity, lower immunogenicity, and higher cell variability , it becomes one of important sources considering its targeted tissue microenvironment. Therapeutically, lumbar puncture or subarachnoid injection of the stem cells can be achieved in 3rd /4th lumbar intervertebral space . The relAnother example from earlier research includes eight Parkinson’s patients receiving a therapy of MSC injection through carotid artery, with similar design tested in animal models . The resNR4A2). Nurr1 is believed to become the differentiation initiator of neural progenitors into dopaminergic neurons eventually [Enhancement strategies of cell-based mechanisms have been investigated in past 10 years for the design that particularly uses transgene vectors committed to novel stem cell transplantation therapies . To overentually . With inentually , 34. Immentually . Other tentually .For PD (and other) neurodegenerative disease treatments, research to date can identify neurotrophic mechanisms behind promotion of neurological recovery by mesenchymal stromal cell transplantation . MSC thaIn an improved microenvironment, cell replacement effect with the transplanted stem cells may be still dependent on a differentiation into dopaminergic neuron-like cells or activated endogenous neural stem cells to replace corresponding types of neural cells in the damaging dopaminergic tissues . It shouAutopsy has indicated identifiable activation of microglia from PD brains in the postmortem study where both local and systemic inflammation  can induce the proliferation effect of microglia. Transplanted particular stem cell type should inhibit kinds of proliferation triggers with reduced expressions of microenvironmental pro-inflammatory factors. Elevated levels of the pro-inflammatory cytokines detected in the brains of Parkinson’s patients have included IFNγ, IL-1β, and TNFα . AnotherThe need for cell replacement with dopaminergic neurons to the Parkinson’s patients gradually increases over time during disease progression (xellsmart.com). Under the advanced use of small molecule series combined with certain circumstances, adult cell (including stem cell) can be expected reprogrammed towards specified stage of neurons . In the Researchers have long reported in studies using immunosuppressants to treat animal models of PD. Immunomodulatory effects on the increased levels of pro-inflammatory cytokines  have been observed in the Parkinson’s patient brains with a large number of proliferating activated immune cells . For exaIn PD and other neurodegenerative conditions as one combinatorial target, their glutamate-induced excitotoxicity , a reactIn patients with PD, cell metabolism can be improved by mitochondria (the energy factories for the cells) as a means of improving processes for cell oxidative phosphorylation, energy supply, cell differentiation, oxygen free radical formation, and apoptosis control. Toxic substances or mutations in mitochondrial DNA may disrupt the oxidative respiratory chain within the mitochondria. Theoretically, those can lead to the damage of the oxidative respiratory complex function in the target region/transplantation site of diseased conditions, which aggravate oxidative stress and cause damage to dopaminergic neurons. Fortunately, transplanting stem cells has demonstrated the ability of alleviating those mitochondrial dysfunction . Via traDepression, mainly characterized by significant and lasting depressive symptoms, is a common mental illness that endangers the physical and human mental health. The WHO reports that about 5% of the world’s population  are suffering from depression. Unfortunately, the first-line treatment of depression with antidepressants of SSRIs, SNRIs, NaSSA, and SARIs shows limited effects for relieving symptoms in depressed patients. Pre-PD depression (a history of depression prior to an onset of PD motor symptoms) demonstrates an association with poorer cognitive abilities. As of 2020, depression may become the world’s second-most common disease after heart disease. PD-NMS and the depression history are detectable and correlated to the Beck Depression Inventory (BDI) and the Hamilton Anxiety Rating Scale (HAM-A), by which an incidence of depression in women is twice more than that of men. For depressive disorder, it is the main health trend of its global burden of disease study for women aged 15 to 44. Depressive symptoms not only cause a decline in the quality of life of affected individuals and a deterioration in their social functions but also lead to increasing expenditures on health service resources . Since 2Indeed, many kinds of antidepressants promote proliferation of the adult hippocampal neuroblast and further upregulate neurotrophic factor expression of BDNF, VEGF, and some others. It is usually maintaining for the curative effect of antidepressants in several weeks since the start of treatment and supporting hippocampal neuronal regeneration as the basis of their encouraging activities . Due to Since the neuronal damage in the hippocampus is pathologically significant and extremely sensitive to stress, the dentate gyrus should become a unique therapeutic target for the depression as well as in PD with impaired cognitive performance. Taking into consideration of cognition pathways across dentate gyrus and other parts of the hippocampus, the regeneration within these functional neuronal circuits can be constructive. By location, the granular cells in the dorsal dentate gyrus can significantly inhibit contextual learning activities, while the granular cells in the ventral dentate gyrus can inhibit the anxiety and other stress reactions . TypicalOur aforementioned hypothesis can be further explained that the hippocampal neural stem cells are largely inhibited accomplished with any depressive symptoms in PD conditions. Normally, hippocampal formation of synaptic input from a neuronal cell can extend all the way to the pyramidal cell layer of CA3 region, where established connections of synapses integrate into the trilaminar loop of the hippocampus. PD conditions disrupt their neuronal development by affecting neuronal cell identity with the developing stem/progenitor cell stages, thereby inhibiting majority of differentiating and proliferating neural cells  integrated into the loop. In addition, the hippocampus develops one of anatomical inputs to the hypothalamus, modifying stress responses under inhibitory negative feedback controls. HPA axis hyperfunction has been presented in around 70% of patients linked to hippocampal changes [In our research hypothesis, relief against the involved psychosocial dysfunction of pre-PD depression is probably based on evidence in adult mammalian models with the decrease (or increase) of hippocampal neuronal regeneration leading to the occurrence of (or recovery from) the depressive-like behaviors. In early PD/pre-PD brains, there are limited regions showing potential pathological changes, as well as showing neurogenesis, e.g., to the glossopharyngeal nerve, the olfactory bulb, or the vagal nerve. Once the symptoms appear, indicated as reparative damaged functions to the hippocampus, cellular protection for hippocampal neurons should be implemented. By study models, it allows direct critical cellular/pathobiological measure underlying anti-depression after promoting hippocampal neuronal regeneration. For the use of chronic continuous intensive stimulation to establish depressive mice, with antidepressant drugs such as fluoxetine or imipramine demonstrated anti-depressive, it is considered appropriate to study the hippocampal involvement. As in the previous study of their hippocampus destroyed by X-ray radiation experimentally, there has been few relieving effects with the same antidepressive intervention . It is tAn application on cell therapies may be helpful to meet the expectations for the relief of depressive symptoms in PD . In geneStem cell transplantation into the depressive brain has been explored/proved in different study models. One example is the hippocampal orthotopic transplantation of MSC populations for the treatment of depression in the mouse model. Their survival time for the stem cells after transplantation may have been relatively short, but the mechanism of its action involves vasculature recruitment  to promote endogenous neuronal regeneration. It does improve anxious behavior, depression, learning and memory, and other executive functions . VasculaDepressive-like symptoms after inferior cavity hemorrhage are relieved with increased expression of tyrosine hydroxylase in the substantia nigra and basal ganglia , a new tRecently, our and others lab’s published results support hypothesis with intranasally delivered stem cells  to improve the depressive-like symptoms as a consequence of inhibiting brain inflammation and/or promoting endogenous neuronal regeneration and repair. Specifically, the 8-week-long chronic unpredictable mild stress (CUMS) of mice is established, which induces stable depressive-like phenotype under hyperactive HPA axis but no later than 36 days without any further CUMS treatment . In ordeThe study reported is considered important and relevant for update on Parkinson’s interventions as well as for biotherapy-related education. Among them, stem cell therapy may be one particularly effective outcome-improving strategies in PD-NMS treatment at their early PD/pre-PD stages via cell replacement or vascular mechanisms. Underlying PD-NMS such as depression and neuroimmune modulation can be accompanied alongside the host microenvironmental improvement by mesenchymal stromal cell (including exosome) biotherapy within the brain of PD conditions of disrupted neuronal connections and vascular homeostasis. Consequently, neuro-replacing and vascular component repairs are expected as linked between objectives and the performance of its beneficial effects.


Rapid progress in nanotechnology has advanced fundamental neuroscience and innovative treatment using combined diagnostic and therapeutic applications. The atomic scale tunability of nanomaterials, which can interact with biological systems, has attracted interest in emerging multidisciplinary fields. Graphene, a two-dimensional nanocarbon, has gained increasing attention in neuroscience due to its unique honeycomb structure and functional properties. Hydrophobic planar sheets of graphene can be effectively loaded with aromatic molecules to produce a defect-free and stable dispersion. The optical and thermal properties of graphene make it suitable for biosensing and bioimaging applications. In addition, graphene and its derivatives functionalized with tailored bioactive molecules can cross the blood–brain barrier for drug delivery, substantially improving their biological property. Therefore, graphene-based materials have promising potential for possible application in neuroscience. Herein, we aimed to summarize the important properties of graphene materials required for their application in neuroscience, the interaction between graphene-based materials and various cells in the central and peripheral nervous systems, and their potential clinical applications in recording electrodes, drug delivery, treatment, and as nerve scaffolds for neurological diseases. Finally, we offer insights into the prospects and limitations to aid graphene development in neuroscience research and nanotherapeutics that can be used clinically. This enin vitro because of their physical and chemical properties  materials have been extensively explored for biomedical applications . With suPrevious studies have shown that graphene can aid in the adhesion and differentiation of neurons and can act as a neuroprotective agent. Graphene is a potential carrier to control drug release at specific disease sites, making it a valuable treatment strategy for neuronal diseases. Moreover, graphene and its derivatives can be used as scaffolds to enhance nerve repairing, establishing it as a next-generation candidate for advancing medical bioengineering. However, the safety of nanomaterials is essential to protect human health and ensure environmental safety. Thus, further studies are warranted to fill the knowledge gap on the limitations of carbon nanomaterials, including their toxicity and biocompatibility .In this review, we focus on novel graphene applications in the nervous system. Graphene-based materials may be used clinically in the future; however, this article is limited to pre-clinical applications. We aimed to describe the properties of graphene-based materials applicable in neuroscience research, including using them as electrodes for neural recording and imaging, neural cell substrates, photothermal effects, and drug and gene deliveries. Furthermore, we aimed to outline the application of graphene-based materials in neurological diseases and neural tissue engineering. Finally, we aimed to discuss graphene’s cytotoxicity and provide an overview of its prospects.2.−1, superior to graphene grown on indium tin oxides. Unfortunately, this technique requires a very high temperature and vacuum environment, as well as exorbitant prices for only small-size devices. Many graphene-based nanomaterials are built up mostly by chemical reactions for covalent bonding, such as graphene oxide [GO; Graphene is a sheet comprising a single layer of carbon atoms formed by stripping highly oriented pyrolytic graphite, like carbon nanofibers, another type of nanomaterial. In 2D, sp2 hybridized carbon atoms give graphene a hexagonal honeycomb lattice pattern with a thickness of approximately 0.335 nm [At room temperature, the interactions between carbon atoms in graphene are strong. Electrons in the carbon atoms are not easily affected or scattered by the movement of surrounding carbon atoms; its conductivity also contributes to electron transfer . Additio3.Neural interfaces are typically implanted with electrodes that can record or stimulate target tissues or cells in the brain, thereby establishing connections with the nervous system . Howeverin vivo microscopy techniques performed on freely behaving animals. However, one of the major problems of using graphene in in vivo studies is that it is ultra-flexible and difficult to implant surgically. Graphene can be mechanically deformed beyond the linear regime ; this isin vivo neural recording by graphene electrodes compared to gold electrodes  for recording cellular activity is challenging. Studies have demonstrated effective ectrodes . In thesectrodes . Recentlin vivo recording validation.The mechanical properties, electrical conductivity, and large surface area of graphene make it perfect material for developing ideal neural interfaces and more reliable electrodes, while highlighting the unique advantages of composites, demonstrating greater application potential in neural interfaces. However, more studies are required to investigate the long-term effects of graphene-based nanoelectrodes during 4.Traditional metal electrodes used for recording electrophysiological activities obstruct the vision field, produce optical shadows, and are more likely to introduce light-induced artifacts during the recording. Researchers have discovered that transparent graphene could solve this problem due to the unique band structure in the transmission spectrum of graphene that prevents the mentioned limitations. Theoretically, graphene allows the hot ballistic charge carriers produced by light  to transin vivo. in vivo. Moreover, these cellular-scale methods enable the recording of electrophysiology and calcium fluorescence in CA1 pyramidal neurons in hippocampus seizures  region . Recentlin vivo and achieved ultra-efficient tumor ablation after laser treatment. Similarly, in vitro. However, the rGO-PEG showed concentration-dependent cyto- and geno-toxicity. No toxic signal was found in the histological examination of GO-PEG during PTT by Liu operation. Another transition metal disulfide that is attracting more interest in the biomedical field is Tungsten disulfide because of its photothermal properties, high lubricity, and catalytic activity. Its high NIR absorption capacity has been applied to photothermal therapy of tumors .PTT has received widespread attention because it can thermally ablate cancer cells in a minimally invasive manner under an NIR laser. The broad optical absorption spectra are important in photothermal medical applications. Nanomaterials with strong absorption in the NIR regime can act as effective PTT absorbers that achieve therapeutic temperatures with less total light energy, reducing heat escaping from the target to healthy surrounding tissue and avoiding tissue damage. GO and rGO have been widely exploited as effective absorbers for NIR-based PTT in cancer therapy . Clinicaf tumors . The devPhotoacoustic and fluorescence imaging can also be combined with PTT for diagnosis. Photoacoustic imaging has few specific signals and cannot image deep tissues. Fluorescence imaging does not use ionizing radiation and has low tissue penetration. Thus, further research is needed to fully understand how to apply carbon nanomaterials better and fully in neurology, as their functions involve the interaction between their various properties. Therefore, improving the functionalization of nanomaterials, which can create better and less risky options for patients during clinical treatment of diseases, remains challenging.5.9 W m−2 K−1. −1 K−1.Graphene has high thermal conductivity (κ) and great thermal management potential, indicating its development in nanoscale engineering for heat transport and management. Researchers have suggested various effective approaches for modulating the thermal conductivity of graphene. Depositing nanoparticles on the surface of graphene can mediate its thermal management . Chemicain vivo  accumulation. Recently, As thermal stability improves, graphene use has risen in the fields of clinical biomedical application and tailored nanomedicine for neurologic diseases. For example, graphite lattices in the graphene family of nanomaterials fuse to form disparate forms of molecules, with a potential role in drug and gene delivery for treating neurological diseases. Using two complementary strategies for conferring static or spatial stability can improve the thermal stability of graphene and allow cells to occupy stable graphene sheets . In 2008in vivo . The comThese findings provided new ideas for combining PTT and graphene-based drug/gene delivery therapy and demonstrated a significant improvement over traditional surgical treatment method. Graphene-based materials showed high potential for the novel and efficient treatment of neurological disorders. However, the interaction between graphene and cells may interfere with the different cell viability reduction rates in thermal therapy . Future 6.The first aspect to consider when developing graphene-based biomedical applications is their biocompatibility to avoid any adverse effects on living tissues . GraphenGraphene biocompatibility and its effects on the performance of living cells have shown that graphene on top of matrix substrates does not impede the interaction between neurons and substrates, thus maintaining neuronal viability . Li et ain vivo studies have demonstrated that the immune system is highly sensitive to graphene and quickly responds by increasing anti-inflammatory cytokine synthesis (in vivo zebrafish model (in vivo settings. Fabricating graphene with poly(ε-caprolactone) caused an acceptable degree of immunological response in vivo  in in vivo mouse experiments did not accumulate in main organs with fast kidney clearance, probably due to their ultra-small size  and low (up to 70 kΩ−1) conductivity favored neuron spreading without clear alteration, indicating that the high electrical conductivity of graphene is not the crucial property and the efficient range of its electric conductivity for graphene-based neuronal interfaces is broader than expected  to ensure the delivery of sufficient dosage to kill cancer cells. Second, the delivery should specifically target cancer cells, avoiding harmful effects on normal tissues. For example, subarachnoid hemorrhage occurs in severe neurological diseases, and studies have demonstrated the feasibility of using functionalized GO (FGO) for effective drug delivery in such cases. The content of FGO nanotablets loaded with pirfenidone, which is used to treat subarachnoid hemorrhage, gradually increases with an increase in drug concentration , making 1-42 (Aβ1-42) is an important biomarker in AD and PD  in neuroscience in e repair , we founin vitro and in vivo studies suggest that current studies have several limitations. First, compared with the number of in vitro studies, in vivo studies exploring the basic properties of graphene, specifically its biocompatibility and toxicity are limited. Lack of consistency between in vitro and in vivo results may downgrade graphene into only a research tool. Regarding in vivo study models, nearly all animal models are limited to mice, and no other species have been considered in exploring graphene applications. Second, most studies suggest that nanomaterials induce a mild immune response. However, researchers are yet to identify the standard method of administration. Furthermore, whether nanomaterials act the same way when eliciting an immune response via different routes of administration in vivo remains unknown, although the toxicity and pharmacokinetics of nanoparticles are closely dependent on the administration route. Third, most studies, whether in vitro or in vivo, did not perform in-depth investigations on the underlying mechanism involved in the relationship between cell differentiation and graphene-based substrates, such as induced morphologic interplay and cellular modulation pathways. Without these fundamental definitions, determining the role of graphene in biomedical applications may be challenging.The observed discrepancies in in vivo because the results from in vitro studies cannot be directly applied to humans. Moreover, the graphene-based implant as an electrode or scaffold should also be further investigated to observe its long-term effect in vivo. More animal models should be considered to clarify the relationship between different species and graphene. Second, graphene can exhibit long-term and short-term toxic effects on the same cell and can potentially cause long-term damage to cellular structures. This review highlighted that graphene toxicity is highly dependent on excretion, functionalization, size, and structural dimensions. Therefore, comparing graphene toxicity between different experiments is difficult, given its diversity in shape, surface, size, and fabrication. Thus, the fabrication and terminology of graphene and the examination of toxicological methodologies must be standardized. This will better aid the understanding of the physicochemical properties and potential toxicity of graphene in vivo and in intro, allowing its practical application in humans. Third, future studies should incorporate graphene with various optical components to probe deeper brain tissues and understand how brain circuits process information because a graphene electrode with a two-photon microscope can record the whole cortical surface in neuroscience investigation. Moreover, electrical stimulation has been clinically applied in rehabilitation after nerve injury. Graphene-based conduits have shown great potential in nerve repair as scaffolds. Future studies should measure the potential of combining electrical stimulation and graphene-based conductive scaffolds in clinical nerve repair.The following are proposals for future experiments for improving graphene and its derivatives development: First, the main consideration of nanomaterials in biomedical application is their biocompatibility. Further studies must focus more on biocompatibility and toxicity This review sheds light on the safety and biological properties of graphene that must be considered before the utilization of graphene-based therapy for human benefit. This may pave the way for using graphene and its derivatives as powerful materials with versatile scientific and clinical applications.TY and YY wrote the first draft of the manuscript. L-YM and YL wrote sections of the manuscript. JB, F-YW, and LZ reviewed the literature. All authors contributed to manuscript revisions as well as read and approved the submitted version.This work was supported by the National Natural Science Foundations of China , the Innovation Team Project of Yanbian University (China), and the International Cooperation Project of Science and Technology Department of Jilin Province (20210402004GH).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ebola virus (EBOV) is one of the deadliest viruses causing severe diseases with high mortality. After the outbreak in West Africa in 2014, many specific treatment measures were tested experimentally; among these, countermeasures based on neutralizing antibodies (NAbs) have been considered one of the most practical means. However, some investigators have reported that patients with critical Ebola virus disease (EVD) could experience a deleterious recurrence of EBOV from immune-privileged organs with concomitant NAbs treatment, most prominently in the brain , ocular fluid, and seminal fluid has been documented in West Africa since 2004 . This std plexus . In addid plexus . This red plexus , 6. Furtd plexus , 8.Although treatment with NAbs produces positive results in EVD patients, particular attention should be paid to the possible recurrence of EBOV from immune-privileged organs in these critical patients, who generally cannot survive the infection. The proposed solutions  from this research may be insufficient to eradicate the persistence of EBOV since fatal cases of EBOV infection usually have low antibody levels and an unbalanced T-cell response . ReconstAs one of the foundations of immunology, the success of the rabies vaccine is an ideal example of contingency vaccination. The first treatment strategy is to inject attenuated rabies viruses into the victims as soon as possible. Although successful rabies treatment in the 19th century was comparatively low and lacked rigorous control, attenuated viruses from the spinal cords of rabbits that had succumbed to rabies have emerged as the first reliable treatment for rabies, with a mortality rate of nearly 100% . MoreoveContingency vaccination can be an alternative treatment for critical cases of EVD patients. A choice for this purpose has already been offered by successful vesicular stomatitis virus (VSV)–based EBOV vaccines , 16. PreMeanwhile, EVD patients are in critical condition. Consequently, developing a subunit-based protein or an messenger RNA (mRNA) vaccine is a possible treatment option that can be combined with NAbs and antivirals. Moreover, since the relapse of EBOV from the immune-privileged site usually takes time, a VSV-based EBOV vaccine could be administered to recovered patients for approximately a month or other suitable time intervals. In this way, vaccines can serve as a “boost” to elicit protective immunity. Subsequent antibodies and T-cell responses could serve as an arsenal that protects patients who cannot exert a protective immune response during EBOV infection, thus preventing possible EBOV recurrence from immune-privileged organs. One critical point is the identification of EVD patients with an elapsed potential. As described in the case reports , upon adAnother interesting question is that not all patients who received NAbs treatment experienced EBOV recurrence. A more comprehensive study may be required to clarify which patients are inclined to relapse. Aside from the evidence of the brains of antibody-treated non-human primate survivors , the NAbTo avoid a potential recurrence of EBOV in immune-sensitive organs, treatment with NAb in patients with critical EVD may be supplemented by an EBOV GP subunit vaccine or an mRNA vaccine to help reduce the possibility of relapse. NAb treatment could prevent fatal outcomes, and vaccine inoculation could elicit B-cell and T-cell responses, which may help reduce virus persistence and disease relapse Figure 1YL and FZ provided administrative support and financial support. WY and JL conceived of the study and wrote the manuscript. CY prepared the figure. WY, CY, JL, YL, and FZ checked and finalized the manuscript. All authors contributed to the article and approved the submitted version.


Amide proton transfer-weighted (APTw) imaging is a non-invasive molecular MRI technique with a wide range of applications in neuroradiology and particularly neuro-oncology imaging. More than 15 years of pre-clinical experiments and clinical studies have demonstrated that APTw metrics are reproducible and reliable, leading to large-scale clinical acceptance. At present, major vendors of MRI scanners provide APTw sequences upon request. However, most neuroradiologists are unfamiliar with this advanced MRI contrast, its related metrics, and its established added value to patient care. In this manuscript, we present the APTw contrast and illustrate its clinical potential for glioma patients, before and after tumor therapy. We also show common artifacts of APTw imaging and discuss potential limitations and future refinements. Our goal is to suggest how this emerging technique can aid in diffuse gliomas work-up. During the saturation period, proton exchange is continuously repeated to enhance the detectability of low-concentration molecules. In APTw imaging, the chemical species of interest are amide protons of mobile peptides and proteins: thus, the effect detected by water signal intensity larger when the protein concentration is higher.Amide proton transfer-weighted (APTw) imaging is an MRI technique based on the principles of chemical exchange saturation transfer (CEST). In CEST imaging, molecules in the millimolar range with exchangeable protons are detected indirectly by measuring the intensity changes of water protons before and after the application of a radiofrequency (RF) pulse train. The RF pulse train is selectively applied to the resonance frequency of the proton group of interest leading to a depletion of the magnetization of these protons, also called saturation. This saturation is transferred  partially explaining their APTw hyperintensity pletely non-invasive. This contrast mechanism provides information at the subcellular protein level, justifying the term “molecular” technique, and does not require an exogenous contrast agent, so it is completely non-invasive. It should be noted that amide protons are not the only contributors to APTw contrast, but several confounding effects compete with saturation transfer , and disentangling their contributions is complex. Therefore, the term “weighted” is preferred to the solely “APT imaging” and APTw acquisition protocols should strictly follow expert recommendations for APTw brain tumor imaging at 3 T to avoid improper qualitative or quantitative evaluation of APTw maps.Tumors have an increased protein content compared to normal brain tissue, Concerning data processing approaches, magnetization transfer ratio asymmetry relative to the water frequency (MTRasym) is the recommended metric to compute APTw contrast, even though alternative methods have been proposed. MTRasym can be complemented by fluid-suppressed metrics to improve image readability  and higher in high-grade gliomas (HGGs), and it also differs in Grade 2 from Grade 3  and APTw signal intensity. In addition, APTw signal intensity correlates with other MRI tumor proliferation and density markers, as choline/creatine ratio in magnetic resonance spectroscopy (MRS), and apparent diffusion coefficient (ADC) in diffusion-weighted imaging (DWI)  .When compared to four advanced DWI models, APTw imaging shows increased diagnostic accuracy.Current advanced MRI protocols cannot replace the full relevant prognostic information provided by biomolecular analysis on surgical tissue samples. Nevertheless, preliminary non-invasive information can be very helpful for promptly orienting therapeutic strategies. For example, a lesion may be surgical inaccessible , patientImportant diagnostic and prognostic biomarkers have been shown to correlate with APTw contrast, most notably IDH mutation and MGMT methylation status. Therefore, APTw may, highlight different subtypes of underlying tumor malignancy. In the modern era of neuro-oncology, accurate stratification of patient outcome is essential to tailor treatment strategy and therefore prediction of patient survival is of strong clinical relevance , it also appears to provide a more accurate diagnosis. Additionally, a voxel-wise analysis between APTw maps and MET-PET shows a significant correlation in LGG but not in HGG, suggesting that even though these techniques are both a surrogate for protein metabolism, they reflect different biophysical processes.Compared to multiparametric MRI protocols that include DWI and MRS, APTw imaging has higher diagnostic performance in the distinction between glioma progression and treatment effects. as it enables earlier distinction between responders and non-responders patients  of the same geometry is acquired, allowing to realign water center frequency. A ΔB0 map is usually implemented in the APTw protocol as a separate acquisition and B0-corrected APTw maps are usually provided by vendors. Advanced post-processing approaches should also correct inhomogeneities, motion, noise and, importantly, artifacts due to fluid components. Indeed, large vessels, cystic or hemorrhagic areas and liquefactive necrosis demonstrate high APTw signal intensities. The mechanism beyond these hyperintensity artifacts is complex, and mostly due to spillover dilution effects that dilute CEST effects in solid tissue, but to a lesser amount in more fluid environments. In the future, the validation of fluid-suppressed metrics is advisable, as it will allow more accurate pre- and post-therapeutic tumoral assessment.BThe delineation of the region of interest in a tumor and its normalization with the contralateral normal-appearing white matter  should a1 inhomogeneities (0 and B1 inhomogeneities, affecting APTw signal repeatability. Currently, no robust B1 correction approach has been proposed for APTw imaging. Therefore, caution should be taken when analyzing anterior frontal lobe and infratentorial lesions.Finally, the anterior frontal lobe is more sensible to Beneities , while tAPTw imaging may allow to improves glioma patient care and might offer complementary information to conventional and advanced MRI approaches. Attention should be paid to adjusting APTw parameters to the recently standardized guidelines, in order to correctly interpret APTw signal intensity. In the future, emerging techniques for fluid suppression will increase APTw accuracy and furtherly refine molecular correlations.


The high susceptibility of carnivores to Suid Alphaherpesvirus 1 , renders them inadvertent sentinels for the possible occurrence of Aujeszky’s disease (AD) in domestic and wild swine populations. The aim of this study was to epidemiologically analyse the occurrence of PrV infections in domestic and wild animals in Germany during the last three decades and to genetically characterise the causative PrV isolates.PrV in dogs was detected using standard virological techniques including conventional and real time PCR, virus isolation or by immunohistochemistry. Available PrV isolates were characterized by partial sequencing of the open gC reading frame and the genetic traits were compared with those of archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in the domestic pig population.During 1995 and 2022, a total of 38 cases of AD in carnivores, e.g. dogs and red foxes, were laboratory confirmed. Sequencing and subsequent phylogenetic analysis of PrV isolates established a strong connection between AD cases in carnivores and the occurrence of PrV infections in European wild boars in the end phase of and after elimination of AD from the domestic pig population. While PrV infections occur at low numbers but regularly in hunting dogs, interestingly, PrV was not observed in grey wolves in Germany. In none of 682 dead-found grey wolves and wolf-dog hybrids tested from Germany during 2006–2022 could PrV infection be detected by molecular means.Although PrV has been eliminated from domestic pigs, spillover infections in domestic and wild carnivores should always be expected given the endemic presence of PrV in wild pig populations. Since detection of PrV DNA and virus in carnivores is sporadic even in areas with high seroprevalence of PrV in wild pigs, it may not reflect the full diversity of PrV. Varicellovirus within the subfamily Alphaherpesvirinae of the Orthoherpesviridae family [Pseudorabies virus (PrV), an enveloped double-stranded DNA virus and member of the genus e family , is the e family . Althouge family . While oe family , 4. In ge family , 6.AD has been eradicated from populations of domestic swine through use of culling and/or vaccination programs in many countries in Europe, in North America, Australia and New Zealand , 7–9 butCanis lupus familaris) may be the first indication that the virus is present in a swine herd or a local wild swine population [Because subclinical infections and nonspecific clinical signs are common in swine, the high susceptibility of carnivores renders them inadvertent sentinels for virus occurrence. Often detection of PrV in dogs (pulation . Dogs thpulation –24. Oncepulation .Vulpes vulpes) [Puma concolor couguar) [Canis lupus) [Lynx pardinus) [Lycaon pictus) [Ursus arctos) [Although PrV spillover infections are observed most commonly in farm dogs, in recent years cases of PrV in hunting dogs after direct contact with wild boars have been repeatedly reported from Europe , 26–33,  vulpes) –42, endacouguar) , grey wos lupus) –46, Iberardinus) , African pictus)  and capt arctos)  after coIn this study, we describe occurrence of PrV infections in domestic dogs and wild carnivores in Germany during the past three decades, with a subsequent effort to genetically characterize isolates based on partial sequence analyses. Furthermore, we wanted to elucidate whether PrV infections in wild boar pose a threat to the rising, highly protected population of grey wolves in Germany.Because AD is a notifiable infectious disease in Germany , any sushttps://www.dbb-wolf.de/). Georeferencing of data and map visualization was done using the ArcGIS 10.8 package.As part of a research project of the Leibnitz Institute for Zoo and Wildlife Research (IZW), Berlin, on the causes of mortality in grey wolves, brain samples from wolves and wolf-dog hybrids in Germany from 2006 to 2022 were submitted to the NRL at the Friedrich-Loeffler-Institut (FLI) and tested for PrV. Master data for wild carnivore samples such as date and geographical origin (Gauss-Krueger coordinates) were obtained from TSN or the IZW and DBBW  database  using a polyclonal PrV rabbit hyper immune serum  or an in-house polyclonal rabbit antibody against PrV gB . PositivFor the characterisation of genetic traits and for phylogenetic analysis, archived PrV isolates from carnivores and domestic pigs from Germany before the elimination of AD in 2003 were also included.Sequencing PrV isolates or DNA was done essentially as described . BrieflyBetween 1995 and 2022, PrV infections were reported in a total of 35 dogs and three foxes in Germany  , three golden jackals (Canis aureus), 15 stone and pine martens , four European polecats (Mustela putorius), four raccoons (Procyon lotor), and three badgers (Meles meles) were tested. In none of the animals could PrV be detected.In the frame of the research project on causes of mortality in grey wolves in Germany, during the period 2006–2022 at total of 682 grey wolves and wolf-dog hybrids, 31 lynxes  after onset of clinical signs. On necropsy, the dog showed hemoperitoneum, hemothorax, black-coloured stomach content, gastric mucosal hemorrhages, melaena as well as hemorrhages of the mediastinum and mesenterium. Histopathologically, moderate mixed-cellular encephalitis with vasculitis and perivasculitis, gliosis, scattered neuronal necrosis and extensive hemorrhages was found in the brainstem. Mononuclear infiltrates, neuronal necrosis and hemorrhages were also detected in the trigeminal ganglion. Immunopositive neurons and glial cells were detected within and adjacent to the affected areas , ing Fig. C.The PrV positive red fox from Saarland was found in January 2019 in an agonal state showing extreme pruritus, and was humanely euthanized in a local veterinary clinic. While rabies could be excluded by routine diagnostics, PrV was confirmed by standard virological methods. For the remaining PrV positive fox from Schleswig-Holstein no further information on the history and possible source of infection was available.Sequence analysis of PrV isolates from Germany identified different variants according to a limited number of sequence variations, i.e. SNPs and deletions and insertions Fig. .Phylogenetic analyses revealed that AD in carnivores in Germany was caused by different PrV strains. Carnivore PrV isolates originating from prior to the elimination of AD from domestic pig populations in Germany group together with PrV from pigs Fig. . More reAlthough AD has been eliminated from the domestic pig population in Germany , PrV infWild boar associated PrV cases in dogs are more likely after direct contact during hunting than oral ingestion of offal. Likely, increased stress levels in latently infected wild boar may cause reactivation of virus replication and active shedding without eliciting clinical signs , eventuaAgainst this background it is interesting that despite other reports none of the 682 investigated free-ranging wolves and hybrids submitted between 2006 and 2022 tested positive for PrV Fig. B. Certaihttps://www.dbb-wolf.de/totfunde/statistik-der-todesursachen). Since infection of grey wolves with PrV would inevitably lead to a clinically visible manifestation and eventually death, the absence of PrV-infected grey wolves in our sample suggests that PrV infections are very rare in wolves and the few occasional infections may have gone undetected.Studies of scat samples from Europe, on the other hand, indicate a certain flexibility of the wolf as a predator depending of the availability of prey, which significantly increases the chances of becoming infected with PrV, as is the case with hunting dogs. While in northern plain lands of Europe wolves seem to rather avoid wild boar as prey , in the In contrast, we report three cases of PrV in free-ranging red foxes. In one case, the fox was shot by a hunter as it showed atypical behavior suggestive of encephalitis, which was confirmed by immunohistopathology Fig. C, while While the virus found in one red fox GER 641) from the western federal state of Saarland was identical in its partial DNA sequence to other PrV isolates from the previously established “clade B” , isolate1 from thFurthermore, sampling biases and surveillance gaps may suggest epidemiological links where in reality there are none. As regards isolate GER 642, this red fox isolate serves as an indicator for the presence of a yet unknown PrV variant in this part of Germany bordering Poland, a country where no information on PrV characterization in wildlife is available. These results are similar to findings in Austria where the genetic diversity of PrV was only evident after investigating PrV from hunting dogs . AlternaWith their prominent clinical picture, PrV infections in carnivores are likely to be discovered and virus can be characterized, as seen in this study and others , 29, 31.


Glioblastoma multiforme (GBM) is a primary malignant brain tumor characterized by a high grade of malignancy and an extremely unfavorable prognosis. The current efficacy of established treatments for GBM is insufficient, necessitating the prompt development of novel therapeutic approaches. The progress made in the fundamental scientific understanding of GBM is swiftly translated into more advanced stages of therapeutic studies. Despite extensive efforts to identify new therapeutic approaches, GBM exhibits a high mortality rate. The current efficacy of treatments for GBM patients is insufficient due to factors such as tumor heterogeneity, the blood–brain barrier, glioma stem cells, drug efflux pumps, and DNA damage repair mechanisms. Considering this, pharmacological cocktail therapy has demonstrated a growing efficacy in addressing these challenges. Towards this, various forms of immunotherapy, including the immune checkpoint blockade, chimeric antigen receptor T (CAR T) cell therapy, oncolytic virotherapy, and vaccine therapy have emerged as potential strategies for enhancing the prognosis of GBM. Current investigations are focused on exploring combination therapies to mitigate undesirable side effects and enhance immune responses against tumors. Furthermore, clinical trials are underway to evaluate the efficacy of several strategies to circumvent the blood–brain barrier (BBB) to achieve targeted delivery in patients suffering from recurrent GBM. In this review, we have described the biological and molecular targets for GBM therapy, pharmacologic therapy status, prominent resistance mechanisms, and new treatment approaches. We also discuss these promising therapeutic approaches to assess prospective innovative therapeutic agents and evaluated the present state of preclinical and clinical studies in GBM treatment. Overall, this review attempts to provide comprehensive information on the current status of GBM therapy. There is a dearth of efficacious treatment modalities for persons diagnosed with GBM ,2. GBM, The foundation of glioma treatment depends on the tissue diagnosis, which is further boosted by molecular analysis to complement the histopathologic examination. The attainment of gross total resection remains the preferred outcome, where feasible. The degree of tumor resection at the onset plays a pivotal role in treating disease and OS outcomes for patients with high-grade glioma, surpassing all other currently accessible therapies . The ext®, a medical device that produces low-intensity and alternating tumor-treating fields. This approval was explicitly for treating newly diagnosed GBM when combined with TMZ [In 2015, the FDA approved the use of Optunewith TMZ . DespiteCombinatorial approaches are increasingly being used to treat patients with multiple metastases. In conjunction with surgery, stereotactic radiosurgery and brachytherapy can be utilized to enhance local control for brain metastases . ContempCombining immunotherapy with another targetable mechanism has a similar attraction. The appeal of anti-CD276 studies in combination with bevacizumab stems from the documented association of CD276/B7-H3 with angiogenesis.The extracellular domain of EGFR, which is only present in the EGFRvIII mutation, makes it the perfect particular antigen for both vaccines and CAR-T treatment. Given that single-target therapy causes recurrence and later resistance to the initial treatment, as was previously discussed. Recent research has provided evidence that extracellular vehicles (EVs) possess advantageous characteristics such as excellent biocompatibility, a high capacity for carrying drugs, extended circulation duration, efficient crossing of the blood–brain barrier (BBB), targeted delivery to affected areas, and effective transport of diverse cargos for the treatment of GBM . SignifiThe poor prognosis in GBM is due to unique treatment limitations, including the intertumor and intratumor heterogeneity, which facilitates the selection of resistant subpopulations, the fortified location of the tumor, which hinders the delivery of therapeutics, as well as the induction of a strong local immunosuppression. The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) can repair the alkylation damage of TMZ by eliminating O6-methylguanine adducts. As a result, the MGMT gene promoter’s methylation state has significant clinical implications ,20. SeveThe current approaches include surgery, chemotherapy, and radiation therapy in conjunction with treating brain tumors . Recent Surgical resection has conventionally been seen as an essential element in treating brain tumors. The evidence so far suggests an associative correlation between maximizing surgical resection and increased life expectancy in patients diagnosed with low- or high-grade gliomas. However, it is essential to note that data are scarce about the specific influence of the extent of resection on patient survival . Moreovep < 0.001) [The fluorescence-guided approach leads to an enhanced extent of resection . Multipl< 0.001) . Another< 0.001) . This te< 0.001) . NeverthThe management of GBM has experienced a significant paradigm shift due to the high incidence of isolated brain metastases in individuals with non-small-cell lung cancer (NSCLC) who acquire targetable mutations . The eff®  and Visualase®  [Here a heat-delivering probe is used in laser interstitial thermal treatment (LITT), directed by MRI thermometry . HeatingMN, USA) . Real-tiMN, USA) . DigitalMN, USA) .Since LITT is equally helpful for radiation-related alterations (such as radiation necrosis) and recurrent malignancies, it is particularly promising for recurrent brain metastases . AdditioIn addition, LITT can be used to treat high-grade gliomas that cannot be surgically removed. Ivan et al.’s meta-analysis showed that an average % percentage of tumor ablation volume of 82.9% resulted in OS and PFS of 14.2 and 5.1 months, respectively . AblatioThe following section overviews the most frequently used chemotherapeutic drugs for treating primary brain tumors. The underlying tumor type and the presence of systemic illness influence the choice of chemotherapy strategy for metastatic brain cancers. It is outside the purview of this article to explore each of the many agents in detail. A comprehensive list of FDA-approved drugs used in treating brain tumors has been listed in A DNA alkylating drug called temozolomide (TMZ) results in poor DNA repair and cell death. TMZ is an oral medication that can be given in a variety of clinical settings, including inpatient, outpatient, or at home . The sidCombination therapy with procarbazine, lomustine, and vincristine is recommended in gliomas ,69. A viSimilarly, some side effects of the nitrosourea alkylating drug lomustine are myelosuppression, nausea, tiredness, and pulmonary fibrosis. Combined radiation treatment with PCV significantly improved PFS compared with radiation and Temozolomide for IDH-mutant AA. However, radiation and TMZ were found to be better tolerated.Bevacizumab is a monoclonal antibody to vascular endothelial growth factor and is known to prevent the formation of tumors . It is tp = 0.0008) [Bevacizumab plus re-surgery improved OS, according to a retrospective study by Yamaguchi et al. in 2021  , but a s 0.0008) .The use of bevacizumab for treating patients with recurrent GBM has been rejected by the European Medicines Agency (EMA), with one of the reasons being a lack of positive benefit–risk for bevacizumab .A nitrosourea DNA alkylating chemical called carmustine blocks DNA replication and transcription—the intravenous administration of carmustine. Myelosuppression, weariness, nausea, vomiting, danger of pulmonary damage, and other side effects make carmustine much less attractive than temozolomide. With the addition of carmustine or other comparable nitrosoureas, surgery and radiation therapy be beneficial by meta-analysis . HoweverDihydrofolate reductase is irreversibly bound and inhibited by methotrexate (MTX), which prevents DNA synthesis, repair, and cellular replication. MTX can be administered orally, intravenously, or intrathecally for oncologic therapy. A potentially effective treatment for patients with GBM is intrathecal MTX administration mixed with systemic chemotherapy. This, treatment has mild treatment-related side effects . NumerouAs summarized in Wu et al.’s  review, The utilization of whole-brain radiation therapy (WBRT) is on the rise for individuals who are not eligible for surgical or Stereotactic radiosurgery (SRS) interventions since it has been shown to significantly enhance the survival rates of patients with brain metastases. Although WBRT has been found to be successful in attaining control over intracranial conditions, some studies have indicated a correlation between WBRT and a loss in neurocognitive function. This decline has been shown to impact the long-term cognitive status and overall quality of life of individuals undergoing WBRT ,95. OverLike the surgical resection approach, systemic medication is employed in conjunction with SRS to attain localized CNS control and impede the dissemination of metastatic disease. According to a recent study , it is rRecent studies have indicated no significant disparities in local control outcomes when comparing surgical resection and SRS as standalone treatment modalities ,98. NeveThe current body of literature supports the utilization of several fractions to enhance the treatment effectiveness for brain metastases despite the feasibility of deploying either single-fraction or three-fraction SRS. As a result of these considerations, our standard protocol entails administering a total dose of 27 Gy in three fractions to metastatic brain sites, excluding lesions located in the brainstem and tumors exceeding 3 cm in size or exhibiting significant vasogenic edema. According to a published study, implementing a 2 mm margin surrounding the resection cavity in SRS significantly decreases local failure rates from 16% to 3% for 12 months .Individuals who would typically not be considered suitable candidates for SRS due to large tumor sizes or lesions located in the eloquent cortex can experience advantages from using hypofractionated stereotactic radiosurgery (HF-SRS). It has emerged as a viable therapeutic option for patients requiring high radiation doses, with the added benefit of reducing negative neurocognitive effects. This is achieved by administering a sequence of low-dose treatments, resulting in a higher cumulative radiation dosage. According to a study conducted in 2019, administering a dose of 30 Gy for these lesions is generally considered safe and beneficial, divided over 5–6 sections . This trAccording to recent research, there is evidence suggesting that radiation therapy possesses immunomodulatory properties, which can alter the microenvironment of tumors and consequently lead to the reactivation of the immune system . OngoingThe Gamma Knife (GK) utilized a stereotactic radiotherapy technique that enhances radiation delivery precision for localized malignancies, whether primary or metastatic. A multiheaded cobalt unit is used to distribute GK. One benefit of GK is that it just requires one treatment session and has fewer radiation effects on nearby healthy tissue. Given the focused beam field, greater accuracy is required for immobilization; patient immobilization is accomplished by using external frames (with an accuracy of 1–2 mm) or custom-fit masks (with an accuracy of 2–3 mm) . GK-mediBrachytherapy, which uses brain implants for radiation sources, enables very targeted radiation delivery to tiny cancers. Iodine and iridium are the most frequently used radiation sources, and they can be positioned utilizing stereotactic guidance for either permanent or transient periods . EarlierCompared to traditional RT, photon beam therapy (PBT), which uses photon irradiation, may provide more localized radiation delivery , and ProRT has been used for decades to manage benign and malignant brain tumors. Adverse side effects of RT are frequently classified as acute, early-delayed, or late-delayed, depending on when the symptoms first appear . An immeEarly-delayed effects often manifest within 1–6 months after RT . LethargMore than six months after RT, late-delayed problems emerge and are typically irreversible . RadiatiFurther, Yamanaka et al. have systemically reviewed the characteristics and outcomes of radiation-induced gliomas. They collected information on 296 cases of radiation-induced gliomas . They obThe NovoTTF-100A System, approved by the FDA, is a novel therapeutic approach developed by Novocure, Ltd. in Haifa, Israel. The first-generation tumor-treating field (TTField) device (NovoTTF-100A System), which was renamed Optune later, was authorized by the FDA for recurrent GBM therapy in the year 2011 . The TTFDespite the advantages of TTF in the field, there are several limitations, including high price and availability issues .The CNS is an immune-privileged site with limited T cell access to perform its functions. This is primarily attributed to the presence of the BBB, the absence of dedicated lymphatic channels, the low basal expression level of Major Histocompatibility Complex (MHC) class II molecules, the scarcity of antigen-presenting cells (APC), and the continuous production of immunosuppressive cytokines like tumor growth factor beta (TGF-ß). These factors collectively contribute to the significant impact on the ability of T cells to exert their immune functions . Recent In recent years, there has been a substantial increase in the utilization of immunotherapy as a significant contributor to therapeutic improvements in cancer research. This is supported by several scholarly articles ,129,130.Furthermore, it is commonly acknowledged that molecular heterogeneity inside GBM is a crucial factor in the emergence of resistance to treatment therapies. As a result, the imperative to tackle this heterogeneity has emerged as a fundamental clinical goal in the endeavor to develop efficacious immunotherapeutic approaches that are tailored especially to GBM . AdditioDespite encouraging outcomes, immune checkpoint inhibitors therapy ICIs are ineffective for all solid tumors, some cancers have a low response rate, and there are significant side effects . An analCAR T treatment offers the distinct benefit of circumventing the requirement for MHC presentation of antigens and the successive establishment of an adaptive immune response . The fieThe utilization of cancer vaccine therapy has demonstrated significant potential in both preventative and therapeutic contexts ,152. In A limited quantity of particles can traverse the BBB, necessitating the development of innovative technologies and delivery systems to effectively transport medications into the brain. The utilization of nanocarriers and nanotechnology in drug delivery has the potential to surmount the BBB owing to its inherent attributes such as biosafety, sustained drug release, improved solubility, enhanced drug bioactivity, BBB penetrability, and self-assembly, as supported by the scientific literature ,157. NanLiposomes are lipid-based vesicles that have gained significant attention in the field of drug delivery. The composition of liposomes bears resemblance to that of cellular membranes, given its construction of a hydrophilic core encased by an external phospholipid bilayer. This attribute enhances the lipophilicity of molecules and facilitates the passage of lipophilic macromolecules across the BBB. Liposomal nanoparticles have numerous advantages, such as their straightforward manufacturing, capacity to encapsulate a diverse array of anticancer medications, excellent biocompatibility, high effectiveness, lack of immunogenicity, enhanced solubility of anticancer agents, and widespread commercial availability . LiposomCertain receptors or antigens overexpressed on GBM cells have been identified as promising targets for advancing innovative nanotechnology in tumor treatment. For example, studies have examined the effects of IL-13-conjugated liposomes and IL-4 receptor-targeted liposomal doxorubicin in m models. These investigations have demonstrated notable reductions in tumor size when compared to liposomes that were not conjugated ,165. TheNotwithstanding the widespread utilization of liposomal nanoparticle GBM therapy, several drawbacks necessitate resolution. Variations in the effects of liposomal nanoparticles are observed in different brain regions, and their ability to permeate the BBB is influenced by the specific medication or surface chemicals they carry. Recent nanocarrier and GBM therapy advancements have been summarized in The presence of chemoresistant/radioresistant cancer stem cells (CSCs) and biological barriers like the blood–brain barrier (BBB) extend hindrance to the efficacy of conventional therapies against GBM ,172,173.Targeting solid tumors using NPs for diagnostic and therapeutic purposes is a popular concept that has been explored for a very long time. The delivery of NPs to tumor regions largely relies on EPR-based passive targeting or their functionalization using suitable ligands targeting specific biomarkers (active targeting) overexpressed on the cancer cells. Over the years, several organic and inorganic NPs have been utilized for drug delivery; however, delivery efficiency could not be increased beyond 0.7% . MNPs haOver the years, NPs such as MNPs or their composites have proved their mettle as efficient drug/radiosensitizer/photosensitizer delivery platforms and in performing simultaneous therapeutic  and imaging functions , demonstrating incredible potential against GBM. A summary of various MNPs developed in the treatment of GBM is presented in detail in a previous review by Dhar et al. .Four subgroups of GBM tumors were discovered using transcriptional profiling data from bulk tumor tissues: mesenchymal, classical, proneural, and neural. A recent study revealed 18 driver genes with varied expression profiles in several molecular subtypes, including MGMT, ATRX, H3F3A, TP53, EGFR, NES, VIM, MIK67, and OLIG2 . It was Despite significant efforts dedicated to understanding the intricate factors contributing to the development and nature of brain tumors, the outlook for individuals diagnosed with these tumors continues to be discouraging. Heterogeneity inside and among tumors is attributed to genetic and non-genetic variables operating at various lengths and time ranges. This results in a distinctiveness observed in each tumor and patient. Therefore, adopting a comprehensive approach encompassing multiple levels of biological systems is imperative to comprehend and effectively tackle the intricacies of tumors. Adopting a personalized precision medicine strategy that considers a patient’s tumor’s distinct characteristics and biological makeup is imperative to address these obstacles. To achieve this objective, it is imperative to adopt a systems biology approach that encompasses a comprehensive understanding of the various levels of organization involved in illness causation and progression. The comprehension of illness characteristics at a systems level might aid in categorizing patients into clinically significant subtypes and provide insights into potential targets for therapeutic intervention, hence improving treatment outcomes .The resistance of GBM cells and glioma-initiating cells (GICs) against current treatment techniques is facilitated by their robust DNA repair mechanisms and ability to self-renew. Therefore, developing novel treatment techniques will be necessary to manage GBM long-term effectively. The profound comprehension of the GBM microenvironment and the potential to manipulate the patient’s innate and adaptive immune system to combat the tumor constitutes the foundation of immunotherapeutic approaches that now hold promise for the battle against GBM. Although the immunotherapeutic strategy has demonstrated efficacy in various solid and hematologic neoplasms, its application in treating GBM is hindered by immune resistance and an immunosuppressive environment . The futNanoparticles possess distinct physical attributes, including size, shape, and surface qualities, enabling them to effectively encapsulate and transport therapeutic compounds to the brain . NanoforMagnetic nanoparticles (MNPs) have emerged as an auspicious nanoscale material for applying intratumoral hyperthermia therapy in GBM. Nevertheless, the replication of the effects shown in various nanoformulations on GBM cell models in actual clinical trials is hindered by the unpredictable nature of tumor heterogeneity, which is a significant barrier to the successful treatment of GBM. The available evidence on immunotherapy for GBM is generally limited, hindering its potential for major therapeutic efficacy. However, the utilization of combination therapies may offer more promising outcomes. Despite obstacles and unsatisfactory clinical outcomes in developing immunotherapy for GBM, this approach is warranted due to the therapeutic promise of this treatment and the rapidly advancing pace of research in this field.Furthermore, the therapeutic implications of the role played by the BBB in the lack of success in treating GBM necessitate a renewed focus on enhancing technologies that disrupt the BBB, creating agents that can penetrate the BBB, and improving implanted drug delivery systems that circumvent the BBB . Lastly,Despite the advancement in the field, GBM remains the deadliest tumor type, with restricted therapy options and poor survival. Given the genomic intricacy, cellular heterogenicity, and diverse signaling pathways, a monotherapy will unlikely be successful in GBM. An efficient treatment methodology for GBM will require combining multiple therapy approaches considering diverse oncogenic pathways. To overcome the therapy challenges, we may require an inclusive comprehension of the detailed molecular mechanisms of the therapy resistance. For instance, a recent study identified a Syx-RhoA-Dia1 signaling axis as a DNA damage regulator and therapy resistance in glioblastoma . The com


The liver is a major store of glycogen and is essential in maintaining systemic glucose homeostasis. In healthy individuals, glycogen synthesis and breakdown in the liver are tightly regulated. Abnormal glycogen metabolism results in prominent pathological changes in the liver, often manifesting as hepatic glycogenosis or glycogen inclusions. This can occur in genetic glycogen storage disease or acquired conditions with insulin dysregulation such as diabetes mellitus and non-alcoholic fatty liver disease or medication effects. Some primary hepatic tumors such as clear cell hepatocellular carcinoma also demonstrate excessive glycogen accumulation. This review provides an overview of the pathological manifestations and molecular mechanisms of liver diseases associated with abnormal glycogen accumulation. The liver plays a central role in carbohydrate metabolism. It is regarded as a “glucostat”, maintaining systemic glucose homeostasis through glucose uptake and storage post-prandially and glucose production and release during fasting. The physiologic regulation of hepatic glycogen synthesis, glycogenolysis, gluconeogenesis, glycolysis, and other enzymatic reactions is mediated through various mechanisms, including the balance of hormones such as insulin and glucagon, allosteric control by metabolites such as glucose, acetyl-coA, and glucose-6-phosphate, the availability of substrates, and the cellular redox state . Dysregulation of glycogen metabolism in systemic conditions such as diabetes mellitus (DM), hepatic conditions such as non-alcoholic fatty liver disease (NAFLD), as well as genetic diseases (glycogen storage diseases) can lead to histological manifestations in the liver and cause significant hepatic dysfunction. When there is clinical uncertainty as to the cause of an abnormal liver enzyme profile, a liver biopsy can be helpful in guiding towards the correct diagnosis and excluding other diseases. The pathological changes, however, are often under-recognized or can be incorrectly attributed due to unfamiliarity with different patterns of injury as well as histological overlap between entities. In addition, glucose metabolism is often altered in cancer cells in order to support their rapid proliferation and expansion; this is part of metabolic reprogramming, a recognized hallmark of malignancy ,3,4. MosThis review focuses on the histological manifestation of diseases associated with abnormal glycogen accumulation in the liver and briefly explores possible molecular mechanisms behind these changes.The liver is a major store of glycogen. Glycogen is a branched polymer of glucose that is synthesized through the actions of glycogenin, glycogen synthase, and glycogen branching enzyme . GlycogeGlycogen synthesis is regulated by phosphorylation/dephosphorylation and/or binding of allosteric ligands . GlycogeThe liver contains two types of glycogen: small β particles and larger α particles; the latter is a composite aggregate of β-particles . α-partiIn a normal state, all hepatocytes contain glycogen. The amount of glycogen is largely homogeneously distributed through out the liver lobule, although there can be zonal variations depending on the fasting or post-prandial state, corresponding to zonal variations in metabolic enzymatic activity . On lighExcessive glycogen accumulation within hepatocytes occurs in diseases caused or accompanied by the dysregulation of carbohydrate metabolism. In these pathological conditions, the excess glycogen is distinctly visible on H&E; the hepatocytes usually exhibit cytoplasmic pallor and rarefaction or may show cytoplasmic-glycogen-filled inclusion bodies. Some hepatocytes may also demonstrate glycogen-filled nuclear vacuoles without a delimiting membrane (“glycogenated nuclei”) ; these aGlycogenic hepatopathy is a distinctive, reversible clinicopathological entity caused by excessive overloading of hepatocytes with glycogen, classically seen in patients with poorly controlled type 1 diabetes mellitus. While first documented as a component of Mauriac syndrome in 1930 , it was Glycogenic hepatopathy typically occurs in children and adults with marked or prolonged hyperglycemia, followed by exogenous administration of insulin in the setting of type 1 DM. Most patients have hepatomegaly and elevated serum aminotransferases, which improve after improved glycemic control . Rarely,The excessive glycogen accumulates in hepatocytes because of the insulin-independent passage of glucose into hepatocytes during the period of hyperglycemia, followed by insulin-mediated conversion into hepatic glycogen; both elevated glucose and insulin are required  Figure . PatientApart from type 1 DM, glycogenic hepatopathy has also been described in other clinical conditions, such as following the administration of short-term high-dose steroid therapy , dumping1c levels [A case–control study confirmed that patients with glycogenic hepatopathy had poorer glycemic control, as evidenced by a history of recurrent episodes of diabetic ketoacidosis and elevated HbAc levels . Howeverc levels . Althougc levels , this doc levels .Histologically, glycogenic hepatopathy is characterized by a striking diffuse hepatocellular change—the hepatocytes appear pale with cytoplasmic rarefaction and accentuated cell membranes, due to cytoplasmic accumulation of glycogen . PAS andFibrosis is usually not a feature of glycogenic hepatopathy. Although occasional cases may have mild portal or pericellular fibrosis , only veRecently, it has been recognized that excess accumulation of glycogen within hepatocytes can also be seen in the context of non-alcoholic fatty liver disease. In these cases, the dominant clinicopathological picture is not that of the typical glycogenic hepatopathy patient ; instead, patients are typically older, have type 2 DM and other features of metabolic syndrome, and steatohepatitis. These glycogenic hepatocytes can be seen in up to 54% of NAFLD cases ; the glyPPP1R3B, a hepatic glycogen metabolism regulatory protein involved in glycogen synthesis, have been reported to have reduced hepatic steatosis [The presence of focal/patchy hepatocyte glycogenosis in a significant proportion of NAFLD patients underscores the complexity of glucose regulation. Insulin resistance, which is a major hallmark of NAFLD, impairs hepatic glycogen synthesis through dysregulation of glucokinase translocation from the nucleus to the cytoplasm, a rate-controlling step in insulin-stimulated hepatic glycogen synthesis . Hepatocteatosis , possiblteatosis . Indeed,teatosis . Glycogen storage diseases (GSDs) are genetic disorders of glycogen metabolism, many of which present as an abnormal accumulation of glycogen or lipid in the liver. They are classified based on enzyme deficiency and the affected tissue (muscle and liver); the GSDs primarily affecting the liver are types 0, I, III, IV, VI, and IX . The hepThe glycogenosis seen on liver biopsies in patients with hepatic GSD can look indistinguishable from that seen in glycogenic hepatopathy, although in some cases of GSD, there may be subtle but increased cytoplasmic clumping of glycogen, and in type IV there are distinctive ground-glass type inclusions . The cliHepatic steatosis may also be present in patients with GSD. The dysregulation of liver glycogen metabolism often leads to compensatory increases in lipolysis from extrahepatic sources and mitochondrial fatty acid oxidation in GSD types associated with fasting ketotic hypoglycemia . In contrast, the hyperlipidemia seen in GSD type I results from the intracellular accumulation of glucose-6-phosphate, leading to increased glycolysis, increased production of acetyl-CoA, and increased lipogenesis .Abnormal glycogen accumulations can sometimes also be seen in conditions not primarily driven by altered carbohydrate metabolism, such as urea cycle defects and drug-induced changes. The urea cycle occurs primarily in the liver and refers to a series of mitochondrial and cytosolic enzyme reactions that remove excess nitrogen from the body through the conversion of toxic ammonia into urea; urea is then excreted into the urine. Interestingly, foci of glycogenesis and glycogenated nuclei can be seen in patients with urea cycle defects, sometimes in a nodular manner; concomitant steatosis and varying degrees of fibrosis may also be present ,53. The pathogenesis of increased glycogen accumulation in patients with urea cycle defects remains to be determined. In one study, the activity of glucose-6-phosphatase, debranching enzyme, and phosphorylase appeared normal or only mildly abnormal. It was therefore suggested that the excess glycogen accumulation could be due to dietary treatment, wherein the high proportion of leucine could have stimulated excessive insulin secretion and glycogen synthesis . More reGlycogen pseudoground glass changes have been reported in patients taking numerous medications ,57. PseuThese drug-induced cytoplasmic changes may persist or resolve; their clinical significance, apart from being a histological mimic of other causes of pseudoground glass changes, is unclear at this time. Liver masses can have abnormal glycogen accumulation which leads to a clear cell appearance. These include cases of hepatic adenomas (particularly sonic hedgehog hepatic adenoma ) and cleMetabolic reprogramming, the alteration of a cancer cell’s metabolism, is important in carcinogenesis; it facilitates cell growth, proliferation, and other cancer-associated hallmarks ,3,4. Altisocitrate dehydrogenase 1 (IDH1) R132C mutations in 5 out of 20 clear cell HCCs (25%) by pyrosequencing; this mutation was absent in 28 HCCs with other morphologies in their study, although the authors did find 3 out of 193 HCCs (1.6%) from The Cancer Genome Atlas with non-clear cell morphologies that also had IDH1 mutations [+-dependent enzyme that is highly expressed in the liver [The metabolic reprogramming underlying the accumulation of intracytoplasmic glycogen that typifies clear cell HCC has been challenging to study. There is currently no uniform morphologic definition for clear cell HCC; neither has any clear genetic correlate been identified . One stuutations . Isocitrhe liver  and catahe liver . In normhe liver  and alsohe liver . More rehe liver . IDH1 gene expression has been shown to correlate with mRNA levels of canonical sterol regulatory element binding protein (SREBP) gene targets in several different human cancers, including HCC [IDH1 mutations demonstrate a greater proportion of intracytoplasmic lipid rather than glycogen to impart the optically clear cytoplasmic appearance. Certainly, some clear cell HCCs appear to have foci of microvesicular or even macrovesicular steatosis that is evident on H&E. Some degree of shunting of substrates across the closely interrelated glucose and lipid metabolic pathways is not unexpected. Contrary to what is observed in glycogenosis in NAFLD patients, however, some studies have noted an increase, rather than decrease, in the incidence of fatty change within HCC with an increasing proportion of clear cells [Increased ding HCC . The SREding HCC . Of noteding HCC ,76,77. Iar cells . While tar cells , this asThe metabolic alterations and genetic basis underlying cases of clear cell HCC with predominant glycogen accumulation and little/no steatosis may be different from those tumors with some fat. It is thought that, at least in some settings, glycogen synthesis and accumulation is an adaptive response to hypoxia through the effects of hypoxia-inducible factor (HIF), in anticipation of an unfavorable environment deficient in glucose during which rapid mobilization of these glycogen stores can then occur . In cleaIn clear cell renal cell carcinomas, glycogen synthase 1 (GYS1) was found to be overexpressed and promoted tumor growth . IncreasThe metabolic phenotypes of tumors may change as cancers progress from preneoplastic lesions to established malignancies. Recent work has suggested that glycogen accumulation is important in liver tumor initiation, possibly through sequestration of Hippo kinases Mst1/2 and indirect activation of the oncoprotein Yap ; in thisIt is likely that the clear cell subtype of HCC (as currently defined by pure morphology) represents several biologically different entities, with overlapping morphology but differing underlying molecular mechanisms. It remains to be determined whether the accumulated glycogen in clear cell HCC is truly essential for tumor initiation and/or growth  or a disGlycogen in hepatocytes is increasingly understood as not just a simple inert macromolecule but a complex molecule with important ramifications on metabolic pathways. The liver, as the main site of glycogen synthesis, storage, and lysis, manifests many of the key pathological changes associated with abnormal glycogen metabolism. Important liver diseases that result from glycogen accumulation include genetic glycogen storage diseases and acquired diseases resulting from insulin dysregulation and/or medication effects. Some primary hepatic tumors may also manifest prominent glycogen accumulation. Future work focusing on the mechanisms behind conditions with dysregulated glycogen metabolic pathways will lead to a better understanding of the significance of these changes on health and disease.


Environmental perturbations are encountered by microorganisms regularly and will require metabolic adaptations to ensure an organism can survive in the newly presenting conditions. In order to study the mechanisms of metabolic adaptation in such conditions, various experimental and computational approaches have been used. Genome-scale metabolic models (GEMs) are one of the most powerful approaches to study metabolism, providing a platform to study the systems level adaptations of an organism to different environments which could otherwise be infeasible experimentally. In this review, we are describing the application of GEMs in understanding how microbes reprogram their metabolic system as a result of environmental variation. In particular, we provide the details of metabolic model reconstruction approaches, various algorithms and tools for model simulation, consequences of genetic perturbations, integration of ‘-omics’ datasets for creating context-specific models and their application in studying metabolic adaptation due to the change in environmental conditions. Haemophilus influenzae RD [Since the first genome-scale metabolic reconstruction in 1999 for enzae RD . Genome-enzae RD , 4 and ain silico gene knockout experiments [The applications for metabolic reconstructions of microbial species are wide-reaching within biology, including but not limited to the design of metabolic engineering approaches , the moderiments , studyineriments , 16 and eriments .During their lifestyle as reproducing cells, dormant cells, commensals or pathogens, microbes encounter multiple different environmental perturbations, both externally and within a host niche, to which microorganisms must adapt accordingly. Such perturbations are often difficult to study in a laboratory, both technically and financially.in vitro with the aim of identifying just how microbes alter the way they metabolize and respire in order to best survive, colonize a host, induce virulence factors, evade the host’s immune system or produce products of biotechnological interest.The ever-increasing availability of ‘-omics’ data for microbial species has allowed the integration of such data with GEMs to create context-specific metabolic reconstructions , detailiSince the late 2000s, high-throughput sequencing technologies have become more frequently applied in the biology field and have been continually developing. Since first-generation Sanger DNA sequencing technologies , next-geab initio, gene prediction methods [DFAST, MicrobeAnnotator and Prokka all utilize homology-based annotation pipelines whereby reference databases or genomes are used to identify sequence similarity facilitating the prediction of protein coding genes . In cont methods , 31, 36. methods , 37. All methods . The out methods , while M methods . While aDue to the revolution in the sequencing field, an enormous number of species have been sequenced and annotated , 39. HigWith whole genome sequencing techniques becoming more accessible, the number of sequenced genomes has been growing exponentially since the first bacterial genomes were sequenced in 1995 . As a remerlin [merlin have inbuilt genome annotation tools  associations, and therefore gene expression data obtained through transcriptomic experiments can be integrated into networks by adjusting reaction fluxes. Gene expression data are mapped onto GPR associations to define inactive and active reactions in the condition of interest, and from this definition context-specific models are extracted . SeveralTo ensure that context-specific models are representative of the organism’s metabolism in a particular context, it is vital to evaluate expression scores while mapping to GPR associations , 121. ThThe GIMME algorithm can also be extended to include metabolomics: the Gene Inactivation Moderated by Metabolism, Metabolomics and Expression (GIM3E) algorithm . IntegraIntegration of microbial GEMs with ‘-omics’ data can enhance our understanding of microbial metabolism during infection such as deciphering the metabolic changes induced in pathogens by antibiotic treatment , exploriEscherichia coli and Saccharomyces cerevisiae integrated with transcriptomics data, pFBA was the most accurate at predicting reaction fluxes compared with experimentally measured fluxes [For models of d fluxes . Howeverd fluxes  when comGenome-scale metabolic models are a valuable tool to investigate the lethality and downstream metabolic effects of gene knockouts or mutations in species , 95, 129Genome-scale essentiality studies have become increasingly popular in the last 20 years, but they are expensive and not always feasible, particularly for less well-studied species. Such datasets are often used to validate phenotypic predictions generated from model reconstructions , 131. Hoin silico can be particularly helpful to determine the effects this genetic perturbation has on metabolism. For example, simulation of in silico single and double mutant strains of Mycoplasma pneumonia, using an experimentally validated GEM, revealed that biomass synthesis in this pathogen has very limited adaptability [Neurospora [Generating mutant bacterial strains, gene knockdowns or knockouts, can be challenging and elucidating any downstream effects can be even more challenging. Creating reconstructions of mutant strains tability . Metabolurospora .Streptomyces coelicolor increase antibiotic production [Streptomyces hygroscopicus [Strain-design algorithms can be implemented with GEMs in metabolic engineering approaches to identify optimal conditions for the maximal production of a particular metabolite of interest. Such approaches have been implemented to identify that the overexpression of genes encoding the enzymes acetyl-CoA carboxylase and branched-chain α-keto acid dehydrogenase in oduction . Similarscopicus , 7. Guidscopicus   is the most common method to predict fluxes across reactions in metabolic reconstructions, but has limitations; therefore, several other methods have been developed to expand upon this methodology, including dynamic FBA, geometric FBA and parsimonious FBA.Microbes will adapt their metabolism in response to several genetic and environmental perturbations, and simulating these perturbations through the integration of ‘-omics’ datasets with metabolic reconstructions and the use of other constraint-based analysis approaches can aid in the elucidation of such adaptations.