Oral lichen planus (OLP) is a chronic inflammatory disorder with increased risk for malignant transformation. Biomarker validation is a pivotal step in moving newly discovered biomarkers towards clinical implementation. We performed a systematic review of studies on biomarkers related to OLP, wherein biomarkers have been described in at least two independent studies. Our aim was to determine whether any of these biomarkers might be promising in predicting the increased risk of malignant transformation of OLP. We searched the following databases until August 2021: PUBMED, EMBASE, and Web of Science. Due to high heterogeneity, a qualitative rather than quantitative assessment was conducted. Only proteins that consistently showed a significantly high level of expression in neoplastic tissues versus OLP in two or more publications were considered as promising markers. Initial database researches identified 1671, of which 24 articles were included in the final analysis. The most frequently reported proteins were p53, Bcl-2 and Ki-67, though there were controversies. PCNA and P21 were the only proteins that showed consistent evidence of clinical usefulness as cancer predictors to be considered as promising markers. Extensive methodological variations in the evaluation of expressions and statistical analyses of the included markers were observed, which hampered comparisons of the results. Multiple levels of heterogeneity with a scarcity of high-quality studies were identified. PCNA and P21 were identified as promising predictive markers for evaluating the risk of malignant transformation of OLP, but they require further validation. The focus of future research on validation of predictive biomarkers of OLP should be considered as a high priority because it will accelerate the introduction of newly discovered markers into the clinical setting. Key words:Oral lichen planus, Immunohistochemistry, biomarkers, malignant transformation, PCNA, P21, p53, BCl-2. Abbreviations: Oral lichen planus (OLP), Immunohistochemistry (IHC), Malignant transformation (MT), Oral epithelial dysplasia (OED), Oral squamous cell carcinoma (OSCC), Malignant transformation rate (MTR).Oral Lichen planus (OLP) is a relatively common disease of the oral mucosa and appears to be an immunologically-mediated chronic inflammatory reaction to an unknown antigen . A recenThe overall malignant transformation rate (MTR) of OLP varies between 0.5 -1.2 % . The MTROne relatively simple and affordable technique to study biopsies of OLP and detect pathogenic molecular abnormalities in gene expressions at the protein level is Immunohistochemistry (IHC). IHC is the most common technique that is used to visualize the intracellular as well as the cell membrane proteins, thereby providing a real reflection of what is going on at the cellular level .There is, indeed a three-level evidence hierarchy for biomarker validation, ranging from exploratory to validated to clinically useful. To qualify as a useful biomarker, it is essential to successfully pass them all. A exploratory biomarker is defined as any biomolecule identified in one discovery publication, whereas validation is based primarily on confirming a discovery biomarker’s finding in at least two independent studies . It is aThis systematic review was conducted and reported following PRISMA-protocol guidelines . However- Search strategyPotentially eligible studies were identified in a search of only indexed databases like PubMed, Embase and Web of science. The following keywords were used: “oral lichen planus,” “OLP,” “oral squamous cell carcinoma,” “OSCC,” “oral cancer,” “oral carcinoma,” “epithelial dysplasia,” “oral normal mucosa,” “oral hyperkeratosis,” “malignant transformation” and all possible combinations. The search results were supplemented with manual searching of reference lists to identify publications not captured by computerized searches. There was no restriction regarding year of publication. The last search was conducted in August 2021.- Studies selectionAbstracts that focused on the malignant potential of oral lichen planus compared to that of the normal oral mucosa, OSCC, and OED, by their ability to express specific proteins, were identified by the first author. Subsequently, full-text articles of the selected abstracts were retrieved and assessed independently and blindly by the first and second authors (AAA and RVS) based on specific eligibility criteria. The list of the selected studies prepared by both authors then compared and only those achieved a good agreement were included.- Inclusion Criteria1) Studies including patients with a confirmed histopathological diagnosis of OLP2) Studies reporting protein expression with a recognized classification/grading system3) Studies clearly comparing protein expression between OLP and neoplastic or dysplastic tissues4) Studies focusing specifically on OLP and its malignant- Exclusion criteria1) Studies that included patients with clinic signs of oral lichenoid lesions2) Studies that also focused on oral potentially malignant lesions other than OLP, such as leukoplakia, erythroplakia, or oral submucous fibrosis3) Studies with a sample size ≤ 10 cases4) Studies published in languages other than English5) Studies using animal models or tissues, or various biological samples rather than human tissue6) Case reports, systematic reviews, unpublished studies, and duplicate publications- Data acquisitionThe following data for each study were extracted from full-text articles by the first and second authors (AAA and RVS): study molecules, biomarkers’ functions, sample size, expression rate, odds ratio if reported clearly, diagnostic criteria, and significance of expression in OLP vs normal mucosa and versus OED or SCC. With respect to the biomarkers that were reported in two or more publications, our focus was to assess the consistency and significance in their clinical relevance. In other words, when two or more studies showed significant differences in the expression of specific biomarkers in OED or SCC compared to OLP, they would be considered as promising markers in predicting the risk of malignancy.- Quality assessmentThe quality of the selected biomarkers studies was independently assessed by the first two authors on the basis of the criteria as formulated in the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK), which comprise 20 items . Each itAs shown in Fig. The REMARK reporting assessment is summarized in Supplement 1. All included studies had an average REMARK score which ranged between 9 and 13 points. The statistical methods varied strongly among the included studies, and only one study clearly estimated the risk of MT and provided the odds ratio . In all Sixteen of these papers assessed the difference in expression of specific proteins including samples of OLP and OSCC, four studies examined expression in OLP, OED, and OSCC, while four studies focused on difference of expression between OLP and OED. A total of 22 biomarkers was evaluated, and the most frequently studied protein was p53 which was reported in 10 studies -24, follAlthough virtually all of the included studies presented merely univariate analyses, there was one study that conducted multivariate regression analyses with measurement of odds ratio or hazard ratio . The stuOf the identified proteins, only PCNA , 19) an an19) anCurrently, the absence or presence of epithelial dysplasia is the gold standard for assessment of the risk of MT in OLP . HoweverIt is well-documented that loss of proper control on cell cycle is the main engine in driving cells to neoplastic formation. Thus, iet al, observed a significantly higher level of PCNA expression in atrophic OLP lesions compared to hypertrophic lesions. This, in turn, correlates with the clinical observations that higher rates of MT were found in patients with atrophic OLP lesions  compared proteomic of OLP samples to de novo OSCC samples that were not the result of MT of OLP, while two longitudinal studies compared proteins profiles of OLP that during follow-up eventually developed OED and an OSCC ,32. ThisOur findings should be understood in the context of some limitations. Firstly, we defined the biomarkers to be promising when their predictive value was independently validated in two or more studies. This might result in missing a promising marker that is not yet confirmed in an independent study. One example of such markers that showed very encouraging odds ratio in expecting risk of MT of OLP, but not reproduced so far, are cancer stem cell markers . SecondlIn conclusion, PCNA and p21 have been identified as potentially promising markers for predicting the risk of MT in OLP, but they are in need for further validation in well-designed studies. These studies should investigate the combination of PCNA and p21 along with other encouraging ones markers like cancer stem cell markers or p53 and thus may identify people with OLP at a high risk of MT. Furthermore, standardization of assessment of biomarkers and statistical analyses with REMARK or other prognostic tools in reporting data would be of great value, helping to quantify biomarkers values in predicting the risk of MT. Lastly, the focus on validation of predictive biomarkers of OLP should be considered as a high priority because it will accelerate the introduction of newly discovered markers into the clinical setting.


Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterised by acute inflammation and subsequent neuro-axonal degeneration resulting in progressive neurological impairment. Aberrant immune system activation in the periphery and subsequent lymphocyte migration to the CNS contribute to the pathophysiology. Recent research has identified metabolic dysfunction as an additional feature of MS. It is already well known that energy deficiency in neurons caused by impaired mitochondrial oxidative phosphorylation results in ionic imbalances that trigger degenerative pathways contributing to white and grey matter atrophy. However, metabolic dysfunction in MS appears to be more widespread than the CNS. This review focuses on recent research assessing the metabolism and mitochondrial function in peripheral immune cells of MS patients and lymphocytes isolated from murine models of MS. Emerging evidence suggests that pharmacological modulation of lymphocytic metabolism may regulate their subtype differentiation and rebalance pro- and anti-inflammatory functions. As such, further understanding of MS immunometabolism may aid the identification of novel treatments to specifically target proinflammatory immune responses. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterised by acute inflammatory demyelination and neuroaxonal degeneration in the white and grey matter of the brain and spinal cord . The preAlthough its aetiology remains incompletely understood, MS is thought to be triggered by an interplay of genetic susceptibility and environmental factors. Thus, a large number of gene variants, mostly linked to immune system function, as well as smoking, low vitamin D levels and Epstein–Barr virus infection have all repeatedly been associated with increased disease vulnerability ,7,8. TheTo date, a range of immunosuppressive and immunomodulatory drugs have been approved for the treatment of MS. These significantly reduce relapse rates in RRMS by counteracting acute inflammation, but do not slow neurodegeneration-induced disease progression in SPMS and PPMS and often have considerable side effects ,15. GainIn addition to inflammation, both metabolic and mitochondrial dysfunctions have recently emerged as important features of MS pathophysiology and restoring normal mitochondrial functioning as well as addressing the balance between glycolysis and oxidative phosphorylation (OxPhos) could represent a viable novel treatment target . To dateEnergy metabolism involves a complex interplay of metabolic pathways, which are highly regulated in order to meet the varying cellular energy requirements arising from rapid changes in the cell’s environment or functions. Neurons have a particularly high energy demand since they require ATP to drive many homeostatic processes such as restoring ionic gradients following axonal action potentials or calcium influx at synapses . SimilarBriefly, glycolysis involves the metabolic conversion of glucose through several intermediates to pyruvate resulting in a net production of two ATP molecules for each glucose molecule, as well as reducing two NAD+ to NADH. Pyruvate then undergoes oxidation before feeding into the TCA cycle, generating in total four NADH, one FADH2 and one GTP per pyruvate molecule. The TCA cycle occurs in the mitochondrial matrix, with pyruvate being carried across the inner mitochondrial membrane from the cytosol under aerobic conditions. Reduced NADH and FADH2 are then used as substrates to drive OxPhos in the mitochondria generating more of the main energy substrate, ATP .Under anaerobic conditions, as well as aerobic conditions when a high energy burden exists, rather than directing pyruvate towards the TCA cycle, it is instead metabolised to lactate by the enzyme lactate dehydrogenase. During this conversion, NADH is consumed generating NAD+, which can be reused to drive further glycolysis allowing more glucose to be metabolised and ATP to be generated. This allows energy to be produced quickly through glycolysis and is used to supply energy during short-term but intense periods of activity, such as rapid cell proliferation. Lactate is subsequently secreted, and eventually converted back to pyruvate in the liver by a process known as the Cori cycle .Mitochondria are complex organelles found in all eukaryotic cells. They consist of an outer and highly folded inner membrane and contain their own DNA (mtDNA) that encodes several RNAs and proteins. Although they are involved in a number of essential cellular processes, including calcium homeostasis and apoptosis, one of their most important functions is the production of energy in the form of ATP via OxPhos .Under normal physiological conditions, electrons donated by the reducing agents NADH or FADH2 (generated during glycolysis and the TCA cycle) are transferred to oxygen via a series of oxidation–reduction reactions taking place in the electron transport chain (ETC). This is located at the inner mitochondrial membrane and consists of a set of multisubunit complexes  acting aA number of experimental approaches can be used to evaluate mitochondrial function, including the assessment of the oxygen consumption rate (OCR), ETC complex activity, mitochondrial membrane potential or mitochondrial ROS production . Cha+/K+) ATPases that restore ionic gradients and thus the resting membrane potential [Neurons, and especially axons, require large amounts of ATP to maintain key functions, such as action potential transduction, which is dependent on the activity of sodium/potassium . RestinT cell metabolism is tightly regulated to support the differentiation and specialised functions of these distinct T cell subpopulations . Naïve TStimulated Tregs, on the other hand, have been shown to require fatty acid oxidation in addition to glycolysis for their proliferation and migration ,57,58,59This is similarly seen in other immune cells including B cells, where upon stimulation rather than increasing glycolysis, both OxPhos and TCA cycle activity, as well as nucleotide biosynthesis, are increased . LikewisAlthough the processes involved in the development of autoimmune diseases are not fully understood, they clearly involve an impairment in lymphocytic activity, with an imbalance in the effector and regulatory arms of the adaptive immune response. According to the ‘outside-in’ hypothesis of MS, acute CNS lesions in early relapsing–remitting disease are populated by peripherally activated autoreactive lymphocytes, including T cells of the CD4+ and CD8+ lineages, as well as B cells that invade the brain via a compromised blood–brain barrier . These cThat effector T and B cells are inefficiently suppressed by Tregs during autoimmune diseases is primarily supported by the role of Tregs in experimental models of autoimmunity. These include their ability to suppress autoimmune responses in murine models of autoimmune diabetes  and collDue to the metabolic alterations involved in the differentiation of lymphocytes according to their functions and the importance of balancing both effector and regulatory lymphocytic immune pathways, it has been suggested that an impairment in lymphocytic metabolism may be responsible for this imbalance. Indeed, altered mitochondrial metabolism in lymphocytes has been observed in several autoimmune and neurodegenerative disorders, including rheumatoid arthritis, systemic lupus erythematosus, Alzheimer’s and Parkinson’s diseases ,77,78,79While mitochondrial dysfunction in the CNS of MS patients is now well established see , recent A number of studies provide evidence for changes in mitochondrial OxPhos activity in peripheral lymphocytes of RRMS patients. For example, a significant decrease in the mitochondrial respiratory control ratio in CD4+ cells of treatment-naïve RRMS patients compared to healthy controls has been reported indicating a reduced coupling efficiency between electron transport through the ETC and ATP production ,81. MitoTo date, only one study has assessed mitochondrial OxPhos activity in progressive MS patients. Here, a decrease in the OCR of naïve CD4+ T cells, and to a lesser extent in memory CD4+ T cells, was seen in PPMS patients compared to healthy controls. This was accompanied by a lower mitochondrial mass and altered mitochondrial morphology. However, there was no reduction in OxPhos activity in CD4+ T cells of SPMS patients .Analysis of these types of data needs to be performed carefully, however, since as outlined in Accordingly, in one such EAE study, an increase in the mitochondrial OCR, accompanied by a decrease in the mitochondrial membrane potential, cytochrome c expression and reduced ATP levels were reported in Tregs isolated from draining lymph nodes of mice . InteresOverall, these findings suggest that mitochondrial OxPhos activity is decreased in PBMCs, especially in CD4+ T cells of RRMS patients, possibly due to a decrease in the expression or activity of several mitochondrial respiratory chain complexes. A similar decrease in OxPhos activity may also be present in PPMS patients, although this requires further investigation. Dysfunctional OxPhos has also been reported in EAE, although these alterations were restricted to the Treg compartment . Since OIn addition to the above evidence that favouring T cell OxPhos over glycolysis may promote the differentiation of T cells towards a less inflammatory phenotype, mitochondrial metabolism is also important for protecting the cell against ROS-mediated damage. ROS, such as superoxide, are by-products of mitochondrial metabolism and are scavenged by antioxidant defences under normal physiological conditions . HoweverIndeed, CD4+ T cells of untreated RRMS patients showed oxidative protein damage that was accompanied by a significant reduction in the activity of two mitochondrial antioxidants, superoxide dismutase and glutathione peroxidase ,81. ReduIn EAE, an increase in mtROS and a decrease in the mitochondrial antioxidant enzymes manganese superoxide dismutase and catalase in Tregs isolated from animals during presymptomatic disease stages compared to naïve Tregs or predisease nonTreg T cells has been found . This waOverall, it is conceivable that reduced antioxidant defences in peripheral lymphocytes of MS patients and in EAE-derived Tregs render mitochondria susceptible to oxidative damage, which may impair mitochondrial respiratory chain function subsequently contributing to the observed immunometabolic alterations. In addition, mitochondrial dysfunction in EAE Tregs and subsequent Treg cell death may further shift the T cell compartment towards proinflammatory subtypes.The reduction in mitochondrial OxPhos activity in PBMCs of RRMS patients outlined above was accompanied by changes in glycolytic activity. It was shown, for example, that the activity of two key glycolytic enzymes, hexokinase and phosphofructokinase, and the expression of the main glucose importer GLUT1, were significantly increased in CD4+ T cells of treatment-naïve RRMS patients compared to healthy controls ,81. ThisIn contrast, other studies reported a significantly decreased ECAR in CD4+ T cells isolated from untreated RRMS patients and from RRMS patients on immunomodulatory therapy during remission ,85,87. ASimilarly, ECAR has been found to be reduced in naïve and memory CD4+ T cells of PPMS but not SPMS patients, although surprisingly, GLUT1 expression and plasma lactate levels were significantly higher in PPMS samples . Taken tWhile human studies have shown evidence of both an up- or a down-regulation in the glycolytic activity of MS-derived lymphocytes, there appears to be a clear consensus that glycolysis is upregulated in T cells isolated from EAE mice, as well as evidence that this might be involved in driving inflammation. This most likely reflects that most animal studies have treated Tregs and Teffs as separate compartments for analysis, unlike in the MS analyses.For example, one study reported an upregulation of genes involved in glycolysis, including HIF-1α, PKM2 and GLUT1, in splenocytes isolated from EAE mice during active disease, compared to naïve control animals . These mGlycolysis has also been shown to be upregulated in T cells isolated from the CNS of EAE mice. For example, it has been reported that proinflammatory Th17 cells isolated from the spinal cords of MOG-immunised mice showed above all an enrichment in pathways associated with glycolysis compared to homeostatic Th17 cells from the intestine . SimilarMany of the studies mentioned above reporting an increase in lymphocytic glycolysis in EAE also demonstrated the therapeutic potential of inhibiting glycolysis through both genetic ablation of key components of the glycolytic pathway or through pharmacological inhibition of key enzymes. Some of the experiments discussed in this review are summarised in For example, inhibition of glucose metabolism by inhibiting the first enzyme in the glycolytic pathway, hexokinase, was achieved through application of 2-deoxy-D-glucose (2-DG) and was shown to reduce EAE severity, as well as to upregulate Treg differentiation . AnotherInterestingly, genetic ablation of the glycolytic pathway enzyme, glucose phosphate isomerase (Gpi1), significantly reduced the number of inflammatory Th17 cells in EAE but had no significant effect on the number of homeostatic Th17 cells and Gpi1 deficient cells were unable to induce EAE indicating that Gpi1 is selectively required for proinflammatory Th17 cells . The autCollectively, these data identify glycolysis as an important metabolic switch that drives T cell differentiation from quiescence towards a proinflammatory phenotype, thus aggravating autoimmunity. Since Teffs are reported to be more dependent upon glycolysis than Tregs ,56, the Several studies have found that common pharmacological treatments for RRMS can restore the observed immunometabolic alterations. Thus, 12 months of treatment with glatiramer acetate, a common first line treatment for RRMS, restored the respiratory control ratio, complex I and IV activity and mitochondrial membrane potential in CD4+ T-cells of previously untreated RRMS patients to levels comparable to those observed in healthy controls . GlatiraAdditionally, PBMCs isolated from RRMS patients receiving IFN-β did not show reduced levels of aerobic glycolysis, mitochondrial oxygen consumption and complex IV activity in contrast to cells of untreated patients, indicating that IFN-β treatment restores normal immunometabolic activity ,91.Finally, teriflunomide, an enzyme that blocks pyrimidine synthesis, was shown to restore glycolytic activity and mitochondrial OxPhos in CD4+ T cells of RRMS patients currently suffering a relapse to levels similar to those observed in healthy controls .In contrast to restoring immunometabolic changes, another study found that in RRMS patients, dimethyl fumarate treatment supressed the antioxidant capacity of CD4+ and CD8+ T cells through modulation of mitochondrial metabolism resulting in an increase in mitochondrial ROS, in turn inducing a mitochondrial stress response and ultimately increased apoptosis, which especially affected memory T cells .Taken together, these results show that several pharmacological treatments for RRMS are able to restore metabolic changes associated with MS. The mechanisms by which these treatments restore immunometabolism and whether this represents part of their therapeutic benefit or is merely a secondary effect  should be investigated further. It is similarly unclear whether an impairment in T cell metabolism precedes the development of MS and thus represents an additional risk factor or whether this is a result of the heightened T cell activation occurring under autoinflammatory conditions.Overall, the research summarised in this review provides evidence that metabolic alterations are a feature of MS and EAE, not only in the CNS, but also in peripheral lymphocytes. Primarily, changes in the balance between glycolysis and other metabolic processes, such as OxPhos, reflect the activation, proliferation and differentiation processes undertaken by T cells and other lymphocytes in order to allow the immune response to adapt to its required functions. As a result, it is harder in autoimmune diseases to determine whether observed metabolic changes reflect an intrinsic, aberrant response perhaps underlying an individual’s increased risk to developing MS or instead occurs as a result of the autoimmune environment already present. However, the fact that immunomodulatory therapies for MS appear to restore metabolic imbalances suggests that these changes may mainly reflect the overactive status present under autoimmune conditions.That said, there is evidence of mitochondrial stress and damage in MS lymphocytes, such as reduced antioxidant capacity and increased oxidative damage, as well as upregulation of genetic programmes involved in mitochondrial oxidative stress, DNA damage and cell death, which are indicative of mitochondrial dysfunction rather than an (adaptive) upregulation of immune cell metabolism under inflammatory conditions. However, further work is needed to determine the influence of lymphocytic oxidative stress on subsequent metabolic switches within autoimmune lymphocytes.In this light, data showing that in animal models of MS the metabolic balance of lymphocytes can be modified pharmacologically to augment the disease progression is interesting. Although this does not answer the question whether the metabolic ‘imbalance’ characteristic of a proinflammatory response is a cause or effect of MS development, its role in the progression of autoimmunity is clear. Increasing the dependency of lymphocytes on OxPhos rather than aerobic glycolysis has been shown by several groups to favour the function of Tregs over Teffs and may prove to be an interesting concept for promoting remission over relapses in MS patients.It should be noted, however, that comparing animal models with MS must be done with care. Firstly, the studies performed have taken different approaches where MS patient studies on the whole have not differentiated between Teffs and Tregs, instead usually analysing pooled PBMCs or total CD4+ T cells, unlike in EAE, where subpopulation distinctions have been taken into account. Secondly, T cell subpopulations can differ significantly between mice and humans ,99 and tFurthermore, variations between MS patient studies render interpretations of results difficult. In contrast to reports of a switch from OxPhos to glycolysis ,81, someFinally, so far only one study has specifically assessed metabolic activity in PBMCs in progressive MS and found that immunometabolism differed between SPMS and PPMS patients . Future Taken together, current research supports the emerging concept of altered mitochondrial function and cellular energy metabolism in peripheral immune cells as a characteristic feature in MS and its animal model. Further research is required to assess whether these immunometabolic alterations differ between distinct forms of the human disease, whether they are specific to CD4+ T cells, whether they influence lymphocytic activation and function and whether they correlate with disease activity in RRMS see .Box 1Do immunometabolic alterations in CD4+ T-cells contribute to disease progression or are they secondary to other MS pathophysiological processes?Are immunometabolic alterations present in all CD4+ T cells or are there differences in Teffs and Tregs as suggested by EAE studies? Can similar changes be observed in other immune cells implicated in MS pathophysiology, such as B-cells?What is the effect of altered metabolism on lymphocytic activation state and function, specifically with regard to Treg suppressive capacity?Is there a correlation between immunometabolic alterations in PBMCs and RRMS disease activity and does it differ between RRMS and progressive forms of the disease?What are the underlying molecular mechanisms responsible for altered immunometabolism in PBMCs? What is the role of ROS?What is the mechanism by which common pharmacological treatments restore altered immunometabolism in peripheral lymphocytes?


Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed , while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing. In the context of cell-free DNA (cfDNA) research, the structural complexity and versatility of the DNA molecule allows at least two unique biological phenomena to occur: first, diverse information can be encoded into the primary structure of DNA as it exists in the nucleus ; second, as the primary structure of DNA is disrupted and then altered by a variety of processes following its movement from nuclei to intracellular space and various extracellular compartments, it not only conserves the genetic and epigenetic information of the original molecule, but sequences from specific genomic regions may be additionally marked by various physico-chemical features that are often unique to and inform about the characteristics and localization of the processes that generate them . As such, a major portion of cfDNA molecules bear a variety of organism, disease as well as tissue-specific genetic and epigenetic physico-chemical features ,2,3,4.By virtue of its constant release and short half-life, the capture and computational reconstruction of the information stored in the total cfDNA population or specific cfDNA subpopulations may give an unprecedented, almost real-time view of genome function from which the biological, physiological and pathological state of individuals may be inferred.The focal point of cfDNA research remains set on the perusal of DNA mutations for the development of routine clinical assays, but at the same time, the field is rapidly expanding towards epigenetic profiling ,3,4,5. GAmong the various epigenetic marks open to interrogation , various features relating to the fragmentation profiles of cfDNA molecules are increasingly scrutinized ,4,12. ThHere, we focus only on cfDNA size signatures. However, because this is still a young area of inquiry in the field, there are currently many factors that challenge a reliable analysis of cfDNA fragmentation and high-fidelity reconstruction of fragmentomics information in general ,29, inclIn this work, we address some of the above factors and circumnavigate some drawbacks inherent to in vivo characterization by evaluating the average fragment length and relative proportion of differently sized cfDNA populations isolated from the cell culture supernatant of a human bone osteosarcoma (143B) cell line using different, increasingly sensitive electrophoresis-based DNA sizing methods. The results reveal new insights related to cfDNA fragmentomics and highlight issues that still need to be addressed.® CRL-8303™). The cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone DMEM/high glucose) , with 25 mM glucose, 1 mM sodium pyruvate and 4 mM L-glutamine. The culture media were also fortified with 1% penicillin/streptomycin  and 10% fetal bovine serum (FBS) . The cells were grown to confluency in 175 cm2 cell culture flasks  under controlled conditions at 37 °C in a humidified atmosphere with 5% CO2. The growth medium was then collected from each flask into 50 mL nuclease-free conical tubes , centrifuged at 1000× g for 10 min at room temperature, and transferred to fresh 50 mL tubes. The supernatant samples were immediately stored at −80 °C until further experiments were performed. The cells were discarded. Control experiments have shown the presence of FBS-derived cfDNA in cell culture supernatant. For this reason, we collected the supernatant only after 28 h of incubation when the cells have reached confluency. Previous studies have shown that cfDNA levels significantly increase over increasing incubation periods [A human bone cancer (osteosarcoma) cell line was acquired from the American Type Culture Collection (143B)  with up to a total of 10 mL of cell culture supernatant. The vacuum was then applied until the total sample had passed through the column. After the binding step, the columns were washed twice by the addition of 600 µL wash buffer NT3 and centrifugation at 11,000× g for 1 min at room temperature. The columns were dried by centrifugation and cfDNA was eluted into 15 μL elution buffer NE.All cfDNA isolations were performed using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit  from 2–30 mL of cell culture supernatant. This kit has consistently been used for the characterization of cfDNA in our cell culture studies ,32,33,34® fluorometer 3.0  and the Qubit™ dsDNA HS Assay Kit . All preparations were made according to the manufacturer’s instructions. Briefly, 1–3 µL of cfDNA was added to 197–199 µL of the Qubit working solution, mixed by vortexing and incubated for 2 min at room temperature. Concentrations were calculated from a standard curve.CfDNA quantification analyses were performed using the QubitThe automated size analysis of cfDNA was performed by means of capillary electrophoresis (CE) using four different assays on two instruments . CE utilThe extraction, quantification and automated sizing methods used in this study are not suitable for the analysis of single-stranded DNA (ssDNA). Concerning the extraction kit, the protocol using binding buffer NTI is capable of isolating ssDNA, but buffer NTC is recommended for ssDNA, especially for nucleotides shorter than 150 bp. In contrast, the Qubit quantification and Agilent DNA sizing assays utilize an intercalating dye and does therefore not measure ssDNA.g for 20 min at room temperature followed by a second centrifugation step at 1000× g for 2 min to collect the concentrates. Size analysis of cfDNA using the Fragment Analyzer (FA) dsDNA 930 assays confirmed that no cfDNA size artefacts were generated by the Amicon filter tubes.Several different configurations of agarose gel electrophoresis conditions were tested for the optimal separation of nucleosomal cfDNA bands. Variables included gel percentage, gel thickness, well size, buffer type (TBE vs. TAE), the volume of sample loaded, concentration of cfDNA, run time and applied voltage. The following protocol gave the highest resolution separation of cfDNA fragments: 10 cm-long TBE agarose gels  were prepared and ran with TBE buffer (1X) for 90 min at 100 V. The staining of the gels was performed using SYBR™ Gold Nucleic Acid Gel Stain , which is 25–100X more sensitive than traditionally used ethidium bromide, for 40 min in the dark with gentle agitation. A total mass of more than 50 ng cfDNA (not less than 10 ng/µL) in each well gave the best results. Such a high concentration of cfDNA was obtained through two steps. First, we implemented a modified protocol for the extraction of cfDNA from large volumes of cell culture supernatant see . Second,p-values < 0.05 were considered statistically significant.All statistics were performed using either the GraphPad Prism 9 graphing and statistical software program or Microsoft Excel 2019. Only The purpose of this study was to evaluate the average fragment length and relative proportion of differently sized cfDNA populations isolated from the cell culture supernatant of a human bone osteosarcoma (143B) cell line using different, increasingly sensitive electrophoresis-based DNA sizing methods.Size analysis using the Bioanalyzer DNA 1000 kit indicated the presence of four distinct cfDNA populations, including three peaks that correspond to short cfDNA fragment lengths which are characteristic of DNA wound around (i) mono-nucleosomes; (ii) di-nucleosomes; and (iii) tri-nucleosomes; and (iv) one peak of longer cfDNA fragments, the size of which could not be accurately determined due to significant overlapping with the upper marker A. The BiThe gel electrophoresis profile revealed the presence of at least seven distinct cfDNA size populations A. As indTo achieve enhanced separation and sizing of longer cfDNA fragments, we evaluated several variations to standard agarose gel electrophoresis procedures in order to establish an optimized protocol see . In compIn an internal investigation of cfDNA fragment size, Agilent researchers found that their Femto Pulse system, which is more sensitive than the FA dsDNA 930 assay, also revealed up to seven nucleosomal multiples (see their application note: “cfDNA Separated on the Agilent Femto Pulse System”). Thus, regarding cfDNA analysis, the experimental resolution limit of the most sensitive electrophoresis assays currently seems to be hepta-nucleosomes. In Taken together, the results discussed above firstly demonstrate that different sizing methods generate different cfDNA size profiles. Second, this comparative analysis shows that additional nucleosomal multiples are revealed, and the size range of the longer cfDNA populations narrows correspondingly, as the sensitivity and resolution of the sizing methods increase incrementally. This indicates that each method reaches a resolution limit, where the resolving power is not sufficient for the separation of longer cfDNA fragments. The technical reason for this is that DNA migrates through a gel matrix at a rate that is inversely proportional to its size, which results in short fragments traveling faster than large fragments. This migration, however, does not follow a linear pattern and can rather be described using a logarithmic scale. Therefore, the migration speed of a DNA fragment is determined by the logarithm of its molecular weight, which causes smaller fragments to move even faster and larger fragments to move even slower. This is also why we see the logarithmic distribution of fragment lengths in electrophoretic DNA ladders. It is therefore expected that the electrophoresis methods used in this study are limited in their capacity to adequately separate fragments larger than a specific cut-off with high resolution. Consequently, the relative abundances of each smaller overlapping sub-population within the cluster peaks are impossible to distinguish accurately, resulting in the reporting of a single “large” peak with an overestimated modal size and concentration. This significant overlap of peaks is illustrated in A further limitation is that none of the sizing methods used in this study yielded sharp peaks for any of the nucleosomal cfDNA size populations. This indicates that the cfDNA fragments that make up a single peak  are not all exactly the same size, and that the allocated peak size is only an average. This is expected as the linker DNA stretches that are attached to the nucleosomes can range between 20 and 80 bp in length, which means that a range of fragment sizes are expected for each population . The resulting peaks/bands are therefore much broader than those expected for a DNA population of precisely one size , therefore causing significant overlaps, making them even more difficult to distinguish. These overlapping, poorly resolved or entirely unresolved peaks/bands (smears) are then misinterpreted by the sizing software on instruments such as the Bioanalyzer and Fragment Analyzer. Due to the compact distribution and overlapping of the larger fragment size peaks, the software groups several of these smaller peaks into one large peak. The software algorithms are also only capable of assigning accurate sizes and concentrations to the peaks of sufficiently resolved fragment populations, and attempt to do the same for large cluster peaks, thereby labelling the large peaks with sizes that are unrepresentative of the grouped sub-populations .The average length of the cfDNA fragments that constitute each of the different cfDNA size populations as determined by dsDNA 930 assay is shown in Mono-nucleosomal cfDNA fragments demonstrate a modal size of 157–158 bp. Assuming that approximately 147 bp of this DNA is wound around the nucleosome core particle (NCP) , a size During apoptosis, the digestion of DNA by Caspase-activated DNase (CAD) generally produces ~167 bp mono-nucleosomes, also known as a chromatosome, defined as 147 bp DNA wrapped around the NCP plus 20 bp linker DNA bound to histone H1 ,31,39. AIn a previous cell culture study, we observed time-dependent changes in the modal size of mono-nucleosomes . For exaThese time-dependent shifts in epigenetic marks and the size of mono-nucleosomes were further mirrored by both changes in the cell cycle, i.e., rapid cell division and increases in the relative proportion of apoptotic and necrotic cell death. This is in line with the observation that both mitosis and apoptotic and necrotic cell death are characterized by the significant condensation of chromatin.In contrast to mono-nucleosomes, di-, tri-, tetra- and penta-nucleosomes demonstrated modal sizes of 357 bp, 532 bp, 712 bp and 923 bp, respectively . DividinBy calculating the theoretical nucleosome size profile that would form under different sets of conditions , the specific modal sizes observed in this study best match a digestion scheme in which the ~31 bp linker DNA between oligo-nucleosomes have an equal probability of being digested 5, 6, 10, 11, 15, or 16 bp up or downstream from the ~147 bp NCP . HoweverIn terms of the absolute concentration of differently sized cfDNA populations as determined by the dsDNA 930 assay, the larger ~1721 bp cfDNA population is significantly overrepresented, constituting 42.56% of the total population A. High mAs alluded to earlier, however, the values measured for the longer ~1721 bp cfDNA population should be interpreted with caution as the dsDNA 930 assay is not capable of resolving cfDNA subpopulations that range in size between 1 and 5 Kbp, especially when these subpopulations differ in size by nucleosomal increments and are present in one sample. This suggests that the concentration of cfDNA fragments with a modal size of ~1721 bp is overestimated while the modal size is an artefact. Instead, the ~1721 bp peak represents several oligo-nucleosomal peaks in a DNA laddering pattern which continues beyond penta-nucleosomes, and wherein each successive size population, which consists of one additional nucleosome, has a lower concentration and constitutes less fragments than the previous population. A hypothetical illustration of such a corrected frequency distribution is shown in Figure 6 of ref. . This hyTherefore, the frequency distribution curve of the differently sized cfDNA populations shows an exponential decay, which is characteristic of a power-law distribution . In termThis means that (1) each cfDNA size population is fed by the degradation of all size populations larger than itself; (2) a relative change in longer nucleosome chains will result in a proportional change in shorter nucleosome chains; and (3) shorter fragments will appear more often. In this study, we did not experimentally investigate which cellular processes may be involved in the generation of this laddering pattern. In a previous study, however, we have shown that both apoptosis and necrosis govern the release of cfDNA from 143B cells under normal physiological conditions, but that necrosis contributes slightly more . We haveTo investigate the extent to which the DNA laddering pattern continues, we extrapolated the exponential decay in the relative contribution of increasingly longer nucleosome populations. We obtained a logarithmic equation by plotting the natural logarithm of the relative concentration (%) of mono-, di-, tri-, tetra- and penta-nucleosomes toward the total population, as determined by the dsDNA 930 assay for two biological replicates and 11 technical replicates E. This lIn this study, the results seem to indicate that a cfDNA population consisting of an array of 12 nucleosomes may serve as the precursor for the generation of shorter cfDNA populations, and that all cfDNA fragments in the sample may share a common origin. More insight into the results presented in this work may be gained by isolating the respective size populations, followed by sequence analysis of each individual cfDNA population.CfDNA fragmentation profiles were investigated under specific conditions, i.e., the use of only one cell line, one extraction kit and one DNA sizing principle. While some insights from this study may be transferrable to other cell lines and in vivo conditions, more research is needed to encapsulate the wide-ranging factors that can affect cfDNA size profiling. For example, (i) recent experiments have shown various recovery efficiencies and size bias for the capture of cfDNA from cell culture supernatant ; and : (i) the Bioanalyzer DNA 1000 kit; (ii) the Bioanalyzer HS DNA kit; (iii) the FA dsDNA 915 kit; (iv) the FA dsDNA 930 kit; and lastly (v) an optimized agarose gel electrophoresis method. This direct comparison generated several interesting observations.By analyzing samples from the same original source, the least sensitive sizing method revealed only three discernible cfDNA size populations, while the most sensitive method was able to distinguish seven different size populations. Interestingly, the incremental uncovering of additional nucleosomal multiples by increasingly sensitive methods was mirrored by a decrease in the estimation of the size range of HMW cfDNA. Furthermore, the dsDNA 930 assay revealed an exponential decay in the relative contribution of increasingly longer nucleosomal multiples in the cfDNA population, suggesting the involvement of a stochastic inter-nucleosomal DNA cleavage process. In this process, shorter populations accumulate rapidly as they are fed by the degradation of all larger populations, resulting in a significant overrepresentation of mono-nucleosomes. However, this is currently a hypothesis, as more experimental research on in vitro degradation kinetics supported by the mathematical modeling of inter-nucleosomal DNA cleavage under various simulated conditions is needed to confirm whether all linker DNA sites are equally prone to cleavage and are truly “chosen” at random in the specific conditions of this study. In line with this, it is not suggested here that cfDNA degradation always occurs in a stochastic manner, and that there are no preferential cleavage sites or protected regions, which may depend on various factors such as tissue type, cell type and genomic origin of cfDNA fragments .Taken together, these results have several implications. First, it demonstrates the significant variation in the resolving capacities of similar electrophoresis-based methods for the sizing of cfDNA. Not only do electrophoresis-based methods have difficulty in separating nucleosomal multiples, but they also do not possess the resolving power to give insight into minor changes in the structure of nucleosomal sub-populations, especially when these populations are co-present in one sample. This highlights the importance of selecting the appropriate cfDNA sizing methods, whether applied as a method in basic research or as a tool for monitoring the quality control in experiments and clinical assays. Second, it indicates that the cfDNA laddering pattern extends well beyond the tri-nucleosomal cfDNA size profile that is typically generated by the most commonly used electrophoresis-based cfDNA sizing method . Third, the modal sizes of HMW cfDNA populations are exaggerated due to the limited resolving power of electrophoresis, and instead consist of several poly-nucleosomal subpopulations that continue the series of DNA laddering.While it was not directly investigated in this study, it seems likely that a significant portion of short and longer cfDNA fragments might share overlapping origins. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. Moreover, while not observed in this study due to the limited resolving capacity of the methods, other researchers have observed sub-nucleosomal fractions of cfDNA. This first seems to suggest that the view that mono-nucleosomal DNA is only produced by apoptosis and is the most important cfDNA fraction is a biased one, and secondly that the notion that all longer cfDNA fragments are genomic contaminations and do not contain valuable biological information needs to be reassessed.


Critical illness syndromes including sepsis and acute respiratory distress syndrome (ARDS) are major causes of morbidity among adults and children admitted to intensive care units (ICU) across the world. Clinical and biological heterogeneity among patients have, however, long confounded the identification of efficacious therapeutics, and have likely contributed to the repeated failure of clinical trials . AccordiCritical Care [Qin et al. recently published in cal Care  on the ical Care  study. Tn = 56) had the highest mortality (34%) followed by PedSep B and C which had comparable rates of death ~ 10–12%; PedSep A had the lowest mortality rate (2%). The hallmark of PedSep D subclass was the preponderance of patients with multiple organ dysfunction syndrome (MODS). In comparison, PedSep B was characterized by severe respiratory failure and central nervous system dysfunction, and PedSep C was composed of patients with predominant cardiovascular dysfunction and low rates of intubation. Biomarkers of systemic inflammation and endothelial activation demonstrated differences across the subclasses. Relative to PedSep A, patients belonging to PedSep D were characterized by increases in concentrations of interleukin (IL)-8, IL-6 and decrease in concentrations of a disintegrin and metalloproteinase with a thrombospondin (ADAMTS) 13 and low platelet counts. The differences between PedSep B and C were more subtle. Finally, non-randomized administration of corticosteroids was associated with lower mortality among PedSep D subclass, but no difference and higher mortality was observed among patients belonging to PedSep B and C, respectively.Among the four sepsis subclasses identified, PedSep D  used to estimate ICU mortality detailed by Wong et al.  and a mo of MODS . Similar of MODS , a subclSecond, the approach detailed by Qin et al. may also facilitate predictive enrichment, i.e., identification of patients subclasses with shared biological pathways that make them more susceptible to respond to a given therapy. The authors highlight the overlap of phenotypes with those detailed by our group , as wellThe authors acknowledge the limitations of the study, including the retrospective nature of the study, relatively small sample size, and lack of a validation cohort—all of which are endemic to studies focused on critically ill children. In addition, patients belonging to PedSep D were demonstrated to represent various clinical phenotypes previously detailed by Carcillo and colleagues . Thus, iIn summary, these observations suggest that biologically speaking, more is common across critical illness syndromes including sepsis and ARDS across the host developmental age spectrum than features that distinguish them . Accordi


Responsive neurostimulation is an evolving therapeutic option for patients with treatment-refractory epilepsy. Open-loop, continuous stimulation of the anterior thalamic nuclei is the only approved modality, yet chronic stimulation rarely induces complete seizure remission and is associated with neuropsychiatric adverse effects. Accounts of off-label responsive stimulation in thalamic nuclei describe significant improvements in patients who have failed multiple drug regimens, vagal nerve stimulation, and other invasive measures. This systematic review surveys the currently available data supporting the use of responsive thalamic neurostimulation in primary and secondary generalized, treatment-refractory epilepsy.A systematic review was performed using the following combination of keywords and controlled vocabulary:  OR ) OR [responsive neurostimulation AND thalamus AND (epilepsy OR seizures)]. In addition, a search of the publications listed under the PubMed “cited by” tab was performed for all publications that passed title/abstract screening in addition to manually searching their reference lists.Ten publications were identified describing a total of 29 subjects with a broad range of epilepsy disorders treated with closed-loop thalamic neurostimulation. The median age of subjects was 31 years old (range 10–65 years). Of the 29 subjects, 15 were stimulated in the anterior, 11 in the centromedian, and 3 in the pulvinar nuclei. Excluding 5 subjects who were treated for 1 month or less, median time on stimulation was 19 months (range 2.4–54 months). Of these subjects, 17/24 experienced greater than or equal to 50%, 11/24 least 75%, and 9/24 at least 90% reduction in seizures. Although a minority of patients did not exhibit significant clinical improvement by follow-up, there was a general trend of increasing treatment efficacy with longer periods on closed-loop thalamic stimulation.The data supporting off-label closed-loop thalamic stimulation for refractory epilepsy is limited to 29 adult and pediatric patients, many of whom experienced significant improvement in seizure duration and frequency. This encouraging progress must be verified in larger studies. Epilepsy, defined as recurring unprovoked seizures,  is a chrApproved in 1995, vagal nerve stimulators (VNS) were the first implantable neuromodulation devices for epilepsy . While tThe field of neurostimulation for TRE has been progressing over the last four decades with the first case of DBS targeting the anterior nucleus of the thalamus (ANT) reported by ® System1  is a closed-loop device that features two 4-contact electrode leads connected to a neurostimulator. One or two leads are implanted at seizure foci to detect epileptiform activity and deliver stimulation aimed at aborting incipient seizures. The detection system monitors (1) EEG signal intensity, (2) changes in wave frequency, and (3) discharges within pre-specified frequency ranges in order to identify epileptiform activity  with partial-onset seizures in one or two foci which fail to significantly improve on ≥2 AEDs  . EligibiAn examination of publications was carried out by searching PubMed from inception through May 2022 for articles published in peer-reviewed journals. The search strategy utilized a combination of keywords and controlled vocabulary terms (MeSH). Search results were restricted to English language only. Search terms included  OR ) OR [responsive neurostimulation AND thalamus AND (epilepsy OR seizures)]. In addition, a search of the publications listed under the PubMed “cited by” tab was performed for all included references from the initial search reports, and manual searches of the reference sections of the included studies were performed as well. All references were exported into Microsoft Excel for duplicate removal, screening, and data extraction.After removal of duplicates, 109 references underwent title and abstract screening resulting in the identification of 7 publications which met the inclusion criteria . A supplWhile most of the studies described the effects of responsive thalamic stimulation after a treatment period of 6 months or greater, In the first report of closed loop thalamic stimulation to treat epilepsy, In total, the 8/11 patients who received closed-loop stimulation targeting the ANT showed at least 50% improvement in seizure frequency after 1 month. These findings are consistent with the success seen in the pilot studies of open-loop, chronic DBS targeting the ANT . The ANTIn the first case series reporting RNS in the thalamic pulvinar nuclei (PVN), This review identified 10 publications where ECoG was coordinated with automated stimulation of thalamic nuclei triggered by epileptic discharges to treat refractory epilepsy. Early observations of decerebrate animals produced evidence supporting both the “centrencephalic” and “telencephalic” theories of epilepsy which state that generalized epilepsy spreads throughout the cortex from nuclei in the brainstem,  or from Several nuclei have been suggested as targets for deep brain stimulation (DBS) due to their connectivity, anatomical location within the thalamus, and positive results in animal models. Preeminently, The Papez circuit connects the thalamus with the mesial temporal lobe, and incorporates additional limbic and neocortical structures into tracts that carry epileptic activity . This paThe efficacy of disrupting thalamic function in controlling seizures has been recognized for decades . Monkey Although limited in quantity, published results of RNS in both the ANT and CMT for generalized epilepsy and absence seizures have been positive. The decision to target one or the other has thus far been operator-dependent, and further progress is needed to predict which target would be most efficacious in each patient . NonetheAs noted previously, animal models of epilepsy have shown that lesions or high frequency stimulation specifically in the ANT, or its afferent projections, reduce seizure frequency . ConsistClosed- and open-loop thalamic stimulation is being increasingly pursued in clinical settings to treat refractory epilepsy . DespiteClinicians have noted several challenges while employing the RNS system to target cortical and deep brain structures . Over moWhile reports of off-label responsive thalamic stimulation in epilepsy are generally encouraging, the evidence supporting this modality has several limitations. First, the evidence is comprised of a small dataset of 29 subjects, of whom 5 were treated for 1 month or less. Second, these reports are themselves limited by small, unmatched cohorts. Third, the 29 subjects described herein received thalamic stimulation at an advanced phase of their illness, after having failed years of medication trials, many of whom had additionally undergone epilepsy surgeries and/or VNS implants. Whereas this stage of illness is characteristic of RNS candidates, it limits the generalizability and predictive value of these results in patients who may consider RNS as an earlier line of treatment for TRE . AccordiThe data supporting off-label closed-loop thalamic stimulation is limited to 29 adult and pediatric patients with TRE, many of whom experienced significant improvement in seizure duration and frequency. This encouraging progress must be verified in larger studies.CF: conceptualization, methodology, investigation, data curation, writing–original draft, and project administration. EE: conceptualization, writing–review and editing, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


The ocular surface, comprised of the transparent cornea, conjunctiva, and protective tear film, forms a protective barrier defending deeper structures of the eye from particulate matter and mechanical trauma. This barrier is routinely exposed to a multitude of naturally occurring and engineered nanomaterials (ENM). Metallic ENMs are particularly ubiquitous in commercial products with a high risk of ocular exposure, such as cosmetics and sunscreens. Additionally, there are several therapeutic uses for metallic ENMs owing to their attractive magnetic, antimicrobial, and functionalization properties. The increasing commercial and therapeutic applications of metallic ENMs come with a high risk of ocular exposure with poorly understood consequences to the health of the eye. While the toxicity of metallic ENMs exposure has been rigorously studied in other tissues and organs, further studies are necessary to understand the potential for adverse effects and inform product usage for individuals whose ocular health may be compromised by injury, disease, or surgical intervention. This review provides an update of current literature on the ocular toxicity of metallic ENMs in vitro and in vivo, as well as the risks and benefits of therapeutic applications of metallic ENMs in ophthalmology. Demand for engineered nanomaterials (ENMs) has dramatically increased in recent years , with pr2) that are commonly incorporated into commercial sunscreens and cosmetics [The eye, particularly the cornea, experiences similar exposure to ENMs as skin and airways but may react to exposure in unique ways that differ from other tissue and organ types due to the immune privileged status of the cornea and the necessity of transparency for proper visual function. For example, a common response to ENMs exposed through the airway is inflammation ,16. Howeosmetics ,20. ENMsosmetics ,23,24,25Metallic ENMs are nanomaterials based on stable metals in the Periodic Table with at least one external dimension between 1–100 nm . MetalliNumerous in vivo studies have demonstrated that metallic ENMs can induce toxicity in a variety of organs including brain, heart, lung, liver, skin, kidney, and reproductive organs ,36,37,38Metallic ENMs have received considerable attention because they have attractive antimicrobial properties through the generation of reactive oxygen species (ROS), metal-ion release, particle internalization into bacteria, and direct mechanical destruction of bacterial cell wall and/or membrane . UnsurprEyes are sensory organs specifically designed to provide vision. The eye is comprised of three layers—an outer, protective fibrous coat (cornea and sclera), a middle, vascular layer , and inner, neural coat (retina) . The antThe precorneal tear film covers the ocular surface and serves as the first functional barrier by diluting and slowing the penetration of any agent, including drugs or toxic agents, that contact the eye. Any exogenous material, including ophthalmic solutions, are diluted by tears, and typically removed during the first 30 s after instillation from the ocular surface via reflex tearing, blinking and drainage through the nasolacrimal duct . For topThe outermost fibrous coat of the eye, the cornea and sclera are in direct contact with the external environment and provide protection for the middle vascular and inner neural structures. The transparent cornea is a trilaminar sandwich comprised of a hydrophobic multilayered epithelium, a hydrophilic stroma and a hydrophobic single-layer endothelium with its specialized extracellular matrix, Descemet’s membrane . The outIn addition to serving as a barrier against foreign material, the cornea protects against mechanical trauma; thus, each layer must be able to heal with tight junctions reforming following wounding. In vivo corneal wound healing can be easily visualized and monitored using multimodal imaging techniques that specifically target each layer . The uniWithin the eye, two blood ocular barriers exist to protect the eye from toxic agents within systemic circulation, a function that can be circumvented  Figure . The BABDue to their small size, systemically administered metallic ENMs can circumvent these barriers and subsequently accumulate in various tissues and fluids in the eye . While o2) have been raised in the literature [Intravitreal injection is a common delivery route for therapeutics to the posterior segment of the eye. Consequentially, the bulk of existing research regarding toxicity and therapeutic applications of metallic ENMs has focused on this administration route as well as being limited in focus to gold, silver, iron oxide, and cerium nanoparticles (NPs) ,92,93,94terature , highligHerein, we discuss the peer-reviewed literature regarding the use of metallic ENMs in ophthalmology and their toxic effects to the eye.AuENMs have been broadly studied as a therapeutic for the eye as well as for potential toxicity to ocular tissues . InteresFew studies have examined the impact of AuENMs on the anterior segment of the eye, but the impact of particle size and nature of functionalization on toxicity remain central. One study using primary human corneal fibroblasts demonstrated the relationship between functionalization and size of AuENM on cellular toxicity. Corneal fibroblasts treated with polyethylenimine-conjugated AuENMs (~4 nm) exhibited a maximum decrease in cellular viability of 13% . These ABMP7 gene using PEI2 coated AuENMs modulated corneal wound healing in a rabbit photorefractive keratectomy (PRK) model with minimal cytotoxicity and inflammation [However, not all AuENMs are toxic. In contrast to the above study, transfection of the ammation . In anotammation . The broThere has been a large focus on the use of AuENMs in the posterior segment of the eye, and in the retina specifically. Interestingly, several of the studies on the posterior segment of the eye demonstrate that trend of higher surface area correlates with higher toxicity. As an illustration, in two solutions with the same mass of small or large ENMs, the solution with small particles has more particles overall and, therefore, more surface area than the solution with large ENMs. Additionally, the greater the ratio of surface area to volume, the higher the chemical reactivity ,107. TheThe anti-angiogenic properties of AuENMs have made them prime candidates for evaluating ocular therapies for various retinopathies, resulting in several in vivo studies in the posterior segment. While not explicitly focused on toxicology, these studies demonstrate that the size and functionalization of AuENMs impact their toxicity to various ocular structures. Large ENMs <100 nm) that were administered intravenously were unable to cross the BRB  and an i nm that Aggregate these studies, and others   AgENMs was investigated in vitro with corneal stromal cells, with equal concentrations of rod-shaped ENMs exhibiting the most toxicity and spherical-shaped ENMs the least . The difStaphylococcus aureus-induced keratitis as an antiangiogenic and bacteriocidal treatment. They found that spherical AgENMs induce the highest bacterial killing while rod shaped AgENMs suppressed deleterious blood vessel development more than the other shapes, albeit with unfavorable side effects such as corneal thickening [These shape-based toxicity consistencies could potentially serve as predictors of ENM toxicity in a variety of cell types. The same researchers took these three shapes of AgENMs into an in vivo rabbit model of ickening . In aggrickening . In an iickening . This stMarkedly less research has focused on the cytotoxicity of AgENMs in the posterior segment of the eye. However, several sizes of AgENMs have been investigated, one of which utilized an organotypic tissue culture model of the murine retina to demonstrate neuronal toxicity with oxidative stress, apoptosis vacuole formation, and pyknotic cells being observed when treated with AgENMs (20 and 80 nm) . ConsistWhile there is a lack of consensus regarding the impact of size, shape, and functionalization of AgENMs on cytotoxicity, hints of their influence can be seen, specifically with regards to smaller ENMs being more toxic. Further studies are needed to elucidate these relationships for the safe and efficacious development of ophthalmic therapeutics using AgENMs.2), titanium dioxide (TiO2), zinc oxide (ZnO), and magnetic ENMs (mENMs), have also been investigated in ophthalmology  were observed to reduce single cell migration using human corneal limbal epithelial cells [2 ENMs (100 and 25 nm) showed no toxicity to hTCEpi cells but did inhibit migration at higher concentrations [2 ENMs, significantly greater apoptosis was observed [2 ENMs inhibited lens cell growth and induced excessive ROS generation that ultimately led to irreversible cell damage and death. In an in vivo study, topical application of TiO2 ENMs (~140 nm) demonstrated transient, reversible conjunctival redness using the acute ocular irritation test in healthy rabbits [2 ENMs (<45 nm) caused ocular surface damage and resulted in a reduction in conjunctival goblet cell area compared to control eyes [2 ENMs warrants further investigation in disease conditions, such as dry eye.Despite the widespread incorporation of TiOnscreens ,20 routial cells . Similartrations . When huobserved . Further rabbits . Anotherrol eyes . These r2 ENMs (12 nm) in their water did not delay development or induce retinal neurotoxicity [2 ENM in an in vivo mouse model showed a lack of toxicity [2 has been demonstrated to be readily taken up by ARPE-19 cells, as measured by flow cytometry and dark field microscopy [2 ENM may not directly reduce cellular viability; they do induce degradation of claudin proteins, resulting in increased retinal endothelial cell migration in vitro. A single intravitreal injection of TiO2 ENMs has been shown to disrupt the BRB and impair normal retinal electrophysiology in the mouse [2 ENMs on cell behavior while not exhibiting direct cytotoxicity. In aggregate, these results indicate that an ENM need not be directly cytotoxic to disrupt normal eye function, and warrant further investigation given the abundance of TiO2 ENMs routinely applied near the eye via cosmetics and sunscreens.In the posterior segment of the eye, exposing embryonic zebrafish to TiOtoxicity . Experimtoxicity . This lacroscopy . Howeverhe mouse . These rhe mouse , furtherIn comparison to the previously discussed metallic ENMs, ZnO ENMs tend to be directly toxic to various tissues and cells of the eye . In the 2+, are likely to be the primary contributor to cytotoxicity of both ZnO ENMs and ZnS ENMs. A recent study demonstrated particle dissolution and release of toxic Zn2+ from ZnO in the cell culture medium with subsequent accumulation in the lysosomal compartment of mouse macrophage and human bronchial epithelial cell lines that triggered ROS generation and oxidative stress [In the posterior segment of the eye, an in vitro study demonstrated that ZnO (5 nm) reduced cellular viability of both HCECs and ARPE-19 cells , and a me stress . Consiste stress .2 being the most well studied. As free radical scavengers, CeO2 ENMs show promise as a treatment for oxidative stress [2 (100 μg/mL) demonstrating putative genotoxicity in vitro in human lens epithelial cells [2 (10 and 30 nm) in hTCEpi cells and did not observe a decrease in cell viability, but did observe reduced epithelial cell migration at higher doses [Cerium containing ENMs (CeENMs) have been evaluated in vitro and in vivo for a diverse array of ocular applications, with CeOe stress . A variee stress , neuropre stress , to a noe stress ,141 where stress , and lase stress . Only a al cells ,145. We er doses . In aggr2O3, Fe2O3, CuO, V2O5, MgO and WO3 [2O3, CuO, and WO3 induced cytotoxicity at higher doses, with V2O5 significantly reducing cellular viability starting at a dose of 5 µg/mL. All of the materials, other than WO3, inhibited cell migration. This in vitro screening process allowed us to select promising candidates for in vivo testing, where V2O5 did not delay epithelial wound healing in a rabbit model [We have previously screened a wide variety of metal oxide ENMs for in vitro toxicity using hTCEpi cells, include Al and WO3 . Of the it model . Other ait model . As the Xenopus and zebrafish embryos [The magnetic properties of iron based magnetic ENMs (MENMs) are increasingly attractive for targeted drug and cell delivery to specific structures within the eye. The consequences of magnetic field exposure on cells are not well defined. In one study, MENMs were used to deliver mesenchymal stem cells to the trabecular meshwork as a treatment for glaucoma, with no adverse impact on cell viability and multipotency . Additio embryos ; however embryos . This is embryos  it is cr embryos . Additio embryos .In summary, these results demonstrate a putative role of MENMs as an ocular drug delivery platform; however, it is unclear how long these materials persist in the targeted tissue and the potential for degradation of any functionalized coatings could leave cells vulnerable to damage by the core of the ENM. Further studies are needed to fully characterize the toxicity of MENMs, especially under the disease states that are being treated.We have reviewed the available peer-reviewed literature regarding ocular nanotoxicology and a handful of ophthalmic applications of metallic ENMs. Despite their promising utility for the treatment of a myriad of ocular diseases, numerous metallic ENMs showed variable and often unpredictable toxicity to cells and ocular tissues. Despite the push for metallic ENM therapeutics, there is a clear knowledge gap in the use, toxicity, and mechanistic evaluation of the biological effects (if any) of metallic ENMs in the eye. Therefore, comprehensive studies of the ocular nanotoxicology of metallic ENMs are clearly needed with the goal of identifying the underlying physicochemical properties of metallic ENMs that may be driving toxicity. Furthermore, specific to ocular surface applications, studies that focus on the effects of ENMs on corneal wound healing, barrier integrity, and tear film stability are critical, since the ocular surface is the front-line defence against environmental contaminants as well as a primary route for ocular drug delivery. The rise of ENMs in clinical ocular applications, such as imaging and treatments for diabetic retinopathy, also highlight the need for a better understanding of the biological effects of these ENMs on the eye. Deeper insights into the mechanism of toxicity in the eye will improve formulation of ENMs for therapeutic use, improving the safety and efficacy of metallic ENM-based therapies and result in better outcomes for our patients.


Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among patients with refractory epilepsy. While the exact etiology of SUDEP is unknown, mounting evidence implicates respiratory dysfunction as a precipitating factor in cases of seizure-induced death. Dysregulation of breathing can occur in epilepsy patients during and after seizures as well as interictally, with many epilepsy patients exhibiting sleep-disordered breathing (SDB), such as obstructive sleep apnea (OSA). The majority of SUDEP cases occur during the night, with the victim found prone in or near a bed. As breathing is modulated in both a time-of-day and sleep state-dependent manner, it is relevant to examine the added burden of nocturnal seizures on respiratory function. This review explores the current state of understanding of the relationship between respiratory function, sleep state and time of day, and epilepsy. We highlight sleep as a particularly vulnerable period for individuals with epilepsy and press that this topic warrants further investigation in order to develop therapeutic interventions to mitigate the risk of SUDEP. Epilepsy is one of the most common neurological disorders. One in 26 Americans will develop epilepsy during their lifetime  being reduced to 73% of its level during wakefulness  occurs during NREM stage 3 (N3) sleep—although this is likely driven by the reduction in VT. This results in an end-tidal carbon dioxide (ETCO2) concentration that is 1–2 torr higher than waking levels  and oxygen (O2) can trigger oscillations in heart rate, blood pressure, autonomic nervous system activity, and upper-airway resistance. This may create a feedback loop whereby these oscillations in turn affect ventilation and increase the length and symmetry of these periodic breathing cycles  breathing physiology revealed time-of-day differences in breathing; however, the effect was limited to COT and VE were observed. The highest levels were observed during the dark phase, coinciding with elevated temperature and activity. These findings were further expanded in a later study, demonstrating that O2 consumption (a measure of metabolic activity), inspiratory time, and expiratory time also varied across the day  is likely driven by other physiological variables oscillating throughout the day, such as temperature and oxygen consumption.The first clear evidence that respiratory function demonstrated daily oscillations came from Seifert et al. . Adult rT, and VE were shown to vary depending on the time of day. However, recordings were only obtained hourly, and it is unclear if sleep-state was controlled for.Using similar methods to Seifert et al., 2, O2 consumption, and CO2 production were all shown to oscillate throughout the 24 h day, with highest levels in the morning. Interestingly, there was also time-of-day variability in HCVR magnitude, a finding previously demonstrated in awake, adult rats  drops below 90%  may be one of the underlying mechanisms behind cardiorespiratory failure in SUDEP. In Numerous other potential mechanisms underlying ictal respiratory dysfunction and failure have been proposed. A leading hypothesis is that seizures activate inhibitory subcortical projections to brainstem respiratory centers  of SUDEP incidents in EMUs found that all recorded cases of SUDEP featured terminal respiratory arrest prior to terminal asystole  mice and SCN1AR1407X/+ mice, which experience progressive breathing dysregulation  gene, which encodes for an activator of Bmal1 transcription, have been linked to intellectual developmental disorder with or without epilepsy or cerebellar ataxia (IDDECA)  is a technique used to treat refractory epilepsy 2 whereas midbrain 5-HT neurons mediate non-respiratory responses to CO2, such as arousal  plays an important role in sleep-wake regulation and respiration Jouvet, . It is a2 receptor is sufficient to suppress S-IRA in DBA/1 mice , almorexant, decreases the incidence of severe seizures, improves O2 saturation, and increases overall longevity , R01NS095842 (to GB), the Beth L. Tross Epilepsy Professorship from the Carver College of Medicine at the University of Iowa (to GB), and the National Institute of Health/National Institute of General Medicine Sciences T32GM0073367 (to BK).