Amyotrophic lateral sclerosis (ALS) is a rare neurological disorder that affects the motor neurons responsible for regulating muscle movement. However, the molecular pathogenic mechanisms of ALS remain poorly understood. A deficiency in the antioxidant tripeptide glutathione (GSH) in the nervous system appears to be involved in several neurodegenerative diseases characterized by the loss of neuronal cells. Impaired antioxidant defense systems, and the accumulation of oxidative damage due to increased dysfunction in GSH homeostasis are known to be involved in the development and progression of ALS. Aberrant GSH metabolism and redox status following oxidative damage are also associated with various cellular organelles, including the mitochondria and nucleus, and are crucial factors in neuronal toxicity induced by ALS. In this review, we provide an overview of the implications of imbalanced GSH homeostasis and its molecular characteristics in various experimental models of ALS. Cu/Zn-superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9orf72), optineurin (OPTN), p97/valosin-containing protein (VCP), TAR DNA-binding protein (TDP-43), fused in sarcoma (FUS), Ewing sarcomabreakpoint region 1 (EWSR1), and TATA box-binding protein-associated factor 15 (TAF15), have been linked to the sporadic and familial forms of ALS . Ac. Ac80]. ic oxide . Further pathway . They alnic mice . Nrf2 ovith Nrf2 . Such a G93A-expressing mice during disease progression -GSH, and found that [35S]-GSH binding in the spinal cord was increased in patients with sALS [Previous studies have identified binding sites for GSH in the synaptic membranes of the brain and spinal cord ,110. In ith sALS . Similarith sALS . In factith sALS . GR actiith sALS . Anotherith sALS . GPx catith sALS . Thus, iWeiduschat et al. investigated the in vivo levels of GSH in the motor cortex region of patients with ALS using the J-edited spin-echo difference magnetic resonance spectroscopy (MRS) technique . ConsistIn 1998, Schiffer’s group studied the effect of GSH treatment on the rate of progression of ALS in patients and found that GSH treatment did not have a significant effect on ALS progression . Some cyGSH exerts important functions in the central nervous system as an antioxidant, enzyme cofactor, redox buffer, and neuromodulator. GSH deficiency and the dysfunction of GSH metabolism are common to several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Although accumulating evidence suggests that aberrant GSH homeostasis is linked to the development and progression of ALS, the precise role and mechanism of redox imbalance in neuronal cells in this disease remains to be determined. In this review, we first summarized the experimental evidence supporting the role of GSH in the development and progression of ALS. Many studies on ALS have focused on the decreased GSH content and imbalanced GSH redox status in the nervous system, which result in ALS progression. Furthermore, several GSH-related enzymes that target oxidative stress and regulate GSH homeostasis have also been studied. The mechanism of ALS progression may involve decreased GSH, increased GSSG, and an impaired GSH-associated antioxidant system, suggesting that an imbalance in redox status beyond its physiological limit may be detrimental to motor neuronal functions and survival that regulate disease initiation and conditions. Although several studies provide indirect or direct evidence that the dysregulation of GSH homeostasis and metabolism in the nervous system are associated with the pathogenesis of ALS, it remains unclear whether reduced GSH is a causative factor in ALS or whether various mutations in ALS-causing genes are responsible for the impaired GSH redox status observed in ALS. In addition, the molecular mechanisms and pathways related to the synthesis, transport, and degradation of GSH in neurons and astrocytes are complex, and our understanding of the imbalanced GSH redox status in the brain of ALS patients remains incomplete. Therefore, future studies should aim to intensively investigate whether treatments that increase the GSH/GSSG ratio in motor neurons and astrocytes by activating GSH biosynthesis have clinical efficacy in the treatment of ALS. In addition, there is a need to study and develop effective pharmacological agents that may enhance GSH functions to reduce oxidative stress and damage cellular organelles, including the mitochondria, in motor neurons.


Cancer, but also its treatment, can lead to a reprogramming of cellular metabolism. These changes are observable in metabolite abundances, which can be unbiasedly measured via mass spectrometry metabolomics. However, even when the metabolome changes strongly, a (mechanistic) interpretation is difficult as metabolite levels do not necessarily directly correspond to pathway activities. Here we measure the changes of the cellular metabolome in colorectal cancer cell lines sensitive and resistant to the ruthenium-based drug BOLD-100/KP1339 and the platinum-based drug oxaliplatin. We map these changes onto a cancer-specific genome-scale metabolic model, which allows us not only to compute intracellular flux distributions, but also to disentangle drug-specific effects from growth differences from differences in metabolic adaptations due to resistance. Specifically, we find that resistance to BOLD-100/KP1339 induces more extensive reprogramming than oxaliplatin, especially with respect to fatty acid and amino acid metabolism.Background: Mass spectrometry-based metabolomics approaches provide an immense opportunity to enhance our understanding of the mechanisms that underpin the cellular reprogramming of cancers. Accurate comparative metabolic profiling of heterogeneous conditions, however, is still a challenge. Methods: Measuring both intracellular and extracellular metabolite concentrations, we constrain four instances of a thermodynamic genome-scale metabolic model of the HCT116 colorectal carcinoma cell line to compare the metabolic flux profiles of cells that are either sensitive or resistant to ruthenium- or platinum-based treatments with BOLD-100/KP1339 and oxaliplatin, respectively. Results: Normalizing according to growth rate and normalizing resistant cells according to their respective sensitive controls, we are able to dissect metabolic responses specific to the drug and to the resistance states. We find the normalization steps to be crucial in the interpretation of the metabolomics data and show that the metabolic reprogramming in resistant cells is limited to a select number of pathways. Conclusions: Here, we elucidate the key importance of normalization steps in the interpretation of metabolomics data, allowing us to uncover drug-specific metabolic reprogramming during acquired metal-drug resistance. A reprogramming of metabolism is a hallmark of multiple diseases, including cancer . ChangesIn silico methods have the potential to integrate existing experimental data and to generate new hypotheses about the underlying mechanisms associated with metabolic reprogramming. Genome-scale metabolic models (GSMMs), which capture the known biochemical reactions of a given system, have previously been applied in various cancer studies ,6, such Metal-based drug treatments involving oxaliplatin are a standard therapy for colorectal cancer, the third most commonly diagnosed cancer ,18,19,20Acquired resistance against platinum drugs is thought to be mainly based on elevated DNA-repair mechanisms, detoxification, evading apoptosis and autophagy . HoweverMetabolomics aims to directly measure metabolite abundance from a global and unbiased perspective and has the potential to not only detect metabolic alterations but to discover diagnostic and prognostic markers and to generate hypotheses that can be validated with genetic experiments . Recent While metabolomics studies provide an effective interrogation window for the cellular changes that occur in response to a change in conditions, they do not necessarily provide mechanistic insights into the reprogramming of metabolism. Metabolite pools do not inform about pathway activity, ergo corresponding metabolic fluxes are sometimes measured. Measuring metabolic fluxes, however, also suffers from several practical limitations. For example, a prolonged time for peripheral pathways to reach isotopic steady-state, the fact that simple linear pathways can only be investigated with non-stationary labelling or an increased number of samples, and the complex data analyses required for nonstationary labelling experiments often hinder a successful and comprehensive application of isotopic labelling methods .Recent trends in metabolomics have shown it is always possible to measure more metabolites at more time points and to analyse the obtained results in combination with other ‘omics data sets ,32,33,34Typically, constraint-based modelling (CBM) is employed to study GSMMs and to explore metabolic phenotypes in the form of steady-state fluxes ,44. FluxIn this work, we integrate experimentally determined absolute concentrations of intracellular metabolites and medium-based metabolites and growth rates of the colorectal cancer cell-line HCT116 into a cell-line specific, thermodynamic, genome-scale metabolic model (GSMM). We consider two different models of acquired resistance in colon cancer: oxaliplatin resistant (OxR) and BOLD-100/KP1339 resistant (RuR) HCT116 cells as well as their sensitive controls to generate four model instances. To identify metabolic differences between resistant and sensitive cells, we normalize the calculated flux values by their representative growth rates. As oxaliplatin and BOLD-100 are prepared in different solvents (water versus dimethylsulfoxid (DMSO)), OxR and RuR cells were grown in the same media, but RuR and its respective control were exposed to a low DMSO background equivalent to the drugs’ stock solution solvent. To account for metabolic differences that are the results of a difference in solvent, we further normalized the results obtained for the resistant cells by their sensitive controls. Eliminating both differences in growth rate and solvent background, we are able to draw drug-specific conclusions about the metabolic changes that occur upon resistance. As a result, we are able to identify specific changes in flux that are the direct result of an acquired resistance to either oxaliplatin or BOLD-100/KP1339 treatment.Mycoplasma contamination.HCT116 colon cancer cells were generously provided by Dr. Vogelstein from John Hopkins University, Baltimore. Cells were cultured in McCoy’s medium  supplemented with 10% fetal calf serum  and 2 liplatin ,53 for 2Cells were seeded at densities of HCT116 cells, HCT116 cells with acquired oxaliplatin resistance, and HCT116 cells with acquired BOLD-100/KP1339 resistance were seeded , acetyl-coenzyme A, palmitoyl-coenzyme A, malonyl-coenzyme (A below LOD) was carried out in an additional measurement series of the same samples and on the same instrumental setup but with a dedicated LC-MS method. The same separation was used with the same gradient and eluents, but flushing of the column started at 6 min instead of 7 min, shortening the total measurement time from 15 min to 14 min. The OrbiTrap MS settings were changed with regard to the mass range to 750–1100 m/z, the capillary temperature was lowered from 280 The applied extraction and centrifugation resulted in a pellet containing the high-molecular fraction and non-polar metabolites. This pellet was dissolved in 0.2 M NaOH overnight, diluted 1:10 in the same NaOH solution and determined for total protein content with the Thermo Micro BCA kit, according to manufacturer’s instructions.https://skyline.ms/project/home/software/Skyline/begin.view, accessed on 12 August 2021) extracting the [M-H]Targeted analysis of the data was done with Skyline 20.2  above 30% were removed from the dataset. This was based on the repeated injection and measurement of a pooled quality control sample. Furthermore, metabolites which had mean concentration below the determined lowest limit of quantification (LOQ) according to the validation of the LC-MS method described in  were remDatasets were combined by joining the metabolite data acquired in both positive and negative mode, as well as coenzyme data in the negative acquisition mode. A further calibration was measured in positive mode for several carnitines  with the method for metabolites, since these compounds were not contained in our original calibration mixture. Also the calibration row for coenzymes was prepared freshly in this mixture to avoid degradation by storage. The external calibration of the different coenzymes  was measured in negative mode. For every primary thiol in the dataset  its N-ethyl maleimide adduct was used for quantification after it was made sure that the conversion was quantitative.The cell dry weight measurements of HCT116 sensitive (Sen), oxaliplatin-resistant (OxR) and BOLD-100/KP1339-resistant (RuR) cells were carried out .Robinson et al.  provide soft_l1 for the loss function and Growth rates  and exchper cell . The fitsurement  and the surement  was calcAll applied model constraints are based on data obtained during the exponential growth phase and as such, all model results are specific to this growth phase.https://github.com/EPFL-LCSB/pytfa, accessed on 12 August 2021, formulates thermodynamic flux analysis (TFA) of GSMM as a mixed-integer linear programming problem that incorporates metabolite concentrations as thermodynamic constraints into a traditional flux balance analysis (FBA) model. Masid et al. [The pyTFA package , https:/d et al.  have recd et al. . Absolutf fluxes , we calcf fluxes . Becausef fluxes .find_blocked_reactions in cobrapy (Version 0.19.0) with default parameters, resulting in a model with 4530 reactions and 4492 degrees of freedom. Upon calculating flux values for each model instance using pTFVA as described, we divided each set of flux values by the outgoing flux to biomass production of that model instance, effectively normalizing for difference in growth. We checked for reactions for which both the upper and the lower bound differed by at least 15%. Furthermore, we feature-scaled all flux values to lie between 0 and 1 and divided the flux values obtained in the drug-resistant instances by the corresponding flux values obtained for their respective controls. Having thus normalized for differences in the medium composition, we were able to compare the flux profiles of the two metallodrug resistances to another, again checking for which reactions both the upper and lower bounds differed by at least 15%.We constrained four different instances of the HCT116 model: oxaliplatin-resistant cells (OxR) and their sensitive parental counterpart (HCT116) and BOLD-100/KP1339-resistant cells (RuR) and their sensitive parental counterpart in a DMSO-based medium (HCT116-DMSO). Model instances were constrained using the condition-specific exchange fluxes  and growTo investigate the metabolic changes associated with metallo-resistance in colorectal cancer, we compared the metabolic profiles of resistant and sensitive cells. Using the HCT116 colorectal cancer cell line, cells with resistance to either oxaliplatin (OxR) or BOLD-100/KP1339 (RuR) were compared to their sensitive counterparts. The two acquired resistence models are largely independent of one another: while OxR cells show moderate cross-resistance for the ruthenium-based drug, RuR cells display no cross-resistance and remain sensitive to oxaliplatin treatment . This imWith the aim of investigating whether the observed changes in cellular metabolite concentrations  translatThe maximum relative standard error observed across the metabolite measurements was less than 15%. Thus, when integrating the data into the GSMM and comparing flux differences between condition-specific instances of the model, we used a cutoff of 15% to determine whether fluxes were significantly different across conditions. Comparing resistant cells to sensitive cells, we identify pathways with the most prominent changes in flux upon acquired resistance . DiffereInitially, the oxidative phosphorylation pathway shows the highest amount of flux changes in response to OxR. Upon growth normalizing, however, it is RuR that shows a higher number of flux changes in this pathway. Furthermore, what initially appears to be significant differences in flux through the cholesterol and lipid metabolism, largely disappears upon growth normalization. Changes in the subsystem reactive oxygen species (ROS) detoxification seem minimal prior to growth normalization; the normalized results, however, indicate significant changes in flux with regards to detoxification. While the number of reactions that appear to be affected in starch and sugar and tricarboxylic acid (TCA) metabolism appears to be drug resistance-specific prior to growth normalization, this effect disappears upon growth normalization. The comparison of non-normalized and growth-normalized results in Integrating metabolite measurements into GSMMs allows for growth rate normalization of the calculated fluxes which in turn allows for a direct flux comparison between resistant and sensitive cells. The reprogramming of energy metabolism to support cell growth and proliferation is a major hallmark of cancer  and has In the growth conditions considered here, glucose acts as the primary carbon source . GlucoseWe observe that OxR cells convert less pyruvate into lactate, but in turn consume a higher relative amount of fatty acids compared to their parental sensitive cells. RuR cells, however, show a high glycolytic flux and a high oxygen consumption as well as higher fatty acid consumption than their sensitive counterparts . NotablyWith the four instances of the HCT116-specific GSMMs, further conditions encountered in the tumor environment can be simulated. Simulating the effect of hypoxic growth conditions, we first set the oxygen influx for each model instance to the minimum possible value and then observe the minimum required fatty acid influx as we iteratively increase the oxygen influx, thus plotting the growth normalized production envelope of oxygen versus minimum fatty acid influx for each of the four conditions . While RWe then set a minimum possible fatty acid influx and iteratively increased the total fatty acid influx to the model while calculating the minimum required oxygen influx . We repeCrucially, while a direct comparison between resistant and sensitive cells for each drug respectively can be made, we cannot make a direct comparison between the two drug resistance models . BecauseIn order to be able to compare the metabolic profiles of the two metallodrug resistance phenotypes directly, we finally normalized the flux results obtained from the metallo-resistant model instances against their respective parental sensitive counterparts see  cells hiNotably, when comparing the OxR and RuR model instances to their respective parental HCT116 drug-sensitive counterparts, prior to growth normalization, we identified 1039 (OxR) and 1180 (RuR) fluxes that were significantly different. Upon growth normalization, these numbers reduced to 743 (OxR) and 883 (RuR), highlighting that hundreds of differences observed in the non-normalized results are simply the result of a difference in growth rate. The OxR versus RuR comparison upon growth-media DMSO-background normalization highlighted 670 different reactions, suggesting that another 73 of reactions were initially observed as significantly different because of presence of 0.5% DMSO.Genome-scale metabolic models (GSMMs) provide a platform for integrating omics data sets and for analysing them in the context of metabolic fluxes. As we have shown, GSMMs can be constrained using both extracellular and intracellular metabolite concentrations to study metallodrug resistance in colon cancer. Approximately one-hundred metabolite constraints were applied to study the effect of changes in their concentrations in thousands of reactions. Colorectal-specific GSMMs have previously been constructed ,48,58,71In this study, we investigated various pathways in silico including glycolysis, the tricarboxylic acid cycle, fatty acid and amino acid metabolism, beta-oxidation, the pentose phosphate pathway. A comprehensive stable isotope resolved metabolic flux analysis considering such a diverse set of pathways would require the application of multiple different positionally labelled isotopic tracers ,72. In aThere is no simple relationship between changes in metabolite concentrations and changes in flux . This noAccurate comparative profiling of metabolic changes observed across heterogenous conditions remains a challenge. Differences in growth rates and impact of solvent necessities will result in observed differences in metabolite concentrations but are not causal to a reprogramming of metabolism . ConsideAs a prerequisite to produce accurate, quantitative metabolomics data , we normIntegrating the metabolite data as part of a thermodynamic flux analysis allows us to normalize the calculated reaction rates by the growth rate observed under the corresponding conditions. We showed that growth-normalization reduces the number of reactions that are different between resistant and sensitive model instances and changes some of the conclusions about altered metabolic pathways entirely. Growth normalization is therefore a critical step when looking for drug-specific metabolic phenotypes. A second limitation to studying non-normalized metabolite concentrations is that data obtained from heterogeneous conditions cannot be directly compared. Using the growth normalized flux results we further normalized each resistant model against its sensitive counterpart which was grown in an identical medium and solvent composition. Hence, we were able to do a direct comparison between the two metallo-resistances and to identify drug resistance-specific responses.A limitation to our approach is that we first normalized our flux results to differences in growth rate and then normalized each resistant model against its respective counterpart. This means that we are unable to capture emergent properties that result from both differences in growth rate and solvent impact; we assume that a combined effect of the two is minimal. Furthermore our flux analyses assume metabolism to be in steady-state, such that intracellular concentrations are constant. Nonetheless, we have clearly demonstrated that a growth and medium/solvent normalization is non-trivial as it allows for comparisons across heterogeneous conditions. We expect this method to be of wider applicability in studies where the effects of medium compositions, such as the availability of carbon sources to a cell, are of interest.Time-dependent changes of metabolite profiles have previously been considered ,82, but Defense mechanisms and acquired resistance are well known phenomena when applying metal-based drugs as anticancer agents. Reduced efficacy due to acquired resistance remains a major challenge in systemic anticancer therapy. The complexity is increasingly recognized, as the contribution of epigenetic and metabolic effects will be uncovered. Drug-specific and tumor tissue specific mechanisms have been described, and more recently the tumor microenvironment has come into focus . AccordiIn this work, we consider an in vitro study of colon cancer. Gastrointestinal cancer cell lines, including colorectal cancer cell line HCT116 activate beta-oxidation as a response to oxaliplatin treatment and conversely become more sensitive to oxaliplatin upon inhibition of fatty acid catabolism . A seminThe high metabolic plasticity of cancer cells enables efficient detoxification and protection strategies . NormaliDespite shared commonalities like the production of ROS, it is expected that RuR and OxR models display different metabolic phenotypes, because of known differences in their modes of action ,92,93. OThe comparison of fluxes through key energy metabolism reactions  shows thDrastic changes in oxygen and fatty acid availability are known stress conditions in a tumor microenvironment, and are assumed to be managed with metabolic adaptations . Lipid dAdditionally, the investigation of minimum oxygen requirement at various fatty acid influxes  revealedThere are different ways to capture the metabolic phenotype of a cell. Metabolic profiling via metabolomics provides an interrogation window of the intracellular concentrations at a given point in time. Extracellular concentrations measured over time provide insight to the cellular uptake and excretion rates of cells. Together they can be integrated to constrain the solution space of a genome-scale metabolic model. The calculated flux values can then be normalized according to growth rates and environmental conditions, allowing for drug resistance specific metabolic responses to be identified across heterogenous conditions. We find the outlined normalization steps to be crucial in the interpretation of the results and show that metabolic reprogramming is more extensive in BOLD-100/KP1339 resistant cells than in oxaliplatin resistant cells. We identify pathways, such as fatty acid and amino acid metabolism, to be upregulated in response to a resistance acquired to a ruthenium-based drug when compared to a platinum-based drug. All in all, genome-scale metabolic modelling provides a valuable platform for putting observed changes in metabolite concentrations in the context of metabolic fluxes.


Oxidative stress is a key pathological feature implicated in both acute and chronic liver diseases, including drug-induced liver injury (DILI). The latter describes hepatic injury arising as a direct toxic effect of administered drugs or their metabolites. Although still underreported, DILI remains a significant cause of liver failure, especially in developed nations. Currently, it is understood that mitochondrial-generated oxidative stress and abnormalities in phase I/II metabolism, leading to glutathione (GSH) suppression, drive the onset of DILI. N-Acetyl cysteine (NAC) has attracted a lot of interest as a therapeutic agent against DILI because of its strong antioxidant properties, especially in relation to enhancing endogenous GSH content to counteract oxidative stress. Thus, in addition to updating information on the pathophysiological mechanisms implicated in oxidative-induced hepatic injury, the current review critically discusses clinical evidence on the protective effects of NAC against DILI, including the reduction of patient mortality. Besides injury caused by paracetamol, NAC can also improve liver function in relation to other forms of liver injury such as those induced by excessive alcohol intake. The implicated therapeutic mechanisms of NAC extend from enhancing hepatic GSH levels to reducing biomarkers of paracetamol toxicity such as keratin-18 and circulating caspase-cleaved cytokeratin-18. However, there is still lack of evidence confirming the benefits of using NAC in combination with other therapies in patients with DILI. Undoubtedly, the increased use of drugs in medical care has become the prominent cause of liver injury , 2. ThusDILI broadly describes any injury to the liver that might occur as a result of prescribed medication or even dietary supplements that may develop from asymptomatic liver test elevations to induce acute liver failure . The latIndeed, oxidative stress plays a key role in the pathogenesis of DILI , 14, 15,Great strides in industrialization and drug development have come with benefits as well as inevitable risks. Some of the risks include the development of drug compounds that have a toxic effect on humans, and animals, post-market. A major contributing factor that continues to torment the pharmaceutical industry is the clinical safety issues post-market which are often associated with adverse side effects related to liver injury and causes costly drug withdrawals , 25. In Perhaps the major setback with DILI is the limited information regarding its incidences in population studies. Despite that, a few population-based studies have been conducted and reported around the world. For example, a study by Björnsson and coworkers has reported that in Iceland, between 2010 and 2011, crude annual DILI incidence rate was 19.1 cases per 100,000 inhabitants, with 75% cases caused by a single prescription medication . While iThe pathogenesis of DILI typically implicates the involvement of a toxic drug/metabolite that either directly affects the biochemistry of the cell or elicits an immune response. In any case, the resultant cell death is the event that leads to the clinical manifestation of DILI symptoms. The exact cellular and molecular mechanisms involved in the pathogenesis of DILI are currently poorly understood; however, several studies have divided the mechanisms into direct toxicity which is from drugs or their metabolites directly damaging the cytoplasm membrane, ER, or other organelles of the liver cells. While some studies propose an indirect toxicity injury where toxins first interfere with the metabolic pathways or metabolism of macromolecules, such as proteins or DNA, thus provoking generation of oxidative stress, hinder mitochondrial function that eventually changes the cell structure and eventually causes cell death , 50, 51.in vivo microenvironment, 3D experimental models have become a useful tool to study pharmacodynamics, which is essential for the early detection of possible toxic effects of potential drugs before reaching human testing [Clearly, early research has indicated that different animal species, including guinea pigs, hamsters, and rats, were used to study the pathogenesis of DILI (reviewed evidence ). Extend testing –62. Beyo testing –64. Howe testing –54. Nota testing –54. More testing –54. NoneOxidative stress is well-recognized as a predominant pathogenic process for the development of acute liver failure. As a result, there is already increased evidence describing the implications of oxidative stress in the pathophysiology of diverse liver conditions , 66, whiGenerally, oxidative stress is a well-studied pathological feature caused by an imbalance between the generation of highly reactive molecules and their detoxification by the intracellular antioxidant defence systems. Under disease conditions, the immoderate production of radical molecules such as reactive oxygen species (ROS) usually cause direct damage to the biomolecules and eventually impair biochemical processes and cause cellular injury . AlthougApparently, enhancing intracellular antioxidative systems remains a feasible strategy to counteract oxidative stress to alleviate cellular damage. As such, liver cells respond to oxidative stress by expressing antioxidant and detoxification enzymes to protect against the degenerative effect of free radicals. The expression of these cytoprotective factors is in part regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2), which senses increased ROS production  to produAs evident from From Others, like Wong et al. , 91, wenμg/kg subcutaneously every 12 h for 5 consecutive days. Perhaps a significant study by Nabi et al. [Besides paracetamol overdose, other studies reported on other forms of liver injury, such as that induced by excessive alcohol intake. For example, Stewart et al.  showed ti et al.  demonstrFrom the evidence summarized in Currently, it is understood that depletion of intracellular antioxidant defences, especially the levels of GSH remains the critical factor implicated in the worsening of hepatic injury, in response to drug overdose. GSH is regarded as one of the major cytoprotective antioxidants through its direct and indirect scavenging effects on ROS, leading to the amelioration of oxidative stress and improved liver function , 100. Fu


Journal of Ophthalmic and Vision Research (JOVR), Delrish et al discuss the use of modified nanoparticles for intravitreal drug delivery in a rat model of retinoblastoma. Retinoblastoma is the most common intraocular malignancy of childhood, with an incidence of 11.8 cases per million children aged 0–4 years in the US and with recent increase in incidence reported in Europe.,3 While developing countries often face more advanced disease and worse survival prognosis, retinoblastoma is considered curable, and mortality is rare in well-developed nations. Although the primary goal of retinoblastoma treatment is to protect the child's life, secondary and tertiary goals of globe salvage and visual acuity preservation have become more achievable with treatment advances, particularly the combination of intra-arterial and intravitreal chemotherapy. With current standard of care therapy, globe salvage can be achieved even for the most advanced group E eyes in about 50% of cases.In the current issue of ,7 Moreover, in vitro experiments show that drug dispersion in the vitreous cavity is driven by advective mass transport rather than molecular diffusion, relying on natural saccadic movements to distribute the drug.,9 Drug distribution may be especially challenging in more formed pediatric vitreous, and speed of dispersion could be important to achieve tumor control in the setting of drugs such as melphalan, which have a short half-life. Thus, improved bioavailability of intravitreal chemotherapy agents could decrease injection burden and retinal toxicity, improve overall tumor control rates, and improve globe salvage for retinoblastoma with vitreous seeding.Delrish et al describe what may be the next frontier in intravitreal chemotherapy delivery, with improved drug bioavailability using thiolated chitosan-carboxymethyl dextran nanoparticles (CMD-TCs-NPs). Although intravitreal injection offers direct drug access to the vitreous cavity and retina, it can take several hours for drug to diffuse across the entire vitreous cavity.,12 Chitosan is highly biocompatible, nontoxic, and mucoadhesive; has favorable enzymatic biodegradability, low immunogenicity, and inherent antibacterial properties; and can disrupt epithelial tight junctions to enhance permeability. Given these favorable properties, combined with easy accessibility, chitosan has become a popular candidate for pharmaceutical development, and the US Food and Drug Administration has already approved several chitosan-containing products.Chitosan is a semi-synthetic cationic polysaccharide produced by partial or complete deacetylation of the naturally occurring polymer chitin, which can be harvested from crustacean shells or fungal cell walls. Numerous factors can influence mucoadhesion, including pH, concentration, ionic strength, temperature, incubation time, surface charges, molecular weight, degree of deacetylation, and hydrophilicity, among others. Chitosan, in particular, has pH-dependent mucoadhesion, and certain modifications have been employed to improve the polymer's mucoadhesive properties at physiologic pH relevant to ophthalmic drug delivery. Trimethyl chitosan has been extensively studied and has strong mucoadhesive properties due to its high cationic charge. Thiolated chitosan derivatives have also been studied, which have free thiol groups to form covalent bonds with cysteine-rich subdomains of mucus glycoproteins. Compared to unmodified chitosan, thiolated chitosan has several advantages, including 6- to 100-fold improved mucoadhesion, 1.6- to 3-fold enhanced permeability, and in situ gelling properties at physiologic pH.,15 Together, these properties improve drug residence time and increase bioavailability. Thiolated derivatives require sulfhydryl groups to react, limiting use to cysteine-rich tissues, but secreted protein acidic and rich in cysteine (SPARC) has demonstrated localization to Muller and ganglion cells in the retina, making the retina a reasonable target tissue.Mucoadhesive polymers like chitosan contain numerous charged molecular groups that can form non-covalent bonds with mucin, allowing bioadhesion to mucosal surfaces. Previous studies have demonstrated that nanoparticle surface properties are a key factor determining distribution in the retina and vitreous following intravitreal injection, and although glycosylated chitosan polymers maintained a positive charge and could reach the retina, these particles were not able to penetrate the internal limiting membrane. Thiolated derivatives, on the other hand, may have improved penetration due to their interaction with cysteine-rich SPARC localized to Muller cells. In this issue of JOVR, Delrish et al specifically compare the biodistribution of thiolated versus methylated chitosan-carboxymethyl dextran nanoparticles (CMD-TCs-NPs vs CMD-TMC-NPs) following an intravitreal injection in a rat model of retinoblastoma. The authors found improved uptake of CMD-TCs-NPs in retinoblastoma cell culture, and in the rat model, 24 hr after injection; the CMD-TCs-NPs demonstrated diffusion throughout the vitreous cavity and retinal penetration, while the CMD-TMC-NPs were immobilized in the vitreous. The authors attributed these differences to improved bioadhesion of thiolated chitosan and prohibitive effects on vitreous diffusion of the greater positive charge of methylated chitosan  compared with the thiolated counterparts (+11mV).In ophthalmology, initial interests aligned with the use of chitosan to improve bioavailability of topically administered medications, but there is a growing body of literature on possible intravitreal applications. Of relevance to the article at hand, Delrish et al recently reported improved efficacy and tumor control using thiolated chitosan nanoparticles containing topotecan compared to free topotecan in a rat model of retinoblastoma. These early studies indicate feasibility and potential benefit of the application of thiolated chitosan derivatives in retinoblastoma treatment. Although further study is required to investigate the safety and reproducibility prior to human trials, there are potential exciting applications of these biocompatible nanoparticles to improve treatment outcomes for retinoblastoma in the future, and this technology could also be applied to a wide variety of other retinal diseases which have traditionally been managed with intravitreal injections.Although this was a small study with 10 rats, Delrish et al concluded that surface charge is a key factor determining vitreous diffusion and retinal penetration for chitosan-derived intraocular drug-delivery mechanisms, and they propose future use of thiolated chitosan derivatives for intravitreal chemotherapy delivery aimed at retinoblastoma treatment.This publication was supported by Grant No. P30 CA015083 from the National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the National Institutes of Health.There are no conflicts of interest.


Prostate cancer still represents an important health problem in men, considering its high frequency. Over the last decade, novel treatment options have emerged, leading to notable clinical benefits. These recent scientific acquisitions are creating the basis to widen the treatment scenario of this tumor, evolving from targeting the androgen receptor axis or the traditional chemotherapy approach.In recent years, the advances in the knowledge on the molecular characteristics of prostate cancer is allowing to explore novel treatment scenarios. Furthermore, technological discoveries are widening diagnostic and treatment weapons at the clinician disposal. Among these, great relevance is being gained by PARP inhibitors and radiometabolic approaches. The result is that DNA repair genes need to be altered in a high percentage of patients with metastatic prostate cancer, making these patients optimal candidates for PARP inhibitors. These compounds have already been proved to be active in pretreated patients and are currently being investigated in other settings. Radiometabolic approaches combine specific prostate cancer cell ligands to radioactive particles, thus allowing to deliver cytotoxic radiations in cancer cells. Among these, radium-223 and lutetium-177 have shown promising activity in metastatic pretreated prostate cancer patients and further studies are ongoing to expand the applications of this therapeutic approach. In addition, nuclear medicine techniques also have an important diagnostic role in prostate cancer. Herein, we report the state of the art on the knowledge on PARP inhibitors and radiometabolic approaches in advanced prostate cancer and present ongoing clinical trials that will hopefully expand these two treatment fields. Excluding skin cancer, prostate cancer (PCa) is still the most common tumor diagnosed in men. Despite the good prognosis in terms of 5-year survival rate considering all stages, reaching up to 98%, prostate cancer can threaten long-term health and still remains the second-leading cause of cancer related death in men, after lung cancer . When apAmong these, radiometabolic approaches have been explored, starting with the introduction of radium-223, a calcium-mimetic alpha emitter with a short range, that selectively binds to areas of increased bone turnover, such as skeletal metastases, considering the enhanced osteotropism of prostate cancer. The high-energy alpha particles can cause several double-stranded DNA breaks, leading to localized cytotoxic effect in the target lesions, with relatively low toxic effects on the nearby bone marrow . It is wBRCA1, BRCA2, and ATM  Ga-PSMA-11 PET/CT 8, in whi. The and177Lu-PSMA-617  PET ligand in men with suspected recurrence or newly diagnosed prostate malignancy  represents a promising strategy against prostate cancer. Many mechanisms of PARPi resistance have been studied so far and some of these combinations could help us to overcome them. Nevertheless, many open issues still remain, that will be central topics for future research. Pivotal importance has to be addressed to the individuation of predictive biomarkers of response and further efforts should be made to identify specific patient’s characteristics that will support the choice of one treatment over the other, especially in the presence of multiple therapeutic weapons with profoundly different mechanisms of action. Moreover, an improved knowledge of the HRD cancers’ biology will lead to further approaches to avoid or delay PARPi resistance, thus gaining better long-term outcomes for our patients. Lastly, the greatest efficacy of PARPis has been shown in mCRPC patients with HRR and 177Lu-PSMA-617 than radium-223, given the available recent results of the related studies. Future efforts are required to better stratify mCRPC patients who may benefit the most from these radiopharmaceutical agents or the related combination approaches.Radiopharmaceutical approaches also seem to be promising therapy options in some patients with metastatic disease. Even though their use is still limited, in the next few years we should witness a progression towards a more frequent use of


The pathophysiology of accelerated atherosclerosis in people living with Human Immunofediciency virus (HIV) is complex. Coronary artery disease (CAD) has become an important cause of mortality in these patients. They often have atypical symptoms, leading to frequently missed diagnoses. We report a case of a 51-year-old male undergoing antiretroviral therapy who was admitted for acute coronary syndrome. He had severe coronary artery disease that involved difficult management. Human immunodeficiency virus (HIV)-infected patients live longer now due to highly active antiretroviral therapy (HAART). Due to their increased life expectancy, they have an increased cardiovascular risk caused by the HIV itself and the specific therapy . Cardiov3). Resting transthoracic echocardiography showed a left ventricular cavity of normal size with mild systolic dysfunction—a left ventricular ejection fraction of 47% and wall motion abnormalities in the left anterior descending artery territory with hypokinesia of the apical segments of the anterior and lateral wall and of the apex, consistent also with the aspect of the polar map of global longitudinal strain (2) and low probability of pulmonary hypertension. The patient was admitted to hospital due to a high-risk cardiovascular profile and wall motion abnormalities, even though he was not presenting with typical angina.A 51-year-old Caucasian man, having lived for 20 years with HIV and undergone antiretroviral therapy (ART) including nucleoside reverse transcriptase inhibitors  and protease inhibitors (PIs—lopinavir and ritonavir) for more than 10 years, presented to the on-call room for atypical chest pain. The patient was a cigarette smoker (10 pack-years), of normal weight, and without a familial medical history of atherosclerosis. His physical examination and laboratory tests were unremarkable  = 507 cells/mml strain . FurtherAfterwards, the patient proceeded to coronary angiography, which revealed a left main coronary artery lesion and extensive triple coronary disease with extensive, diffuse atherosclerotic involvement of proximal and mid segments of all three coronaries and chronic LAD occlusion . In ordeHe was discharged with medical therapy recommendations, which included aspirin, clopidogrel, rosuvastatin, and carvedilol. In agreement with the infectious disease doctor, due to drug–drug interactions (those receiving ritonavir are at risk of diminished clopidogrel response), we decided to stop the protease inhibitors so that an integrase inhibitor  could later be prescribed.Five months after the CABG, due to the recurrence of atypical angina pain, he underwent non-invasive ischemia tests. Therefore, he had a Bruce protocol stress test, which was positive  for ischemia , and furImmediately, a coronary angiography was performed, which showed occlusion of the internal saphenous vein to the first diagonal artery, 95% stenosis of the internal saphenous vein to the left anterior descending artery, and 80% stenosis of the internal saphenous vein to the right coronary artery .Taking into account the risks and benefits of percutaneous coronary intervention (PCI) versus a coronary artery bypass graft (CABG), EuroSCORE II 9.17% mortality risk, and Society of Thoracic Surgeons (STS) score 4.15% mortality risk, PCI with drug-eluting stents for revascularization was performed, with the patient’s consent. We decided to perform staged PCI revascularization. First of all, we implanted three drug-eluding stents (DESs) on the whole left anterior descending artery (LAD) and one DES between the left main artery distality and LAD proximity . After tOne month later, the patient came for right coronary artery (RCA) elective angioplasty, so we performed drug-eluding stent angioplasty on the RCA mid segment, with good results. Interestingly, at diagnosis the internal saphenous vein graft to the right coronary artery was totally occluded this time around . At a 5-Although this patient did not have typical symptoms, taking into account his HIV infection treated with HAART, we considered him to be at high risk of having an acute coronary syndrome. Reduced Global Longitudinal Strain showed us significant ischemic changes and led to further angiography, which revealed severe coronary artery disease requiring coronary artery bypass grafting (CABG). Less than six months later, the patient needed repeat revascularization via percutaneous coronary intervention (PCI) of the native coronary arteries.It is unknown whether people living with HIV (PLHIV) have a higher frequency of atypical symptoms such as silent ischemia that can be observed in chronic diseases (diabetes mellitus or chronic kidney disease) .There are different data regarding the difference between the incidence rates of cardiac vascular disease in PLHIV and in HIV-negative individuals. PLHIV do not differ from the others in terms of risk factors and angiographic characteristics after a first episode of acute coronary syndrome, except for higher rates of illicit drug use and hypertriglyceridemia in PLHIV . Recent The long-term prognosis after an acute coronary syndrome in people living with HIV is unclear , but it The prevalence of left main disease is 6%, which is equivalent to that in HIV-uninfected persons ,12. CABGAntiretroviral-associated endothelial dysfunction has been described in many studies and may be associated with the increased incidence of cardiovascular diseases. Experimental studies demonstrated that in mice, antiretrovirals such as nucleoside reverse transcriptase inhibitors (NRTIs) or PIs induce endothelial injury after only a few days of treatment . These aDrug–drug interactions also represent a problem in this case. The coadministration of clopidogrel or prasugrel and boosted regimens has been evaluated in studies. Prasugrel appears to remain an adequate antiplatelet agent in these patients and could be preferred to clopidogrel, regardless of the metabolic interaction and inhibition of its bioactivation pathways . UnfortuThe spectrum of myocardial disease in people living with HIV ranges from incidental asymptomatic findings to symptomatic disease . Before The pathophysiology of accelerated atherosclerosis in these patients is complex, including traditional atherosclerotic risk factors with a higher prevalence, endothelial damage and dysfunction, hypercoagulability, local and systemic inflammation, altered immune responses, and metabolic disorders from antiretroviral therapy. Coronary artery disease (CAD) has become an important cause of mortality in people living with HIV. Cardiac ultrasonography is a useful tool in standard clinical practice and is able to detect left ventricular systolic dysfunction in patients with acute coronary syndromes .Considering Framingham Risk scores and the atherosclerotic cardiovascular disease (ASCVD) risk algorithm, the cardiovascular disease risk in people living with HIV is underestimated .People living with HIV have accelerated atherosclerosis. It can lead to severe coronary artery disease with unspecific symptoms. Non-invasive tests to detect myocardial ischemia, such as stress cardiac magnetic resonance, myocardial perfusion scintigraphy, and coronary CT angiography, should be studied for detecting early stage disease. Coronary artery bypass grafting using arteries is preferred due to the increased risk of venous occlusion. Percutaneous coronary intervention with drug-eluding stents remains an option, even though the stenting is often extensive and there is the risk of target lesion revascularization failure in the long term. Drug–drug interactions are a real problem in people living with HIV, and treatment must be continuous with a strong collaboration between the infectious disease specialist and the cardiologist.


Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields. Plant growth is based on the ability of plants to convert carbon dioxide into sugars via photosynthesis and metabolize them into a wide range of other biomolecules . The maiThe major constituents of plant cell walls are polysaccharides, which are divided into three different classes: cellulose, hemicelluloses, and pectins –6. Cellull walls , 8. Cellll walls .Hemicelluloses primarily consist of e glucan . These pe glucan –13, by ge glucan , 14, 15.Pectins are also made in the Golgi lumen by GTs and are some of the most complex and dynamic cell wall molecules. Homogalacturonan (HG), a homopolymer of ogenesis –18. HG bogenesis , 20.Unlike the flexible primary cell walls which encase cells that are still growing, thick secondary cell walls are deposited once cells have ceased growth. These strong walls provide mechanical strength as well as creating the vascular tissue needed for water transport and providing resistance to biotic threats . The secBecause of its abundance and extensive crosslinking, lignin is usually the main factor that influences the resistance of cell walls to decomposition. However, the recalcitrance of biomass to processing is still a poorly understood phenomenon that is also influenced by cell wall morphology, porosity, and the abundance of the varying constituent polymers –27. FluoThere are a handful of imaging methods that utilize the intrinsic chemical or mechanical features of cell wall polymers, such as atomic force microscopy (AFM), Fourier-transformed infrared (FTIR) microspectroscopy, confocal Raman microspectroscopy (CRM), coherent Anti-Stokes Raman scattering microscopy (CARS), stimulated Raman scattering microscopy (SRS), Brillouin microscopy, and X-ray computed tomography (CT) , 30. AltThis review is divided into two major sections: the first part discusses options for cell wall visualization by various types of fluorescence microscopy and the second section deals with molecules that enable exogenous or endogenous ‘tagging’ specific molecular targets. All research methods come with their own limitations, drawbacks, and challenges, and we use this opportunity to particularly highlight these. We also suggest how future advances and development in the field can mitigate the current drawbacks and pitfalls.Epifluorescence microscopy and confocal laser-scanning microscopy (CLSM) are well-established microscopy techniques. Epifluorescence involves simultaneous illumination and detection of the entire field of view, allowing low photon doses and quick imaging for effective control of phototoxicity, but large amount of out-of-focus light is collected. CLSM has the opposite limitations, using a bright, focused excitation laser that is scanned across the sample, removing unwanted emission signal by passage of the returned light through a pinhole before detection. This gives crisp images, at the expense of high photon doses and slow imaging times. Basic CLSM or epifluorescence images can be particularly helpful when using superresolution imaging, as comparison of images made with these novel methods and well-understood, traditional techniques allows one to clearly determine what features should be evaluated as potential artifacts or as new discoveries. Spinning-disk confocal microscopy overcomes the disadvantage of traditional CLSM’s long image acquisition times and high illumination intensities while still rejecting a very large fraction of out-of-focus light by using multiple pinholes to project a series of moving, parallel excitation light beams onto the sample. These features make it ideal to study the dynamics of fast biological processes with much lower levels of photobleaching and phototoxicity.Deconvolution techniques serve similar purposes to light restriction by confocal pinholes, reducing the amount of out-of-focus light in the resulting image. However, rather than blocking light, deconvolution reassigns it back to its true source by correcting for the dispersion of the signal via a (relatively difficult) data analysis optimization problem . While iConfocal images do not have perfect out-of-focus light restriction, and datasets can be improved somewhat with appropriate deconvolution methods . Additio-2 limit . On a sp-2 limit . These t-2 limit , or poll-2 limit .Total internal reflection fluorescence microscopy (TIRFM) provides the excitation energy as a thin  can be applied to image developmental processes in plant cells that are hidden under thick layers of plant tissues and that are difficult to physically extract . LSFM uver days  and can  flowers , and thi≈700 nm) .Even the best objective lenses can only laterally resolve light from visible wavelengths to around 200 nm, but individual proteins and glycans are typically one or two orders of magnitude smaller, 2 to 20 nm in width. In order to come closer to resolving the fine structure of cell walls, fluorescent imaging with superresolution techniques that overcome the resolution limit of a microscope’s optical system is becoming widely used in plant cell research . SoIt is essential for the user to understand that many superresolution images are a product of extensive data analysis and fitting and can be fundamentally different to those created with traditional optics. This issue is most pronounced for images produced with localization microscopy techniques, and less so for structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. This is quickly appreciated by considering what a dark area of an image is comprised of for normal or for localization-based images. With widefield, confocal, SIM, or STED fluorescence microscopy, a dark area on an image is physically measured to have no fluorescent signal at the corresponding location in the sample. However, for localization microscopy (as discussed below in "In structured illumination microscopy (SIM) , the illStimulated emission depletion (STED) microscopy is a superresolution technique based on confocal microscopy, but it uses a sophisticated two-laser shaped beam to effectively shrink the spot size of the scanning excitation laser . While fArabidopsis embryo fertilization [Expansion microscopy (ExM) relies on physical expansion of biological structures by flooding a sample with acrylamide and then polymerizing the monomers into a hydrophilic polyacrylamide gel cross-linked to cellular components or fluorescent labels. The sample is then enzymatically digested and the polymer is carefully swollen from 4 to 10 times the original size, effectively allowing a 4-10x higher resolution image to be taken on completely standard imaging equipment. Application of this method to organisms with strong cell walls lags significantly behind its use with softer cell types, but there are a few reported examples. Several fungal species were recently successfully expanded , and thelization . With coA large number of fluorescent superresolution imaging techniques that have been developed in the last inciples , 60. Funinciples , 62. Theinciples  and an ainciples .Penium sp. was visualized in living cells via CLSM after treatment with pectate lyase and labeling with various antibodies. This showed that HG is secreted into certain cell wall areas as a highly methylated form before it becomes demethylated and cross-linked by Live cell imaging is important for studying biological processes, such as cell wall biosynthesis and remodeling, in real time , 65. A went}Ca2+ .While unicellular organisms are relatively easy to image, visualizing cell wall dynamics in living land plants can be challenging. Sample mounting and protection usually requires special growth systems that allow for non-invasive imaging of intact plant organs such as roots. Optimally, such systems provide measures to control the environmental conditions during the experiment. Placing specimens in sealed chambered slides and/or under agar or phytagel is a straightforward measure to control humidity . HoweverMore advanced live cell imaging setups usually require special and/or customized microscopic equipment. As an illustration, plant roots are frequently used models to study various physiological or developmental processes. One way to monitor root biomass formation under a natural gravity vector is to use vertical imaging setups to counteract gravitropism. Customization usually starts from standard inverted CLSM systems, and in recent years, a number of laboratories published their imaging systems and described the customization steps in detail. This includes mounting a microscope on a metal plate, which can be flipped 90°  see Figa, addingin planta with superresolution techniques, 2D or 3D SIM is typically chosen as the most compatible technique with the sample requirements of live cells. STORM and PALM are possible to use as well; however, longer imaging series might be limited due to photobleaching of fluorophores . Excitis et al. . It is bIn plant tissues, autofluorescence can be used for label-free cell wall imaging of phenolic-containing polymers like lignin or ferulates, but it can be obstructive when using exogenous fluorescent probes , 77, as Arabidopsis, which falls mainly in the blue-green and red regions of the optical spectra. Therefore, fluorophores with green excitation laser and an emission peak in the yellow-red region should be preferred when imaging this plant tissue [In photosynthetic cells, chlorophyll is the major source of autofluorescence, joined by the cell wall phenolics and other endogenous fluorophores such as ferulates, flavonoids, etc., recently extensively reviewed by Donaldson . Figure t tissue , 86. It Arabidopsis roots.One way to simply remove autofluorescence from images is using the ClearSee or related protocols, designed to preserve fluorescent protein (FP) signals while optically clearing samples . AlthougExternally applied small fluorescent molecules or conjugates of fluorophores to other small molecules, like oligosaccharides or polyethylene glycol (PEG), can be used for direct labeling and/or real-time imaging of dynamics of cell wall polysaccharides and other non-carbohydrate cell wall components like lignin, suberin, and cutin , 92. ManArabidopsis epidermal cells [The most used fluorescent dyes are traditionally those recognizing al cells , onion pal cells , and in al cells . In addial cells , basic fal cells .There are also several reactive dyes that chemically react with either hydroxyl or aldehyde groups of a glycan moiety. Such an example is 5- aminofluorescein (5-DTAF), which reacts with hydroxyl groups at high pH. This dye was used to label cellulose microfibrils for superresolution microscopy  and to pFluorophores are also available for monitoring changes in physicochemical properties of the apoplastic moiety, such as 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), which was used to study the pH response to auxin in roots . The com targets , 100.Penium margaritaceum intricate extracellular matrix [A new class of oligosaccharide-based fluorescent probes for pectin was introduced recently and exhibits stringent specificity for certain polyionic homogalacturonan (HG) regions. Specifically, fluorophore-functionalized chitosan oligosaccharides (COS) are able to bind to de-esterified HG by means of positive-negative charge pairing  and haver matrix .in muro [Small-molecule dyes can also be incorporated into the cell wall polymers as substrates. For example, fluorescent monolignols can be polymerized into lignin macromolecules, allowing visualization of a cell wall polymer formation by radical coupling in muro . This lain muro .Finally, although they are not usually considered along with small molecules, quantum dots (q-dots) are fluorescent semiconductor particles that are even brighter than most chemical dyes and are extremely photostable. Interestingly, (CdSe)ZnS q-dots naturally bind to hexahistidine affinity tags, which was used to study the distribution of carbohydrate-binding modules (CBMs) on a cellulose microfibril by identification of individual olecules .One of the most serious drawbacks of using small-molecule fluorescent probes is that their low molecular weight is usually associated with only limited specificity to glycan targets. In the case of reactive dyes, specificity is an even bigger issue as they usually target functional groups present in a large number of glycan moieties. In addition, some small-molecule fluorophores exhibit broad emission spectra and long Stokes shifts, complicating their use in multilabel experiments with the already limited window of wavelengths available for use in plants.Can we expect the introduction of new fluorescent probes in the future? Several fluorescent dyes were synthesized in the past as possible new dyes for natural materials in the textile industry, and these chemical libraries may be a valuable resource for novel probes with new specificities or applications, but many have not been characterized in detail. Other inspiration for new types of probes can be found in the animal biology field, where fluorescent glucose derivatives are commonly used as biosensors for tracking metabolic fluxes and glycogen biosynthesis . Unfortuin situ analysis of the plant cell wall structure  remain the most important molecular probes for ructure  or single-domain antibodies (sdAb) are promising smaller alternatives that still use the same binding mode as traditional IgG and IgM reagents . AdditioArabidopsis seed albumin and globulin [Arachis hypogaea glycinin  [Ricinus communis ricin toxin [Chlamydomonas reinhardtii cell walls at affinities of up to 1 nM were reported [Only a handful of examples of nanobodies against plant targets are available, such as those against globulin , ArachisAra h 3) , and Ricin toxin . Additioreported , althougreported –127 willThe substrate recognition moieties of carbohydrate acting enzymes (CAZYmes) are called carbohydrate-binding modules (CBMs) and are the second-most used proteinaceous probes for glycan targets. CBMs are particularly important for dealing with the lack of mAbs reactive towards cellulose. The characterization of the cellulose fraction is an essential step of biomass valorization because features such as crystalinity index (CrI) and degree of polymerization (DP) substantially affect saccharification efficiency . CBMs maBesides CBMs, quite a few other types of non-antibody protein reagents are available for modification into new fluorescent probes. These scaffolds are chosen because of their favorable biochemical properties, such as thermal and pH stability, ease of expression, etc . One typsee Fig. g [119, 1CBMs have been widely applied to visualize the diversity of cellulose arrangements in plant tissues –140. ForAlthough CBMs have a diverse binding repertoire, a common drawback is that their specificity is often low . This isOne popular method to find higher-affinity binding variants of protein domains like CBMs is phage display and directed evolution . TraditiAn interesting class of non-immunoglobulin scaffolds with well-established carbohydrate-binding activities are the variable lymphocyte receptors (VLRs) or “lambodies,” named after their function as antibodies in lampreys and hagfish. In a mechanism conceptually similar to that of mammalian B-cell receptors, the lamprey/hagfish lymphocyte-like cells assemble a lambody gene by selecting variable region domains randomly from a large set of possible sequences encoded by the germline, which code for turns of a leucine-rich repeat (LRR) domain . The newAn underexplored method in understanding biomass utilization is imaging with directly-labeled CAZYmes or whole complexes of CAZYmes, such as cellulosomes. This approach has already been demonstrated with a few examples, such as with research on cell wall biomass deconstruction . Besidesin situ with a process that does not utilize genetic probes, but instead relies on small, metabolically-incorporated functional groups that are later conjugated to a suitable fluorophore. The click reactions that conjugate the fluorescent label are usually done at ambient temperature in an aqueous system with high specificity and reaction rates, and most can proceed without a catalyst. Furthermore, click reactions can be bioorthogonal, leading to low non-specific incorporation and allowing their use in live systems [Click chemistry labeling is a useful tool for imaging of polysaccharides and other cell wall polymers  systems .Classical click reactions, shown in Fig. Three main reaction types are applied to plant cell wall research: (i) the classical Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), (ii) the strain-promoted alkyne-azide cycloaddition (SPAAC), and (iii) the inverse electron demand Diels-Alder  into the pectin RG-II of nt et al . Furtheris roots . Both CuArabidopsis roots [Even though many alkyne and azido sugars were found to be incorporated without any obvious detrimental effect on growth, this is not always the case. For example, McClosky and coworkers investigated the incorporation of 6-deoxy-alkynyl glucose (6dAG) into is roots , where tSeveral studies have also demonstrated click-labeling of lignins with modified monolignols –177, whiFor monosaccharide labels, it is important to consider which position of the monosaccharide ring is modified, and if it supports correct linkage of the desired polymer . AdditioThe type of click reaction used should be critically evaluated. For fixed samples, the small metabolic label size and established bioorthogonality of the CuAAC reaction is ideal, but in non-fixed samples, the CuAAC reaction damages the plant and may not keep the cell membrane intact during the labeling for some tissues , 178–180N-azidoacetylgalactosamine  is epimerized into N-azidoacetylglucosamine (GlcNAz), and the same process seems to happen in plants that are unable to use GalNAz directly [It is also important to determine which polysaccharides the chemical reporter gets incorporated into, as most monosaccharides are utilized for more than one polysaccharide , 182. Modirectly , 183. Todirectly , 185.Arabidopsis roots with differently functionalized versions of fucose, Kdo, N-acetylglucosamine, and N-acetylgalactosamine. Continuing work will be required to establish which of these and other currently available sugars and modification positions are tolerated by plants and can enter their metabolic networks.Moving forward, it appears likely that more chemical reporters and click-labeling reactions will be developed or taken from animal cell research for use in plants. Recently, Wang et al. reported synthesis of glucose, mannose, rhamnose, and sucrose variants with new alkene and alkyne substitutions , and ZhuWe expect that current labeling strategies will be combined and optimized, for example, using two or three click reactions that are bioorthogonal and orthogonal to each other, such as those reported in Simon et al. . Ideallyng steps . We lookng steps .Clostridium thermocellum was characterized with a click-compatible substrate [Click-labeling is not restricted to only biosynthesis and recycling of plant cell walls, but it can also be used to investigate enzyme and hormone activities. For example, the cellulosome of ubstrate , or receubstrate . Furtherubstrate , and simFluorescent proteins (FPs) have become the most important fluorescent probes for live cell imaging (for extensive reviews of FPs see –195) andhttps://www.fpbase.org/.Genetic engineering applications involving or modifying FPs are almost endless, many of which are compatible with fluorescent imaging. They have been turned into subcellular localization markers , biosensA prominent example using FP-tagged cell wall biosynthetic proteins was performed by Paredez et al. , where tAequorea [in vitro [Choosing the best FPs for imaging of cell wall-resident and biosynthetic proteins remains challenging, but it is well worth the effort to replace old FP technology with new variants that are better suited to the task. Newly discovered native fluorescent proteins  or enhaAequorea ) are pubAequorea ). Tools Aequorea  can helpin vitro , 212 or in vitro , 214. Adin vitro .There is no good reference for quantitative comparison of FPs in plants, partly due to the high biological variability in expression levels and silencing. An overview of the most commonly used FPs and other notable newcomers is shown in Figure Excitation/emission spectra Due to strong autofluorescence in different plant tissues, FPs with cyan and far-red FPs emission wavelengths should be avoided , and unfortunately, the field lacks a reporting standard for photostability. If only single images are necessary, bright but fast-bleaching fluorophores like mScarlet can be used; if time-lapse images are required to observe dynamics, dimmer FPs like mCherry are more useful due to their slower photobleaching [leaching .Sensitivity to pH The pKa measurement describes the pH value at which fluorescence intensity drops to 50% of its maximum value due to chromophore protonation. Common FPs like mEGFP (pKa=6.0) and EYFP (pKa=6.9) will therefore loose most of their brightness when targeted to the cell wall [ell wall . On the ell wall , 218. Seell wall , where tNon-fluorescent FP fraction Not all FPs that are expressed in cells will be fluorescent. There is a considerable fraction of non-fluorescent proteins present , which or mEGFP . There aor mEGFP , 221.In the context of targeting FPs to the cell wall of plants, the following criteria should be considered:While the highly active field of FP engineering for mammalian systems is geared towards brighter far-red dyes , this waFPs with large stokes shift like LSSmOrange and LSSmKate2  show proNucleic acid-based probes, called aptamers, are another class of underutilized molecules to be explored for plant cell wall labeling. They are short oligonucleotides (ssDNA or RNA), usually 20-60 bp long, that form unique 3D structures which can bind their target with high affinity and specificity. Aptamer technology was introduced over 30 years ago , 234, anin vitro evolution, a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Various types of selection are available to assure identification of highly specific and selective aptamers, which is effective for challenging targets such as those with low immunogenicity [New aptamers are typically identified by genicity . The nucin situ gene expression reporter system in N. benthamiana and Arabidopsis [Although several carbohydrate-binding aptamers with potential application in plant research have been developed, e.g., galactose, cellobiose, cellulose, and  studies –240. Howbidopsis .Fig. 8ApUnfortunately for cell wall applications, the heavy reliance on hydrogen bonding for standard nucleic acid interactions can lead to unsuccessful selection of aptamers that require binding with more hydrophobic or charged character. Because of this, much of the recent work towards improving aptamer selection is dedicated to increasing the chemical diversity of the component nitrogen bases . One sucCareful consideration of application and optimization of the selection process are crucial for development of high affinity binders, comprehensive overview of the potential SELEX and post-SELEX pitfalls, and possible solutions to overcome them can be found in Wang el al. .A major benefit of using aptamer technology instead of protein-based labeling reagents is that their nucleic acid nature gives aptamers an unmatched versatility in downstream applications. For example, aptamers are modular, which allows them to be linked together for simultaneous detection of several targets, or addition of known fluorophore binding sequences can allow fast synthesis of fluorescent probes . Modern The combination of SELEX-based evolution and PCR-based amplification technology may allow microscopy to be used as an aptamer selection tool, such as with the “Morph-X-Select” technique used to evolve aptamers that associate with target cells isolated by laser dissection from native tissue sections . ApplicaIn this review, we discussed both well-established and state-of-the-art techniques for fluorescent probing and imaging of plant cell walls and plant biomass. We attempted to highlight possible future directions in this dynamic research field. Fluorescence microscopy analysis has been pivotal for our understanding of mutants in biosynthetic genes, the characterization of plant varieties, and for monitoring the stages of biomass deconstruction. However, there are still many gaps in our understanding of plant cell wall diversity, how different cell wall components are synthesized, how these components are then assembled into the cell wall, and finally how these elaborate structures are dynamically modulated during the life of a plant. Filling these gaps would be not only beneficial for understanding plant biology but also for setting directions in bioengineering and designing biomass processing for its efficient conversion into biofuels and high-value chemicals.in situ are still limited, and development of fluorescent probes recognizing such interlinks would greatly help our understanding of how the individual polymer components of cells wall are ultimately assembled into whole, functional tissues and organisms. The problem is not straightforward and likely requires non-conventional approaches, such as in vitro evolution and selection of probes.Many probing tools are focused on selectively detecting the structures built from cell wall components, but very few technologies are able to detect the numerous bonds between the various cell wall components. These bonds are of both non-covalent  and covalent nature and are the ‘rivets’ holding the cell wall structure together. In many instances, these interactions are the key factors determining the level of biomass recalcitrance. NMR techniques have enabled an appreciation of the biochemical and structural nature of interaction of some of the polymers, especially of xylan, lignin, and cellulose. However, the possibilities to study and detect these interactions Although the current immunological toolbox for creating molecular fluorescent probes is relatively extensive, some classes of specificities are largely missing, such as probes to discriminate the level and patterns of acetylation in the cell wall. Additionally, the non-glycan cell wall components are generally underrepresented, such as waxes and suberin. Last but not least, algae are also an emerging potential source of biomass production with a high abundance of polysaccharides, especially of sulfated polymers. However, they are very biochemically diverse and the current repertoire of molecular probes for these less well-studied polysaccharides is still relatively small.Cell wall-resident proteins like expansins, xyloglucan transglycosylases/hydrolases (XTHs), or pectin methylesterases (PMEs) are instrumental for assembly and spatiotemporal cell wall control. However, surprisingly little is known about their precise localization or movement within the cell wall. Probes against some of these cell wall proteins will be instrumental to understand the link between the cell wall protein activity, localization and formation of the cell wall microdomains, and whether modulation of these proteins can be used to design cell walls with desired properties. Fortunately, genetic fusion of fluorescent proteins with these enzymes and proteins is also possible, allowing a powerful, alternate approach to fluorescent imaging for these relatively low-abundance but very important cell wall components.When it comes to imaging technologies, we are certain that superresolution microscopy will be used more in the coming years, in conjunction with new and better fluorescent probes. It is then expected that new features of the cell wall organization will be resolved that were previously out of reach for conventional widefield or confocal microscopy. Additionally, we hope that more effort will be made toward the underexplored combinations of fluorescent microscopy and other visualization techniques such as electron microscopy or atomic force microscopy. Currently, cell walls are typically imaged as 2D scans of a sectioned material, but we anticipate that progress in implementing tomographic methods and 3D fluorescent imaging will give us new information about the spatial organization of plant tissues and the cell wall in 3D, or even the 4D organization through time in live cells.In conclusion, although the field of plant biotechnology is often lagging behind the biomedical field when it comes to imaging technologies, we expect that future implementation of new, exciting, and innovative imaging approaches will bring better understanding of the biology of plant cell walls and will serve the effective utilization of one of the most important renewable energy resource—cell wall-derived biomass.