Coronavirus Disease 2019 (COVID-19) is associated with increased incidence of neurological diseases and neuropsychiatric disorders after infection, but how it contributes to their development remains under investigation. Here, we investigate the possible relationship between COVID-19 and the development of ten neurological disorders and three neuropsychiatric disorders by exploring two pathological mechanisms: (i) dysregulation of host biological processes via virus–host protein–protein interactions (PPIs), and (ii) autoreactivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epitopes with host “self” proteins via molecular mimicry. We also identify potential genetic risk factors which in combination with SARS-CoV-2 infection might lead to disease development. Our analysis indicated that neurodegenerative diseases (NDs) have a higher number of disease-associated biological processes that can be modulated by SARS-CoV-2 via virus–host PPIs than neuropsychiatric disorders. The sequence similarity analysis indicated the presence of several matching 5-mer and/or 6-mer linear motifs between SARS-CoV-2 epitopes with autoreactive epitopes found in Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Myasthenia Gravis (MG) and Multiple Sclerosis (MS). The results include autoreactive epitopes that recognize amyloid-beta precursor protein (APP), microtubule-associated protein tau (MAPT), acetylcholine receptors, glial fibrillary acidic protein (GFAP), neurofilament light polypeptide (NfL) and major myelin proteins. Altogether, our results suggest that there might be an increased risk for the development of NDs after COVID-19 both via autoreactivity and virus–host PPIs. Viruses, since long before the COVID-19 pandemic, have been known to cause diseases. Several viruses are associated as risk factors in the development of various NDs, autoimmune diseases and neuropsychiatric disorders ,3,4. NeuEvidence indicates that patients diagnosed with COVID-19 have increased incidence of diagnosis of neurological diseases and neuropsychiatric disorders 6 months after infection, with patients admitted to the intensive therapy unit having even greater incidence risk . HoweverSARS-CoV-2 infection can also trigger a cytokine storm in some patients with severe infection, which is associated with excessive circulating levels of pro-inflammatory cytokines and increased mortality rates . NeuroinViruses can lead to the development of diseases through both direct and indirect effects with the two main pathological mechanisms being through virus–host PPIs and autoreactivity. Viruses lack their own replication machinery, hence by interacting with the human host, primarily via virus–host PPIs, exploit host components for their own advantage ,16. ThesA prevailing hypothesis is that infectious agents can trigger autoreactivity via molecular mimicry, where immunological memory cells (T and B cells) that recognize viral epitopes mistakenly target self-proteins ,26,27. MHowever, viruses are very common in the general population, hence infection with a virus is not a sufficient factor by itself for disease development . The “muHere, we first use network-based approaches, as illustrated in Our pipeline, which is illustrated in Experimentally validated virus–host PPI data between SARS-CoV-2 and the human host were obtained from VirHostNet 3.0  and IntAString: disease app in Cytoscape to obtain the top 200 disease-associated proteins with the highest disease score, for each of the following neuropsychiatric disorders and neurological diseases: ALS (ID: 332), Prion Disease (ID:649), HD (ID:12858), PD (ID:14330), MS (ID: 2377), CA (ID: 0050753), MG (ID: 437), GBS (ID:12842), AD (ID:10652), Neurotic Disorder (ID: 4964), BD (ID: 3312), Schizophrenia (ID: 5419) and Epilepsy (ID: 1826). The confidence cut-off score of the interactions between the human proteins was set at 0.8. To generate each of the integrated networks we merged the COVID-19-host PPI network with each of the disease-related PPI networks, that contained the 200 disease-associated proteins for each disease/disorder.To identify disease-associated biological processes that might be dysregulated by SARS-CoV-2 infection in existing patients with neurological diseases/neuropsychiatric disorders or possibly lead to their development, we generated thirteen integrated COVID-19-host-disease PPI networks. Using the same approach as in our previous work ,24, we up-value ≤ 0.05 (corrected with Benjamini–Hochberg).To identify pathways that can be modulated by SARS-CoV-2 viral proteins we performed enrichment analysis on their 861 human protein targets by using the ClueGO app  in Cytosp-value ≤ 0.01 (corrected with Bonferroni step-down). For the enrichment analysis of the extracted subnetworks, we used Bonferroni step-down because it provides a stricter result when the sample size of proteins used for analysis is large, whereas in p-value, than in To identify viral-mediated pathogenic mechanisms by which SARS-CoV-2 can possibly lead to the development of the 13 diseases/disorders we first isolated from each of the 13 integrated COVID-19-disease PPIs networks two subnetworks that would allow to identify overlapping biological processes (COVID-19 ∩ Disease). For each integrated networks we extracted the (i) COVID-19 subnetwork, which includes the 861 human protein targets of SARS-CoV-2 viral proteins and their first neighbors and (ii) the disease-related subnetworks, which includes the 200 disease-associated proteins and their first neighbors. Then, we performed enrichment analysis on each of the subnetworks by using the GO-biological processes database, retaining only significant processes that had an adjusted After obtaining the significant GO biological processes for each subnetwork, we removed any false positives enriched GO biological processes results by eliminating enriched terms that only contained term-associated genes that derived from only COVID-19 or only the disease-associated genes. Therefore, each of the GO biological processes terms that were retained for each subnetwork had to contain at least one gene-associated with both COVID-19 and the disease/disorder.ClueGO app, we performed functional enrichment analysis in the isolated GO biological processes (COVID-19 ∩ Disease) to identify the functional groups that these biological processes belong to.Then, by using Venn diagrams, we identified overlapping GO biological processes between the COVID-19 subnetwork and each of the disease-related subnetworks enriched results. Finally, by using the preselected function in To identify common GO biological processes that might be affected by COVID-19 in the thirteen diseases/disorders we classified them into three groups based on their main pathological characteristics: (i) neurological diseases of autoimmune origin (ii) NDs of non-autoimmune origin and (iii) neuropsychiatric disorders. The neurological diseases of autoimmune origin group includes MS, MG and GBS, which are diseases in which autoreactivity represents a main pathological characteristic ,38,39, wIn order to identify common GO biological processes that might be affected by SARS-CoV-2 in the three groups, we first created for each group a GO biological processes- Diseases network. To construct each of the three networks we included the isolated GO biological processes (COVID-19 ∩ Disease) which were identified to be also modulated by SARS-CoV-2 in each disease/disorder. Then, by using these networks we identified the common GO biological processes that might be affected by SARS-CoV-2 in the three groups. Finally, by using the same approach as in Genetic susceptibility is an important risk factor that not only predisposes an individual towards the development of complex diseases, such as NDs, but also determines infectious disease outcome. Host genetics that are involved in viral defenses or the life cycle of the virus can influence viral exposure and disease outcome. Understanding the interaction between host genetics and infectious diseases can aid to elucidate the role of genetic variability in viral pathogenicity and infectious disease susceptibility . GeneticNeurological diseases and neuropsychiatric disorders are complex conditions whose emergence is hypothesized to require the combinatorial action between genetic and environmental risk factors. According to the local impact hypothesis, disease susceptibility genes are located in proximity of viral host targets of viral-associated diseases within a network . TherefoDisGeNET database [DisGeNET, but the HTT was mapped on the COVID-19-host PPIs network to investigate if SARS-CoV-2 viral proteins interact with the HTT gene.Therefore, to identify if genetic susceptibility genes of the thirteen diseases/disorders under investigation interact with SARS-CoV-2 viral proteins, we first collected from database ,48 theirTo investigate the potential of cross-reactivity of SARS-CoV-2 epitopes with human proteins associated with the development of neurological diseases we performed pairwise sequence similarity analysis. Immunogenic epitopes against SARS-CoV-2 found during COVID-19 disease and autoreactive epitopes against human proteins found in the 6 neurological diseases  were obtained from IEDB . No autoThen, we performed pairwise sequence similarity between the SARS-CoV-2 epitopes and the autoreactive epitopes found in each of the 6 neurological diseases, by using the “seqinr” and “Biostrings” packages in R ,51. As iThe enrichment analysis results of the 861 human protein targets of the 15 SARS-CoV-2 viral proteins led to the identification of 28 significantly enriched KEGG pathways. These pathways can be grouped into 11 functional groups, as indicated in Interestingly, the enrichment results also indicated that 10.71% of the enriched pathways belong to the functional group of Amyotrophic Lateral Sclerosis. The group contains the enriched KEGG pathways ALS, Prion Disease and HD. The enrichment results suggest that SARS-CoV-2 interacts with human proteins associated with neurodegeneration and thus can possibly lead to the development of NDs or affect the severity of symptoms in existing patients.Our methodology allowed to extract the most relevant GO biological processes by which SARS-CoV-2 infection might affect existing neurological and neuropsychiatric patients or lead to their development through dysregulation of biological processes via virus–host PPIs. The results, To identify common GO biological processes that might be affected by SARS-CoV-2 infection in the thirteen diseases/disorders, we classified them into three groups based on their main pathological characteristics: (i) neurological diseases of autoimmune origin (ii) NDs of non-autoimmune origin and (iii) neuropsychiatric disorders. Epilepsy did not fall within any of these groups; therefore, it was excluded from this part of the analysis. To identify common GO biological processes affected by COVID-19 in each group we created three integrated GO biological processes-Diseases networks, shown in The integrated GO biological processes-Diseases network of the three neurological diseases of autoimmune origin  revealed 157 common GO biological processes affected by COVID-19, The functional enrichment analysis also indicated that 10.53% of the terms belong to the functional group of virus receptor activity, that involves exogenous protein binding between a virus component and host receptor that mediates the entry of a virus into the cell. It also indicated that 0.88% of the common processes belong to the functional group of positive regulation of catabolic process.The integrated network containing the six NDs of non-autoimmune origin  revealed 156 common GO biological processes between this group of diseases and COVID-19 C. These In addition, 14.71% of the terms belong to the functional group of response to reactive oxygen species (ROS), which suggests that COVID-19 can modulate ROS processes. Our finding is consistent with another study by  that empMoreover, 8.82% of the common processes belong to the functional group of positive regulation of cytokine production, that involves any process that leads to increased production of cytokines. Neuroinflammation is a common feature in NDs involving increased production of pro-inflammatory cytokines ,58. A reFurthermore, our results indicated that 8.82% of the common ND terms belong to the functional group of regulation of protein ubiquitination and 7.84% of the terms belong to the group of response to unfolded protein. Protein misfolding and dysregulation of the ubiquitin proteasome system (UPS) is a common pathological mechanism of several NDs ,61,62. HThe integrated network of three neuropsychiatric disorders  revealed 37 common biological processes E, that bIn addition, 20.0% of the common GO biological processes belong to the functional group of catecholamine metabolic processes, suggesting that SARS-CoV-2 can modulate the production of dopamine, noradrenaline and adrenaline. Catecholamines are hormones that also act as neurotransmitters that are involved in brain functioning, including processes, such as learning, memory and mood . The resCatecholamines are also important for the regulation of emotions, with dopamine being considered as the “feel-good” hormone. Decreased levels of dopamine are associated with depression (neurotic disorder) , whereasFlaviviridae family and a neurotropic virus, can also cause parkinsonism and in rare occasions neuropsychiatric sequels [Various viruses are known to exploit dopaminergic receptors for viral entry into cells ,76. For  sequels ,81. Simi sequels ,83. In a sequels . TherefoCatecholamines are also responsible for the body’s “fight or flight” stress response. Therefore, as SARS-CoV-2 can affect the production of catecholamines it suggests that it might be also able to alter the body’s “fight or flight” stress response system. Overactive or underactive “fight or flight” stress response has been found in young healthy individuals that where infected with SARS-CoV-2, which seems to contribute to “long-COVID-19” symptoms . ChronicMoreover, catecholamines are also involved in the regulation of immune responses, as they can have immune enhancing or suppressive effects depending on their duration and levels . For exaDisGeNET database  has the highest degree of interaction with variant genes as it targets 48 human proteins that have genetic variants associated with the development of these diseases/disorders C, whereaTo investigate the potential of cross-reactivity, via molecular mimicry, of antigenic determinants of SARS-CoV-2 viral proteins that are recognized by the immune system with host “self” antigens, we performed pairwise sequence similarity with autoreactive epitopes found in MG, ALS, MS, AD, PD and GBS. Our hypothesis is that if SARS-CoV-2 epitopes have “sufficient” sequence similarity with known disease autoreactive epitopes then they can potentially cross-react with those proteins, thus leading to the emergence of the associated diseases. For this purpose, we isolated SARS-CoV-2 epitopes that contain matching linear motifs of five AAs or above with autoreactive epitopes found in neurological diseases. Our analysis led to the identification of 5-mer linear matching motifs between SARS-CoV-2 viral epitopes and autoreactive epitopes found in MG, AD, PD and MS. In addition, we identified matching 6-mer linear motifs with autoreactive epitopes found in MS. However, no linear motifs of six AAs or more were found between SARS-CoV-2 epitopes and autoreactive epitopes found in AD, PD, MG. Additionally, no linear motifs of seven AAs or more were found between SARS-CoV-2 epitopes and MS autoreactive epitopes. In addition, no linear motifs of five AAs or above were found in the consensus sequences between SARS-CoV-2 epitopes with GBS and ALS autoreactive epitopes.The identified SARS-CoV-2 epitopes and autoreactive epitopes found in AD, MG and AD which contain matching 5-mer linear motifs in their consensus sequences are indicated in The SARS-CoV-2 epitopes that contain matching 5-mer motifs with autoreactive epitopes found in AD, PD and MG are from Replicase polyprotein 1a and 1ab viral proteins, which are encoded by ORF1a and ORF1ab, respectively. They are non-structural proteins that are only expressed in infected cells and unlike SARS-CoV-2 structural proteins they do not form part of the virion particle . These pIn addition, our analysis revealed 1085 matching 5-mer linear motifs in the consensus sequences of SARS-CoV-2 epitopes with autoreactive epitopes found in MS, which are indicated in Our analysis results also indicated matching 5-mer linear motifs of SARS-CoV-2 epitopes with MS autoreactive epitopes that target the Olfactory receptors 10A4 and 4N2. Loss of smell is a common symptom found during COVID-19 and hyposmia or anosmia can also appear during or after COVID-19 ,102. MorOur analysis also indicated the presence of matching 6-mer linear motifs between SARS-CoV-2 epitopes and autoreactive epitopes found in MS. Pathogenic organisms, including viruses, have been associated as risk factors for the development of several diseases, particularly autoimmune-mediated diseases. The introduction of this new virus, SARS-CoV-2, into the repertoire of pathogens that can interact with the human host has introduced the need to investigate and identify potential diseases that could emerge due to this interaction. Understanding these virus–host interactions could also possibly provide greater insight into the pathogenic mechanisms that lead to COVID-19 disease severity and the emergence of post-COVID-19 symptoms. Most importantly, being able to predict diseases that can emerge due SARS-CoV-2 infection can possibly enable to identify preventive pharmacotherapies that could slow down or even prevent the development of these diseases.In this study, we have developed a bioinformatics-based methodology with the aim to computationally identify pathological mechanisms by which SARS-CoV-2 infection can lead to the development of neurological diseases  and neuropsychiatric disorders . Viruses interact with the human host via virus–host PPIs which allows them to manipulate host processes for their own advantage. These viral mediated effects can result into the dysregulation of host biological processes leading to the development of various diseases. In the first part of our investigation, we explored whether SARS-CoV-2, via virus–host PPIs, can lead to the dysregulation of biological processes associated with the pathogenesis of the thirteen diseases/disorders. In addition, we investigated whether SARS-CoV-2 epitopes can possibly have cross-reactivity with host self-antigens, via molecular mimicry, by comparing them with autoreactive epitopes found in neurological diseases. Moreover, we identified disease-associated susceptibility genes that might alter virus–host PPIs during COVID-19 infection and lead to the emergence of edgetic perturbations, thus resulting in alter virus–host disequilibrium disease states.Our methodology first involved the reconstruction of thirteen integrated COVID-19-host-disease PPI networks. This allowed to identify overlapping GO biological processes (COVID-19 ∩ Disease) and functional groups of biological processes associated with these diseases/disorders that can be possibly modulated by COVID-19 see . Thus, tOur results also suggest that there might be a higher risk for the development of NDs after SARS-CoV-2 infection via the mechanism of virus–host PPIs than the development of neuropsychiatric disorders. It is important to note that HD is a hereditary disease and although SARS-CoV-2 cannot lead to its development, it might cause early age of symptom onset. Early age of symptom onset in HD, due to viral infection has been observed in HD-mutant carriers of at least 36 CAG repeats that were infected with human immunodeficiency virus .In addition, our results indicated that AD has the highest number of overlapping GO biological processes (COVID-19 ∩ Disease) that can be modulated by SARS-CoV-2 through virus–host PPIs, whereas Epilepsy has the lowest number of overlapping processes, Moreover, our results suggest that SARS-CoV-2 can possibly lead to the development/progression of NDs  through several of the known common pathological mechanisms found in NDs D. This iNeuropsychiatric symptoms are common in COVID-19 patients and might persist long after infection, although their severity decreases after COVID-19 recovery . Our anaOur analysis also indicated that a common pathological mechanism by which SARS-CoV-2 can possibly lead to the development/progression of the three neurological diseases of autoimmune origin  is through the positive modulation of cytokine production A. The paHowever, although COVID-19 might possibly confer increased risk for the development of neurological diseases and neuropsychiatric disorders through dysregulation of biological processes via virus–host PPI, other risk factors  could also contribute to determining the outcome and extent of this dysregulation. The combinatorial actions between SARS-CoV-2 infection and these additional risk factors would also possibly determine which disease phenotype might emerge. Therefore, the presence or absence of these additional risk factors during SARS-CoV-2 infection could possibly determine the potential risk level that someone has in developing these diseases/disorders. For example, sleep deprivation causes increased production of beta-amyloid protein which is associated with the development of AD, thus lack of sleep may increase dementia risk . Sleep dThe next part of our investigation involved the identification of potential genetic factors which in combination with SARS-CoV-2 infection could possibly affect virus–host PPIs and lead to edgetic perturbations that would alter COVID-19 pathogenicity and disease outcome, predisposing an individual in the emergence of the thirteen diseases/disorders. Our results showed that AD and Schizophrenia have the highest number of human proteins associated genetic variants involved in their development that are also targeted by SARS-CoV-2 viral proteins B. Thus, Viruses can also lead the emergence of diseases, indirectly through autoreactivity. To investigate whether SARS-CoV-2 epitopes that are recognized by immunological memory cells can cross-react with host “self” antigens, we performed sequence similarity between linear SARS-CoV-2 epitopes and linear autoreactive epitopes found in neurological diseases: MG, ALS, MS, AD, PD and GBS. Other papers have already investigated the molecular similarity between SARS-CoV-2 epitopes and human proteins in various tissues . For exaOur methodology led to the identification of SARS-CoV-2 epitopes that have 5-mer and/or 6-mer linear motifs within their sequences that are also found in autoreactive epitopes associated with PD, AD, MG and MS see . These mHowever, our study is not without limitations. One important limitation of our study, is that non-statistically significant GO biological processes could also be important, as SARS-CoV-2 might exert disease effects through them . In addiRegarding the exploration of the cross-reactivity risk potential of SARS-CoV-2 epitopes, our methodology was designed to highlight matching linear motifs between SARS-CoV-2 epitopes and autoreactive epitopes found in neurological diseases, and exclude discontinued motifs in this study. However, cross-reactivity can also occur between matching discontinued or conformational motifs, therefore additional SARS-CoV-2 epitopes could also possibly cross-react with human proteins. Although our methodology did capture and led to the identification of matching discontinued motifs of more than 5AA between SARS-CoV-2 epitopes and autoreactive epitopes found in NDs (unpublished data), these results can be presented in future work. It is also important to experimentally validate the cross-reactivity potential of the identified SARS-CoV-2 epitopes with their matching human proteins, by using immunoassay methods.Moreover, in this study we choose to focus on a specific list of 10 neurological diseases and 3 neuropsychiatric disorders. The diseases/disorders examined in this study were selected based on the hypothesis that diseases/disorders whose development and/or progression has been associated with viral infections have a higher probability to be triggered by a new emerging virus, in this case SARS-CoV-2, than other diseases, as viruses share several common pathogenic mechanisms. However, our selection list is not exhaustive as other neurological diseases or even other types of diseases, particularly autoimmune diseases, which have a strong association with viral infections can also potentially be triggered by COVID-19. Nonetheless, these diseases are beyond the scope of this paper and may be addressed in future works using the presented computational methodology.In this study, we used an integrative bioinformatics exploratory approach to investigate how SARS-CoV-2 infection can affect existing patients with neurological diseases or neuropsychiatric disorders or whether it can lead to their development. We examined two main pathological mechanisms by which viruses are known to cause disease: (i) virus–host PPIs and (ii) autoreactivity.Our analysis results indicated that NDs  have a higher number of GO biological processes that can be modulated by SARS-CoV-2, via virus–host PPIs, than neuropsychiatric disorders . In addition, our results highlighted the positive regulation of cytokine production as a common pathological mechanism by COVID-19 that can possibly lead in the development of both neurological diseases and neuropsychiatric disorders.Moreover, our analysis indicated that modulation of neuro-immune crosstalk by SARS-CoV-2 might facilitate the development of neuropsychiatric disorders or exacerbate symptoms in existing patients. Based on our results, we suggest that therapeutic strategies that aim to treat COVID-19 mediated neuropsychiatric symptoms should account for neuro-immune crosstalk, and hence a combination of immunomodulatory and neuromodulatory therapies might be more appropriate rather than only the use of neuropsychiatric drugs.Furthermore, the results of our analysis indicated the presence of several matching 5-mer and/or 6-mer linear motifs between SARS-CoV-2 epitopes and autoreactive epitopes found in AD, PD, MG and MS. The results include autoreactive epitopes that recognize APP, MAPT, acetylcholine receptors, GFAP, NfL polypeptide and major myelin proteins.Further studies are required to examine this potential risk with the aim to identify possible preventive measures. In addition, the impact of other indirect pathogenic mechanisms mediated by COVID-19 that might increase the risk for the development of these diseases/disorders should also be investigated. COVID-19 infection was shown to cause the reactivation of latent viruses, including Epstein–Barr virus and herpes simplex virus 1, as well as gut dysbiosis, which are all risk factors for the development of both NDs and neuropsychiatric disorders ,134,135.


Since the corresponding signaling hubs are mainly organized along the cellular membranes, cytosolic proteins like Cdc42 need to be properly targeted and held at the membrane. Here, lipid modifications come into play: Cdc42 can be associated with membranes by different lipid anchors including prenylation (Cdc42-prenyl) and palmitoylation . While Cdc42-prenyl is ubiquitously expressed, Cdc42-palm splicing variant in mainly expressed in the brain. Mechanisms underlying Cdc42 lipidation as well as its regulation are the main topic of this review. Furthermore, we will discuss the functional importance of Cdc42 lipid modifications with the focus on the role of different lipids in regulating defined Cdc42 functions. Finally, we will provide an overview of the possible implementation of Cdc42 lipidation in pathological conditions and different diseases.The protein cell division cycle 42 (Cdc42) is a small GTPase of the Rho family regulating a plethora of physiological functions in a tissue, cell and subcellular-specific manner  When later researchers investigated the interplay between cell cycle events  and cytosolic/membrane events  in yeast Saccharomyces cerevisiae, they identified a mutant that was able to continue DNA synthesis and nuclear division while the emergence of buds was blocked. The responsible gene was named CDC42, in relation to another budding-involved gene, namely CDC24 (p (or G25K) and found that it has the highest degree of sequence identity (more than 80%) with the CDC42. Therefore, the corresponding human placental protein was designated Cdc42Hs. Using different probes, authors not only isolated cDNA for the Cdc42Hs, but also found a second protein with the same size but different C-terminal sequence (Gp’), concluding that these might be another reading frame within the same gene (The protein cell division cycle 42 (Cdc42) was initially purified from the placental membranes during isolation of small molecular weight GTP hydrolyzing proteins . This prly CDC24 . In the ame gene .p’, Cdc42-palm, Cdc42E6, Cdc42b, bCdc42, Cdc42-v2. To simplify the reading, we will mark the first isoform as Cdc42-prenyl and the second one as Cdc42-palm in the further text. The CDC42 gene consists of 7 exons, and the last two exons can be alternatively spliced , whereas Cdc42-prenyl is terminated by a CaaX-box (CVLL)  . In addix (CVLL) . Whereasx (CVLL) . The polx (CVLL) .The di-lysine motif (Lys-183 and Lys-184) within Cdc42-prenyl has been shown to be involved in interaction of Cdc42-prenyl with the γCOP subunit of the coat proteins I (COPI) complex, playing a role in regulating intracellular trafficking . The di-via irreversible thioether bond  and should thus bypass methylation excision , which reside either in the Golgi compartment or at the plasma membrane (see below). Since the CCIF sequence of Cdc42-palm represents a non-canonical CaaX motif, a part of Cdc42-palm will be not recognized by prenylating enzymes, resulting in palmitoylation of both cysteine residues . Since prenylation and palmitoylation possess different affinity for lipid bilayers  and can More recently, we performed a detailed analysis of palmitoylation stoichiometry for Cdc42-palm isoforms by applying acyl-PEGyl exchange gel shift (APEGS) assays . Using tvia palmitoylation. Previously, it has been shown that DHHC5 could be palmitoylated at C-terminal cysteine residues outside of its catalytic core .Since palmitoylation is a dynamic and reversible process, Cdc42-palm can also undergo depalmitoylation. So far, only a few published works investigated the Cdc42 depalmitoylation. Cdc42-palm was shown to maintain asymmetric protein localization during cell division by coordinated interplay with catalytic active acyl protein thioesterase 1 (APT1) in breast cancer cells (MDA-MB-231) . The palCdc42 is highly conserved from yeast to mammals and plays an important role in the regulation of a plethora of cellular processes, including phagocytosis, cell polarization, chemotaxis, cell-migration and division . Cdc42 iAn important physiological function of Cdc42 is the activation of transcription factors such as NFκB, STAT3 and SRF . In partin vivo resulted in a significant increase of Cdc42 palmitoylation   . In the se (ACK) . This idse (ACK) .de novo mutations within the CDC42 gene result in heterogeneous phenotypes with highly variable symptoms including facial dysmorphism, dysregulation of growth, haematological as well as immunological and neurodevelopmental abnormalities such as Takenouchi-Kosaki syndrome (TKS) (Many me (TKS) . HoweverIn 2019, Gernez and colleagues reported about four patients carrying CDC42 gene mutations affecting the last three to five C-terminal amino acids of Cdc42-prenyl . These mutations were associated with severe auto-inflammatory symptoms including anemia, cytopenia, rashes, facial dysmorphisms and are associated with increased ferritin, C-reactive protein, and interleukin IL-18 levels. In addition, patients carrying these mutations were predisposed to the development of macrophage activation syndrome and hemophagocytic lymphohistiocytosis. Of note, the novel C-terminal variants in Cdc42 all occur within or in close spatial proximity to the C-terminal di-arginine motif implicated in membrane binding and lipidation of Cdc42-prenyl. One patient had a mutation within the CDC42 coding region leading to a replacement of the stop codon by a cysteine residue, resulting in a read-through and 24 additional amino acids (*192C*24). Another patient carried the R186C mutation. The other two patients carried a C188Y mutation, which unfortunately were not further characterized in detail . TherefoUsing whole exome sequencing, Bekhouche and co-authors detected a heterozygous mutation in a patient affecting only Cdc42-prenyl, resulting in the exchange of arginine in position 186 to a cysteine residue (R186C). The main clinical characteristics of this mutation include severe neonatal dermatitis accompanied by acute episodes of hepatomegaly with cytolysis, mild facial dysmorphy, and a permanent inflammatory syndrome with occasional monocytosis . DetaileRecently, Nishitani-Isa and colleagues further analyzed molecular details of autoinflammatory pathology evoked by R186C Cdc42-prenyl mutation. Using induced-pluripotent stem cells (iPSCs)-derived myeloid and macrophages cell lines established from the patients carrying this mutation, they reported that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli . NoteworThese combined results not only demonstrate an unexpected association between aberrant palmitoylation of Cdc42-prenyl and activation of inflammatory responses, but also suggest that this pathway might represent a novel target for treatment of autoinflammatory diseases. Moreover, results of these studies suggest that although Cdc42-prenyl is ubiquitously expressed, pathological effects of its aberrant palmitoylation seem to be related to the Cdc42-prenyl population expressed outside of the brain tissue.


Cannabis legalization in many jurisdictions worldwide has raised concernsthat such legislation might increase the burden of transient and persistentpsychotic illnesses in society. Our study aimed to address this issue.Cannabis Act implemented on October 17, 2018) andweekly ED presentation counts of the following ICD-10-CA-defined targetseries of cannabis-induced psychosis  andschizophrenia and related conditions , as well as two comparison series ofamphetamine-induced psychosis  andalcohol-induced psychosis .Drawing upon emergency department (ED) presentations aggregated acrossAlberta and Ontario, Canada records , weemployed Seasonal Autoregressive Integrated Moving Average (SARIMA) modelsto assess associations between Canada's cannabis legalization (via theP = 0.88)]; (2)schizophrenia [24.34 ]; (3)alcohol-induced psychosis .ED presentations for cannabis-induced psychosis doubled between April 2015and December 2019. However, across all four SARIMA models, there was noevidence of significant step-function effects associated with cannabislegalization on post-legalization weekly ED counts of: (1) cannabis-inducedpsychosis [0.34 (95% CI −4.1; 4.8; Implementation of Canada's cannabis legalization framework was not associatedwith evidence of significant changes in cannabis-induced psychosis orschizophrenia ED presentations. Given the potentially idiosyncratic rolloutof Canada's cannabis legalization, further research will be required toestablish whether study results generalize to other settings. CannabisAct—the legislative framework for the legalization of non-medicalcannabis use in Canada. The movement toward cannabis legalization in many jurisdictions worldwide hasraised concerns that such legislation may increase the burden of transient andpersistent psychotic syndromes in society.3 Prior experimental studiesadministering delta-9-tetra-hydrocannabinol  to healthy participants have provided evidence that THC administrationcan cause dose-dependent patterns of positive and negative psychotic symptomology inotherwise healthy individuals.5 In addition, a large body ofepidemiological evidence (utilizing a range of research designs) has demonstrated adirectional dose-response association between cannabis use and incidence ofschizophrenia, with the association being more pronounced among individualsreporting early adolescent initiation, those with a high genetic loading forpsychotic illnesses, and persons reporting prior psychotic symptoms.69 Experimental pharmacologicalchallenge involving patients with schizophrenia has shown that administration of THCworsens psychotic symptoms , while clinical studies  have found that the continuation of cannabis use after the onset of psychoticillness is associated with worse outcomes—including higher relapse rates, longerhospital stays, and more pronounced positive psychotic symptomology, in comparisonto those patterns observed among people with psychosis who discontinue or abstainfrom cannabis use. It is important to acknowledge, however, that only a smallproportion of cannabis users identified in population-based surveys report anypast-year problems related to their cannabis use.On October 17, 2018, the Canadian Federal government implemented the 14 Cannabis legalization may also lead to net societal benefits,such as decreased impacts of a cannabis-related burden on the justice system, reductions in cannabis-related criminalization and stigma, regulated andquality-controlled access to cannabis products, and development of cannabis-relatedtreatment protocols and harm reduction interventions.17 In this context, wetentatively expected that cannabis legalization would increase the number ofhazardous cannabis-use episodes leading to the experience of cannabis-related harmsin the populations of Ontario and Alberta—a pattern which, in turn, would likelycontribute to detectable increases in ED presentations for cannabis-inducedpsychosis and, perhaps, more persistent psychotic syndromes . The Cannabis Act mandates a Parliamentaryreview of the impacts of cannabis legalization on Canadian public health, and thisstudy aims to provide evidence for that review.Cannabis legalization might support increased patterns of cannabis use in societythrough a number of potential mechanisms, such as decreased perceived harmfulness,increased cannabis availability, elimination of criminal penalties as a deterrent tousing, and changes in social norms making cannabis use more sociallyacceptable.The Centre for Addiction and Mental Health (CAMH) Research Ethics Board (REB)provided ethical approval for the current study (REB# 074/2019). The CanadianInstitute for Health Information (CIHI) supplied de-identified data for the project,and patient consent was not required for the release of these data.Data source: National Ambulatory Care Reporting System (NACRS), April 1,2015–December 31, 2019. The lead authors  are bound by data-security and data-privacyagreements with CIHI. As a result, the research team cannot release the raw datafrom the study. Study data are publicly available from the data guardian  upon request and successful application.The current study utilized data from all Ontario EDs  and all Alberta EDs , spanning from April 1, 2015–December 31, 2019, fromthe NACRS database (held and managed at CIHI). Alberta and Ontario are the only twoCanadian provinces which submit 100% of all ED presentations in the province to theNACRS data system and, as a result, we selected only these two provinces for ourcurrent study. Most other provinces and territories submit only a subset of all ofthe ED presentations occurring in their province/territory to the NACRS database. along with optional fields for nine supplemental diagnoses. The primaryand secondary/comparison outcomes in the current study were defined as anoccurrence, in any diagnostic position in a patient's emergency departmentmedical record, of at least one of the identifying ICD-10-CA codes: F12.5(cannabis-induced psychosis)21; F20-F29 (Schizophreniaand related disorders); F10.5 ; or F15.5 (amphetamine-induced psychosis).21 Our ICD-10-basedtransient alcohol- or drug-induced psychosis and schizophrenia outcomes havebeen widely used in prior case-registry studies . Theamphetamine-psychosis and alcohol-psychosis outcomes were chosen to serve asdrug-related comparison series to our primary cannabis-related psychosis series.We included all NACRS records with an identifying ICD-10 code; no exclusioncriteria were applied. Counts of the primary and secondary outcomes wereaggregated across weeks , centered at the date ofCanadian cannabis legalization . All data series wereaggregated across Ontario and Alberta ED presentations.In the NACRS database, each ED medical record included at least oneICD-10-CA-based primary diagnosis,n = 5832; 77.1% male; mean age,28.1 years ]; (2) schizophreniaand related conditions ; (3) amphetamine-related psychosis; and (4) alcohol-related psychosis.The data-series samples consisted of ED presentations occurring during the dataspan  in Alberta and in Ontario for (1)cannabis-induced psychosis ; (2) schizophrenia, SARIMA[52]; (3) amphetamine-related psychosis, SARIMA[52]; and (4) alcohol-related psychosis, SARIMA[52].In the first phase of the modeling, the 185-week pre-legalization series was usedto identify SARIMA models in order to achieve stationarity in each model. In thecurrent study, the seasonal lag term was 52 weeks. We identified suitable modelsto achieve stationarity by examining the autocorrelation and partialautocorrelation functions at different lags, In simple terms, the pre-legalization SARIMA coefficients were used toforecast the predicted patterns of the outcomes across the post-legalizationperiod, and these predicted patterns were compared to the observed patternsoccurring after legalization in order to assess whether cannabis legalizationmight have affected the observed post-legalization data points. Subsequently, wealso estimated more flexible SARIMA models by incorporating a parameter for astep function and a parameter capable of estimating the possible increase orattenuation of the initial step function. We tested for possible innovative outliers and additive outliers inmodels using the framework described in Cryer and Chan (see Section 11.2).After reasonable models were found for the pre-legalization series, the full dataseries were used and step transfer functions were selected as the primaryapproach to estimate the potential step-function effects of cannabislegalization in the four data series. using the package TSA 1.3. The study team conducted the statistical analyses from January-February,2021. The primary research question and associated analyses were notpre-registered on a publicly available platform, and, as a result, the resultsshould be considered exploratory.All models were estimated in R 4.0.2The visual depiction of the time series plots for each of the four primary SARIMAmodels can be found in P = 0.88) visits per week, with the model showing no evidenceof a step-function effect associated with cannabis legalization in relation to thecannabis-induced psychosis outcome (see P = 0.26) presentations per week .Given the null results related to our primary intervention effects across all ofour four SARIMA models, we estimated the SARIMA step-function effect sizes thatour designs would have had reasonable power (80%) to detect through the processof statistical power simulation  occurring 2 years before and 5 months afterlegalization to two Sherbrooke, Québec psychiatric emergency department units, withthe Sherbrooke-based study finding no evidence of any significant difference in thepre- versus post-legalization prevalence of a psychotic-disorder diagnosis inpatients’ medical records. As the authors noted, however, the comparison of the prevalence of pre-versus post-legalization psychotic-disorder presentations may have been confoundedby treatment-service changes occurring during the study period via the initiation ofnew community-based psychiatric-treatment outreach programs , and such alterations may havedifferentially affected patterns of psychotic-disorder presentations to thepsychiatric ED study sites across the pre- and post-legalization periods. In relation to the current study, a number of factors may have contributed tothe null results related to our target series. First, key indicators of patterns ofcannabis use in Alberta and Ontario showed relatively little change afterlegalization. In Alberta, for example, while the prevalence of past-90-day cannabisdid increase significantly between pre-legalization and post-legalization periods(16.4% vs. 19.3%), there was no evidence of post-legalization change in theprevalence among individuals who use cannabis daily/almost daily (7.2% vs. 7.1%)—a pattern of use most closely linked to the likelihood of experiencingcannabis-related harms,32 including persistent psychotic syndromes.31 In Ontario, there was noevidence of pre- versus post-legalization changes in the prevalence of past-90-daycannabis use (15.5% vs. 17.5%) or daily/almost daily use among individuals who usecannabis (6.4% vs. 6.0%). Also, there was a long period of de facto decriminalizationand normalization of cannabis use in Canada prior to legalization. As a result, the legalization of non-medical cannabis use may have had only arelatively small influence on patterns of cannabis use not only in the Ontario andAlberta general populations, but also in those subpopulations potentially at higherrisk for developing psychotic conditions, such as adolescents and young adults,people with psychotic illnesses, or those with a family history of psychosis.Our null results appear similar to those described in a recent medical-chart-reviewstudy comparing the prevalence of pre- versus post-legalization psychotic-disorderpresentations  in Ontario, for example, there were reports that local, unlicensed cannabisdispensaries closed during the period leading up to and shortly followinglegalization in order to qualify for licenses as legal cannabis retailers; (2) government prices for licit cannabis were higher than those prices forcorresponding black market products, which tended to have higher THC potencies3537; (3) thecannabis-legalization rollout faced supply-chain bottlenecks, leading to shortagesand limited selection of cannabis products in government-licensed retailstores3841; (4) while every province andterritory had a government-sanctioned online retail cannabis store open at the timeof legalization, there was a scarcity of brick-and-mortar cannabis stores open immediatelyfollowing legalization, especially in Ontario ; and (5) shortly after legalization, a national rotating postal strikeoccurred—an event which likely would have affected the delivery of cannabis productsordered on government-sanctioned online sales platforms.In addition, the implementation of the Cannabis Act included a two-phasedsequence regarding the availability of cannabis products, with industry-wide rulesand standards regarding the types of cannabis products legally allowed for sale inlicensed retail environments. During the first year of legalization (“Cannabis 1.0”), only dried-leafcannabis products and oils were allowed to be sold in government-sanctioned retailoutlets.43 In the second phase of legalization (“Cannabis 2.0”), cannabisedibles and high THC-potency cannabis concentrates , as well ashigh-potency cannabis vaping products, became legally available in licensed retailoutlets in early 2020 in most provinces and territories.43 Recent research has shownthat daily use of high-potency cannabis is significantly associated not only withthe likelihood of developing a psychotic illness, but also that this likelihood ismore pronounced among individuals residing in geographical regions which had greateravailability of high potency cannabis products. Given the possible link between the daily usage and market availability ofhigh-potency cannabis products and the development of psychotic illnesses, it willbe important for future research to assess the impacts of the rollout of Canada'ssecond phase cannabis legalization (“Cannabis 2.0”) on patterns of psychoticsyndromes in Canadian provinces and territories.It is also possible that Canada's initial legalization of non-medical cannabis useoccurring on October 17, 2018 (“Cannabis 1.0”)—and its concomitant legal restrictionon the sale of high potency cannabis products—may have attenuated the potentialimpacts of legalization on the cannabis-induced-psychosis series and schizophreniaseries in our paper. The  Prior international registry-based studies have found similar patterns: arecent study of all inpatient hospital psychiatric admissions in Denmark estimatedthat the admission rate for cannabis-induced psychosis approximately doubled from2006 to 2016. The authors speculated that this increasing trend in cannabis-induced psychosishospitalizations was associated with the parallel increases in the THC content ofcannabis products in Denmark . At this time, we are not aware of similar THC-potency indicators forcannabis products during the period of our study and, as a result, we are not ableto evaluate the possible statistical association between indicators of THC potencyacross the study period and the observed cannabis-induced psychosis trend in ourproject. It is also important to note that amphetamine-induced psychosispresentations also increased substantially across the 2015–2019 period  at least in ourunderstanding, it is unclear what time period might constitute a more appropriate ordefensible lag period, given the lack of scientific literature regarding the typicallength of cannabis exposure required for exacerbation of pre-existingschizophrenia-related conditions or new-onset schizophrenia conditions vis-à-visemergency department presentation; and (2) the current study incorporated a ratherabbreviated 14.5-month post-legalization period, and the choice of, say, a 6-monthor 12-month lag would make the follow-up even shorter and reduce the statisticalpower of the SARIMA models to detect any potential lagged effects. Future researchmay want to incorporate different research designs to account for a potentiallylonger latency period between legalization-associated changes in cannabis usepatterns and emergent trends in persistent psychotic syndromes. We attempted toexplain, to some extent, the null results in our study by pointing to nationalsurvey data showing no or minimal evidence of changes in the Ontario/Albertaprevalence of past-90-day cannabis use or daily/almost daily cannabis use. It isimportant to note that such prevalence estimates may not necessarily track neatlypopulation-based harms, especially as these prevalence measures do not account forimportant aspects of patterns of use, such as the quantity of use, heavy episodic(binge) cannabis use, THC potency of cannabis products consumed, or typical route ofadministration—all of which may contribute unique components to patterns ofpopulation-based cannabis harms. It may be important for future research toincorporate more detailed assessments of cannabis-use patterns vis-à-vislegalization-associated changes in cannabis-related harms. As a result, thediagnostic validity of our outcome codes is uncertain. The primary ED-based ICD-10outcome series of cannabis-induced psychosis and schizophrenia (and relateddisorders) do not include information capturing quantity, frequency, duration, orthe age of initiation of cannabis use in the ICD-10 diagnostic categories. Also, ouranalyses combined Ontario and Alberta data to maximize statistical powerconsiderations, but future research may want to assess how the different provincialrollout and retail strategies might be associated with cannabis-related andpsychosis outcomes. Also, given the potentially idiosyncratic factors involved inthe Canadian phased rollout of cannabis legalization, our results may not generalizeto other settings.The current study's findings need to be interpreted in light of its potentiallimitations. Given that our SARIMA design is a quasi-experimental approach, it ispossible that unaccounted for variables may have confounded our assessment of theestimated relations between cannabis legalization and the outcome series. The studyalso relied on ICD-10 codes in ED electronic medical records to identify the targetand comparison outcome series. We are not aware of studies assessing the validity ofour ICD-10 classifications for the schizophrenia, cannabis-induced psychosis,alcohol-induced psychosis, or amphetamine-induced psychosis outcomes in our2015–2019 Alberta or Ontario NACRS records in relation to well-validated diagnosticstandards. Nonetheless, prior research has shown that a slightly broader ICD-10classification of psychotic conditions including schizophrenia and other relatedsyndromes  drawn from Alberta hospital-based administrative medical records hadsubstantial levels of accuracy and concordance vis-à-vis expert medical-chart-reviewdiagnoses .Cannabis Act mandated a public review of the healthbenefits and harms associated with legalization, the current study aims not only tooffer initial evidence for this Canadian evaluation, but also to provide preliminarysupport for similar discussions of the cost–benefit calculus in other jurisdictions.Future research will need to collect similar population-based cannabis-consumptionand psychosis-outcome data over longer periods in different settings in order toreach firm conclusions regarding the potential impacts of cannabis legalization ontransient and persistent psychotic syndromes in society.Despite these limitations, we suggest that the study has a number of strengths, whichsupport its contribution to the existing literature. Using population-basedprovincial ED records from Alberta and Ontario  and a quasi-experimental time-series design, thestudy provides a novel examination of the possible association between theimplementation of cannabis legalization and population-based patterns of transientand persistent psychotic conditions—a central topic frequently discussed in reviewsof the potential harms (and benefits) associated with legalization. In conclusion,the project showed no evidence of relations between Canada's first phase of cannabislegalization and ED presentations for cannabis-induced psychosis orschizophrenia-related conditions in the 14.5-month post-legalization period. Giventhat the The Canadian Institute of Health Information provided the study data to the leadauthors  under strict confidentiality and data-security constraints. Asa result, the current study data cannot be released publicly with themanuscript. Nonetheless, study data are publicly available to qualifiedresearchers through an application process to the Canadian Institute of HealthInformation. The lead author (RCC) can provide to qualified researchers detailsfor supporting CIHI data requests for the data contained in this study.Click here for additional data file.Supplemental material, sj-docx-1-cpa-10.1177_07067437211070650 for AssociationsBetween Canada's Cannabis Legalization and Emergency Department Presentationsfor Transient Cannabis-Induced Psychosis and Schizophrenia Conditions: Ontarioand Alberta, 2015–2019 by Russell C. Callaghan, Marcos Sanches, Robin M. Murray,Sarah Konefal, Bridget Maloney-Hall and Stephen J. Kish in The Canadian Journalof Psychiatry


Long-term symptoms are frequent after coronavirus disease 2019 (COVID-19). We studied the prevalence of post-acute myocardial scar on cardiac magnetic resonance imaging (CMR) in patients hospitalized due to COVID-19 and its association with long-term symptoms.In this prospective observational single-center study, 95 formerly hospitalized COVID-19 patients underwent CMR imaging at the median of 9 months after acute COVID-19. In addition, 43 control subjects were imaged. Myocardial scar characteristic of myocardial infarction or myocarditis were noted from late gadolinium enhancement images (LGE). Patient symptoms were screened using a questionnaire. Data are presented as mean ± standard deviation or median (interquartile range).The presence of any LGE was higher in COVID-19 patients  as was the presence of LGE suggestive of previous myocarditis . The prevalence of ischemic scar was comparable . Only two COVID-19 patients (7%) had myocarditis scar combined with left ventricular dysfunction (EF <50%). Myocardial edema was not detected in any participant. The need for intensive care unit (ICU) treatment during initial hospitalization was comparable in patients with and without myocarditis scar . Dyspnea, chest pain, and arrhythmias were prevalent in COVID-19 patients at follow-up  but not associated with myocarditis scar on CMR.Myocardial scar suggestive of possible previous myocarditis was detected in almost one-third of hospital-treated COVID-19 patients. It was not associated with the need for ICU treatment, greater symptomatic burden, or ventricular dysfunction at 9 months follow-up. Thus, post-acute myocarditis scar on COVID-19 patients seems to be a subclinical imaging finding and does not commonly require further clinical evaluation. Myocardial injury is common among patients hospitalized due to coronavirus disease 2019 (COVID-19) , 4. In rCardiac magnetic resonance (CMR) imaging is the gold standard of the non-invasive detection of myocardial diseases due to its ability to detect both myocardial inflammation and scar , 9 accurIn this prospective observational study, we evaluated the prevalence of myocardial scar, myocardial inflammation, and cardiac dysfunction in patients hospitalized due to COVID-19 after nine months of follow-up. We screened study participants for cardiovascular symptoms to evaluate the clinical significance of non-ischemic left ventricular scar suggestive of previous myocarditis in particular. The study’s primary aim was to investigate the prevalence of CMR abnormalities in COVID-19 patients and whether they are associated with a long-term symptomatic burden at long-term follow-up in COVID-19 patients.-type natriuretic peptide (NT-ProBNP), C-reactive protein (CRP) and serum creatinine. All study participants gave written informed consent for participating the study. The study protocol was approved by the Helsinki University Hospital’s ethics board of the Ethics board of Helsinki University Hospital (decision number HUS-1949-2020), and institutional permission was granted by the Department of Anesthesiology, Intensive Care and Pain Medicine (decision number HUS/200/2020). The study protocol is registered at clinicaltrials.gov (NCT04864938). The patients and control subjects were treated according to the principles of the Declaration of Helsinki.This is a prospective observational study conducted between November 2020 and September 2021 in the Helsinki University Hospital. Patients hospitalized for a confirmed (polymerase chain reaction or SARS-CoV-2 antibody test) COVID-19 between March and December 2020 were recruited six months after hospital discharge. Eligible intensive care survivors and patients from a pulmonology ward and follow-up outpatient clinic were invited to participate in the study. A control group with no history of COVID-19 was recruited by media announcements. Our inclusion criteria were Finnish as the language of business and age ≥18. Exclusion criteria for the study were pregnancy, severe renal failure and previous allergic reaction to the gadolinium contrast, severe claustrophobia, and ferromagnetic implants affecting CMR imaging. Due to the concomitant participation in a neuropsychological substudy, previous major neurological comorbidity, neurodegenerative disease, developmental disability, and impaired hearing and vision were also exclusion criteria to the study. Clinical and laboratory variables at the time of hospitalization were collected from the electronic health records. At the time of CMR imaging, patients were screened for cardiovascular symptoms using a questionnaire, and they underwent laboratory testing, including high-sensitivity troponin T (hs-TnT), N-terminal pro BThe imaging studies were conducted using a standard 1.5T CMR-scanner  according to international guidelines . LGE imaVentricular volumes and left ventricular mass were traced from short-axis cine images with papillary muscles included in the blood pool. The Left and right atrial areas were traced on the 4-chamber image. Cardiac chamber volumes were normalized to the body-surface area. Myocardial edema was assessed using short Tau inversion recovery (STIR) imaging and T2-mapping , 12. TheCategorical variables are reported as numbers and percentages. Continuous variables are reported as mean ± standard deviation and median (interquartile range) for normally distributed and skewed data. The statistical comparisons were made in COVID-19 patients with and without different subtypes of LGE and in patients with and without COVID-19 hospitalization. The sample size of the study was assessed post-hoc by the method described by Fleiss et al . The stuThe study population consisted of 95 patients hospitalized due to COVID-19 and 43 control subjects . A totalAll patients underwent CMR imaging after a median of 9 months (range 7–10 months) of their initial COVID-19 hospitalization. Their imaging results compared to the control group are reported in Clinical, laboratory and CMR variables in COVID-19 patients with and without a myocarditis scar are shown in Long-term symptoms in COVID-19 patients and in control subjects were assessed at a median of 9 months (range 7–10 months) after hospitalization at the time of CMR imaging. As shown in This prospective study demonstrates that left ventricular LGE on CMR is a frequent finding in patients previously hospitalized due to COVID-19. Interestingly, almost one-third of these patients had a myocardial scar suggestive of post-acute previous myocarditis, while the presence of an ischemic scar was less prevalent. The myocarditis scar was not related to sustained left ventricular dysfunction, ICU treatment in the acute phase, or higher symptomatic burden by the time of imaging. Moreover, none of the patients had signs of ongoing inflammation on CMR at follow-up. Thus, our results provide reassurance to clinicians since reported myocarditis-like LGE on CMR in COVID-19 patients seems to be mostly a subclinical finding that does not require further evaluation.A few CMR studies predate our own with variable sample sizes and imaging times relative to the COVID-19 infection. As in our study, the non-ischemic LGE pattern suggestive of previous myocarditis has been common in COVID-19 patients. Two larger studies by Puntmann et al. and Kotetcha et al. had 100 and 148 patients with risk-factor matched controls and were conducted 2–3 months after COVID-19 . PuntmanOur study explored the presence of dyspnea, chest pain, syncope, and arrhythmia symptoms after the initial COVID-19 hospitalization and their relation to CMR imaging findings. CMR is an excellent modality for evaluating the cardiac origin of these symptoms, their differential diagnosis, and risk stratification . It can Cardiovascular symptoms and decreased exercise tolerance have been common after COVID-19, which is also a risk factor for developing new cardiovascular diseases –7. AlthoWe did not find signs of ongoing myocardial inflammation in our patients, and a troponin elevation at follow-up was rare. Thus, our study suggests that the clinical yield for CMR screening of unselected COVID-19 population for previous myocarditis would be low. In addition, a positive imaging finding may be unrelated to the clinical symptoms, which might be more commonly explained by other reasons, such as pulmonary sequelae of COVID-19. Sustained troponin elevation or depressed left ventricular function may be more appropriate indications for CMR imaging according to standard clinical practice, and the possibility of COVID-19-induced autoimmune diseases such as cardiac sarcoidosis should not be forgotten .It should be noted that our study population underwent CMR at a post-acute phase, and we were unable to diagnose myocarditis according to the prevailing Lake-Louise criteria . HoweverMyocardial scar suggestive of previous myocarditis was detected in one-third of hospital-treated COVID-19 patients, but it was not associated with greater symptomatic burden, need for ICU treatment, sustained ventricular dysfunction or active myocardial edema at follow-up. Thus, myocarditis-like LGE on CMR after COVID-19 hospitalization seems to be of little clinical significance.S1 Checklist(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S2 Protocol(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Aging is associated with defects within blood stem cells, termed hematopoietic stem cells (HSC), including a loss of their self-renewal potential and a skewed differentiation towards myeloid lineages at the expense of lymphoid cells. Collectively, these HSC defects manifest as anemias, poor response to vaccines and an increased incidence of myeloid neoplasms in older adults. Unlike other somatic stem cells, aged HSCs have been shown to be refractory towards established anti-aging interventions including caloric restriction, exercise, parabiosis and plasma transfer. Thrombospondin-1 (TSP1) was initially discovered as an anti-angiogenic molecule, and recent studies have identified that TSP1 promotes age-related pathologies including chronic inflammation, reactive oxygen species (ROS) generation, and mitochondrial dysfunction. Notably, each of these TSP-1 regulated processes have been shown to critically influence HSC biology, particularly in the context of aging. However, whether TSP-1 directly regulates HSC activity remains unexplored. Here, we sought to determine whether TSP-1 is essential for HSC development, and whether blocking TSP1 signaling could ameliorate age-related HSC defects. Utilizing murine models, we demonstrate that TSP-1 is dispensable for normal HSC development and hematopoiesis. We show that deletion of TSP-1 is sufficient to preserve HSC fitness during aging, as evidenced by preservation of youthful self-renewal potential and balanced lineage reconstitution during serial HSC transplantation assays. Mechanistically, we identify that TSP-1 adversely impacts mitochondrial metabolism within HSCs, and show that loss of TSP-1 prevents the age-related decline in HSC mitochondrial membrane potential. Our findings identify TSP-1 as a pro-geronic factor that can be targeted to preserve HSC healthspan.


Immunotherapy is one of the promising strategies in the treatment of oncology. Immune checkpoint inhibitors, as a type of immunotherapy, have no significant efficacy in the clinical treatment of patients with pMMR/MSS/MSI-L mCRC alone. Therefore, there is an urgent need to find combination therapies that can improve the response rate of immune checkpoint inhibitors. Oncolytic viruses are a new class of cancer drugs that, in addition to directly lysing tumor cells, can facilitate the action of immune checkpoint inhibitors by modulating the tumor microenvironment and transforming “cold” tumors into “hot” ones. The combination of oncolytic viruses and immune checkpoint inhibitors is currently being used in several primary and clinical studies to treat tumors with exciting results. The combination of genetically modified “armed” OV with ICIs is expected to be one of the treatment options for pMMR/MSS/MSI-L mCRC. In this paper, we will analyze the current status of oncolytic viruses and ICIs available for the treatment of CRC. The feasibility of OV in combination with ICI for CRC will be discussed in terms of the mechanism of action of OV in treating tumors. Colorectal cancer (CRC) is one of the most common and deadly cancers in the world community. It is the second most common cancer in women and the third most common in men. In 2018, there were 1.8 million CRC cases and 880,792 deaths worldwide, and CRC incidence is increasing in people under 50 years of age (Immune checkpoint inhibitors (ICIs) are one of the popular immunotherapies for treating tumors in recent years, and they have shown exciting results in treating melanoma . Immune Oncolytic viruses (OVs) are a novel class of tumor therapeutic strategy to reduce tumors through preferential replication in tumor cells and stimulate host anti-tumor immunity to promote the lysis of tumors. There are many DNA and RNA viruses that can be used as OVs. At present, the viruses most commonly used in experimental cancer research are poxviruses, reoviruses, herpes simplex viruses (HSV), and adenovirus . The theWith the marketing approval of the first oncolytic virus, Talimogene laherparepvec (T-VEC), for the treatment of advanced melanoma, oncolytic virus immunotherapy has been used in routine clinical oncology research . The comVaccinia virus (VV) has a safety profile in humans as a smallpox vaccine. Recombinant VV has been used as an expression vector to enhance the tumor lytic effect and has been widely used in clinical trial studies in tumor models and patients with advanced solid cancers .via multiple apoptotic signaling pathways.Researchers  construcColorectal cancer (CRC) frequently causes the spread of tumor cells within the peritoneal cavity, eventually leading to peritoneal carcinoma (PC) . PeritonOncolytic reovirus (pelareorep) is a non-enveloped dsRNA virus that selectively lyses KRAS-mutated colorectal tumor cells. KRAS mutations are prevalent in 40-45% of colorectal cancer (CRC) patients, and treatment options are limited. One study found that , pelareoHerpes simplex viruses can be classified into types I and II . Related researchers have genetically modified HSV-1 to express IL-12. HSV-1 expressing IL-12 promotes the cytolytic activity of natural killer cells and cytotoxic T lymphocytes, infects and kills colorectal cancer cell lines, and promotes anti-tumor immunity . oHSV-2 Adenovirus is the more commonly used oncolytic virus vector, and the genome can be easily genetically modified to achieve better therapeutic results . At presin vivo, so it has been proposed as a potential oncolytic virus vector. Studies have demonstrated that ORFV strain NA1/11 can inhibit CRC growth and metastasis by inducing apoptosis in CRC cells (.An oncolytic measles virus (OMV) encoding IL-12 can effectively induce apoptosis in rectal colon cancer cells by activating the IL-12/IFN-c/TNF-a inflammatory response . Orf virRC cells . In addiRC cells  producedRC cells . On thisRC cells   increase T-cell activity and the ability to kill tumor cells by inhibiting these immune checkpoint activities. At present, ICIs have been shown to have a considerable improvement in the prognosis of patients with microsatellite instability-high (MSI-H)/DNA mismatch repair (dMMR) deficient (MSI-H/dMMR) tumors. In parallel, a series of clinical trials have demonstrated that patients with MSI-H/dMMR mCRC can be treated with Pembrolizumab and nivolumab monotherapy or nivolumab combined with Ipilimumab .Programmed cell death-1 (PD-1) is a transmembrane glycoprotein expressed in activated T cells, and programmed cell death ligand 1 (PD-L1) is expressed on antigen-presenting cells and tumor cells. In the tumor microenvironment, the release of cytokines  can induce high levels of PD-L1 expression. At the same time, the binding of PD-1 to PD-L1 can induce tumor cells to evade immune surveillance by inhibiting T-cell activation and proliferation . TherefoCurrently, the main PD-1 inhibitors used in clinical studies for treating colorectal cancer are Pembrolizumab and Nivolumab. Pembrolizumab is currently approved by the US Food and Drug Administration (FDA) for the first-line treatment of unresectable or metastatic colorectal cancer with high microsatellite instability or mismatch repair defects .A KEYNOTE-164 clinical phase II study enrolled 124 patients with MSI-H/dMMR CRC receiving≥2 standard treatments. The results showed that in cohort A , the ORR was 33%, including 2 CRS; In cohort B (≥ 1 previous treatment), the ORR was 33%, including 5 CRS. This study confirms the anti-tumor effects and clinical efficacy of Pembrolizumab in previously treated MSI-H/dMMR colorectal cancer patients . In the Growing tumors show one of three patterns: (1) little or no tumor-infiltrating immune cell infiltration (“immune ignorance “). (2) the presence of intra-tumoural immune infiltration with minimal or no PD-L1 expression . (3) Immune infiltration that resides only around the outer edge of the tumor cell mass (“excluded infiltrate “). However, most treatment patients showed a lack of PD-L1 upregulation in tumor cells or tumor-infiltrating immune cells . TherefoCytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin-associated receptor family, and the binding of CTLA-4 to its ligand B7-2 protein inhibits T-cell proliferation activation and cytokine production while decreasing the immune response . TherefoIpilimumab, a fully human immunoglobulin G1 monoclonal antibody capable of blocking CTLA-4, was approved by the FDA in July 2018 for the immune combination treatment of MSI-H mCRC after progression on standard chemotherapy. In a large combination immunotherapy study, the CheckMate-142 trial, 119 MSI-H/dMMR CRC patients were treated with Ipilimumab combined with the PD-1 inhibitor Nivolumab and had an investigator-assessed ORR of 55%  and a 12-week disease control rate of 80%. This demonstrates that Nivolumab and Ipilimumab synergistically promote T-cell anti-tumor activity through complementary mechanisms of action, which provides a clinical experimental basis for CTLA-4 inhibitors combined with PD-1 inhibitors in the treatment of MSI-H/dMMR mCRC . TherefoSo far, only immunotherapies with two checkpoint targets, CTLA-4 and PD-L1/PD-1, have been approved for clinical use. However, most cancer patients do not respond to treatment with PD-1/PD-L1 inhibitors or CTLA-4 inhibitors. Therefore, as research progresses, several promising novel immune checkpoint targets are emerging as breakthrough points for cancer immunotherapy.T-cell immunoglobulin and mucin-containing structural domain protein 3 (TIM-3) is a suppressive immune checkpoint molecule. TIM-3 has a suppressive effect on T-cell-induced immune responses. Blocking TIM-3 reverses T-cell dysfunction and restores anti-tumor immunity. Co-blockade of TIM-3 and PD-1 improves anti-cancer T cell responses in patients with advanced cancer . IncreasTIGIT (T-cell immunoglobulin and immunoreceptor tyrosine motif (ITIM) structural domain) is a novel immune checkpoint. TIGIT is expressed on CD4 and CD8 T cells as well as innate lymphocytes, including NK cells and γδ T cells . PD-1 anLAG-3 is an Ig-like structural domain type I transmembrane protein with four structural domains called structural domain 1 (D1) to structural domain 4 (D4). Currently, LAG-3 is a promising therapeutic target for cancer immunotherapy. LAG-3 acts similarly to PD-1 and helps tumor cells to undergo immune escape . One stuCurrently, after the research results of PD-1 inhibitors and CTLA-4 inhibitors in the treatment of CRC, novel immune checkpoints such as TIM-3, TIGIT, and LAG-3 are not only potential biomarkers for diagnosis, prognosis, and survival prediction of CRC patients but also the next breakthrough point in immunotherapy for CRC. There are significant interactions between multiple immune checkpoints in the pathogenesis of CRC. Blocking one immune checkpoint alone may result in compensatory upregulation of other checkpoints. Therefore, combined blockade of multiple immune checkpoints may be a new immunotherapeutic strategy for CRC patients .The tumor microenvironment (TME) refers to the complex multicellular environment in which tumors develop and typically includes T and B lymphocytes, tumor-associated macrophages (TAM), dendritic cells (DC), natural killer (NK) cells, neutrophils, and bone marrow-derived suppressor cells (MDSC) . The proOncolytic viruses are able to induce lysis of tumor cells, which releases tumor-associated antigens and neoantigens (TAAs and TANs) that can be captured by tumor-infiltrating antigen-presenting cells (APCs), resulting in increased T cell infiltration . OncolytIn addition to the release of DAMPs, OV-mediated cancer cell lysis is often associated with the release of various pathogen-associated molecular patterns (PAMPs), including viral components such as nucleic acids , proteins and capsid components. DAMPs and PAMPs are recognized by pattern recognition receptors on antigen-presenting cells (APCs) (such as DC and NK cells) and serve as “danger” and “eat me” signals. This signaling attracts more DCs to the TME, leading to increased recruitment and maturation of tumor-specific T cells into the TME . Thus, o.A clinical trial using transgenic herpes simplex virus type 1  in combination with pembrolizumab in patients with advanced melanoma showed that OVs might make tumor cells more sensitive to immune checkpoint inhibitors by up-regulating PD-L1 expression . In addiTo date, oncolytic viruses as monotherapy have provided only modest anti-tumor effects in patients. Therefore, in order to increase immune stimulation, OVs have been designed to provide therapeutic transgenes to “armed” oncolytic viruses . OVs canResearchers built an “armed” oncolytic vaccinia virus (VV-iPDL1/GM) co-expressing PD-L1 inhibitor and GM-CSF. They found that intra-tumor injection with these engineered OVs promoted tumor infiltration of neoantigen-specific T cells while activating immune cells. VV-iPDL1/GM-secreted PD-L1 inhibitors inhibited neoantigen presentation of PD-L1 on tumor cells, leading to the systemic rejection of both the treated tumor and distant tumors. Therefore, using “armed” OVs co-expressing PD-L1 inhibitors and GM-CSF alone or combined with immune checkpoint inhibitors is a new therapeutic option for treating patients resistant to PD-1/PD-L1 blockade therapy . An M51RIn a study, researchers encoded a humanized anti-PD-1 monoclonal antibody  and designed a novel herpes simplex virus (HSV-aPD-1). Data from this experiment suggest that HSV-aPD-1 further improves the immune microenvironment and plays a crucial role in treating tumors that are not sensitive to ICIs or other immunotherapies . In addiThe degree of inflammation in the tumor microenvironment affects the overall and progression-free survival rates. “Armed” OVs carrying the gene encoding inflammatory cytokines can further modify the tumor microenvironment. A study showed that a recombinant oncolytic poxvirus virus (hIL-7/mIL-12-VV) dually expressing IL-7 and IL-12 completely altered the tumor immune microenvironment by enhancing the inflammatory immune state, showing beneficial systemic anti-tumor efficacy, significantly increasing the sensitivity of solid tumors to systemic anti-PD-1 and anti-CTLA4. This combined IL-7 and IL-12 viral therapy could provide clinical benefit as a single therapy and has the potential to be effectively combined with immunotherapy for various types of solid tumors .The anti-tumor efficacy of an engineered recombinant oncolytic herpes simplex virus (ONCR-177) with five transgenes was enhanced by systemic anti-PD-1 treatment. Experimental data showed that the RR of contralateral tumors treated with combination therapy was significantly enhanced by 40% RR compared with oncr-171 or anti-PD-1 monotherapy. Thus, ONCR-177 enhances tumor lysis tolerance and anti-tumor activity through activation of systemic immunity triggered by transgene expression and can be further enhanced by co-treatment with immune checkpoint inhibitors . It has .In addition, a novel PD-L1 ICI with a cross-hybrid Fc region-mediated effector mechanism of IgG and IgA was designed. It is cloned as a conditionally replicating adenovirus (Ad-Cab) to limit virulence and release only into the tumor microenvironment. Ad-Cab secretes a cross-hybrid IgGA Fc fusion peptide that binds to PD-L1 binding and activates multiple immune pathways. Therefore, designing a novel ICI and expressing it with an oncolytic adenovirus could enhance tumor-killing efficacy while maintaining safety  /DNA mismatch repair specialists (pMMR) (MSS/pMMR), and ICIs have not shown any clinical benefit in treating MSS/pMMR mCRC .The oncolytic virus can enhance the response rate of tumors to immune checkpoint inhibitors. It is an ideal candidate for use in combination treatment strategies. Therefore, immune checkpoint inhibitors combined with the oncolytic virus may be a potential therapeutic option for treating MSS/pMMR mCRC.Currently, experimental studies have shown that the use of oncolytic virus combined with immune checkpoint inhibitors has a better therapeutic effect on colorectal cancer. In an experimental study of triple immune therapy  in a mouse model of CRC, 43% of the mice treated with the triple therapy achieved CR tumors and prolonged OS, and there was no tumor growth in 10 months from the implantation of CRC cancer cells to the end of the experiment. The researchers also observed a similar reduction in tumor load when T cells from surviving mice were treated with triple therapy and fed into the untreated CRC mouse model. Thus, this triple immune therapy therapeutic strategy could potentially overcome low T-cell infiltration and TAMs while overcoming the limitations of PD-1 immunosuppression. This could significantly improve the therapeutic efficacy of anti-PD-1 immunotherapy in colon cancer .in situ rectal tumor model with liver metastases, which was injected intra-tumorally with OBP-502 and treated in combination with PD-1 Ab. The results showed that this combination therapy inhibited liver metastases and rectal tumors. Thus, telomerase-specific oncolytic adenovirus virus led to a celiac effect by activating a systemic anti-tumor immune response, and the combination with PD-1 Ab produced a synergistic anti-tumor effect, even leading to tumor eradication  in which the human telomerase reverse transcriptase (hTERT) promoter element drives the expression of the viral E1A and E1B genes. This genetic modification allows OBP-301 to replicate in tumor cells and induce tumor cell-specific death selectively. In parallel, the researchers developed a CT26 dication .In addition, another trial further enhanced the sensitivity of immune checkpoint inhibitors in a mouse model of dMMR CRC by using a combination of low-dose mitomycin C (mitomycin) and oHSV. It was also found that this treatment strategy promoted the infiltration of activated conventional dendritic cells type 1 (cDC1s) into the tumor .The most common route of oncolytic virus therapy is intra-tumor or intravenous administration, but its use in clinical practice is limited. Researchers have developed an orally available oncolytic reovirus, RC402, which breaks through the limitations of the use of lytic viruses in clinical treatment. Oral RC402 monotherapy significantly increased CD8+ cytotoxic T cells and decreased CD4+CD25+Foxp3+ regulatory T cells in distant colon cancer. At the same time, the combination of RC402 and PD-1 inhibitor further inhibits colon cancer growth and enhances anti-tumor immunity within the tumor microenvironment, leading to complete tumor regression. Therefore, combining RC402 with PD-1 inhibitors could maximize the efficacy of PD-1 immune checkpoint blockade in colon cancer and trigger an effective anti-tumor immune response ..In a clinical trial, 15 patients with refractory colorectal cancer received intravenous oncolytic vaccinia virus Pexa-Vec  every 14 days. This is the first treatment for patients with refractory, metastatic colorectal cancer using repeated doses of Pexa-Vec and administered by intravenous infusion. Ten patients (67%) had imaging stable disease. This clinical trial showed safety against the vaccinia virus in patients given repeated intra-tumor doses every two weeks . In CRC In recent years, immune checkpoint inhibitors (ICIs) have become a promising strategy for treating tumors. However, in treating colorectal cancer (CRC), ICIs have shown promising results only in MSI/dMMR CRC and are very limited for treating pMMR/MSS/MSI-L mCRC. Oncolytic viruses can directly lyse tumor cells and increase T-cell infiltration by activating immunogenic cell death (ICD) and releasing danger-associated molecular patterns (DAMPs) to recruit and promote DC maturation. At the same time, OVs promote the action of immune checkpoint inhibitors by modulating the tumor microenvironment (TME) and transforming ‘cold’’ tumors into ‘hot’’ ones. Thus, combining oncolytic virus therapy with immune checkpoint inhibitors may break through the limitations of ICIs treatment MSS/pMMR mCRC and enhance the sensitivity of colorectal tumor cells to ICIs.Currently, various oncolytic viruses such as vaccinia virus (VV), reoviruses, herpes simplex virus (HSV), and adenovirus have been widely used in experimental studies of colorectal cancer. Studies have demonstrated that OVs can inhibit the growth and distant metastasis of colorectal cancer cells by lysing CRC cells, inducing apoptosis, and reversing TME, and have shown exciting results in treating CRC peritoneal metastases and KRAS-mutated colorectal cancer. The combination of OVs and ICIs has become the most promising strategy for treating solid tumors today. Many researchers have used OVs as a therapeutic platform to achieve better therapeutic results, using viral genome engineering techniques to make OVs carry PD-1/PD-L1 antibody genes or other therapeutic genes, creating “armed” OVs. These “armed” OVs have a complementary mechanism to ICIs in terms of anti-tumor immunity, enhancing the targeted delivery of OVs, further increasing T-cell infiltration, and providing comprehensive regulation of TME and the immune system resulting in a more substantial anti-tumor immune response rate. Therefore, combining “armed” OVs and ICIs may be one option for treating pMMR/MSS/MSI-L mCRC.However, although the combination of OVs and ICIs has shown therapeutic efficacy in animal models of CRC, the specific treatment and prognosis of this combination therapy for CRC patients is unclear due to the lack of clinical trial data on OVs in combination with ICIs in CRC patients. As the immune response is a complex and highly regulated biological process, the efficacy of OVs in combination with ICIs in animal models of CRC cannot be fully replicated in patients with CRC. However, the results of several animal studies and clinical trials in other solid tumors suggest that OVs and ICIs combination therapy remains a very promising strategy in treating tumors. In addition, antagonism and the occurrence of immune-related adverse events (irAEs) need to be considered when combining OVs with ICIs.At present, it remains challenging to get OVs to every primary and metastatic tumor site to achieve the desired effect. In distant metastatic lesions with low T-cell infiltration, the therapeutic effect of OVs combined with ICIs may be minimal. Therefore, there is a need to continue the search for a more accurate and effective immunomodulatory factor to control OV-mediated anti-tumor T-cell responses .In conclusion, the treatment of colorectal cancer is limited by the limited treatment strategies available. Currently, the combination of oncolytic viruses and immune checkpoint inhibitors has shown promise in several clinical trials. This strategy is expected to be a promising treatment option for colorectal cancer. It is believed that in the near future, the combination of OVs and ICIs will bring hope to pMMR/MSS/MSI-L mCRC patients through clinical trial studies.Conceptualization: YR and X-BK; writing—review and editing: YR, J-MM, and Y-YW; visualization: ZF; supervision: X-BK and LY; project administration: GC. All authors have read and agreed to the published version of the manuscript.This research was funded by the Scientific research project of the Tianjin Education Commission, grant number 2021KJ134; Tianjin Municipal Education Commission Scientific Research Project, grant number 2019ZD11.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. 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Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although many reports have detailed a range of neurological symptoms in SARS-CoV-2-infected patients, studies of neuro-ophthalmological manifestations are still scarce.We report a 9-year-old girl with abducens nerve palsy after COVID-19 with no evidence of other neurological disease on neuroimaging. At 2-month follow-up clinical conditions were improved.The palsy may have occurred due to a possible post-infectious immune-mediated mechanism underlying the neuropathy, as opposed to direct viral infiltration. Despite being rare, this complication must be taken into account. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in late December 2019 in Wuhan, China [Clinical presentation and severity of COVID-19 vary significantly from asymptomatic subjects to patients with severe and sometimes fatal disease .Although the majority of patients with SARS-CoV-2 infection have fever and respiratory symptoms like dry cough, fatigue and shortness of breath, as well as myalgia and anosmia , 3, 4, tNumerous evidences support the neurotropic and neuro-invasive potential of SARS-CoV-2 , which cAcute cranial nerve (CN) paresis, including abducens and oculomotor nerve involvement, have been already reported among adults and young adults, but the VI nerve palsy is not well described in children –15.In this report, we describe a 9-year-old female patient who presented isolated abducens nerve palsy after COVID-19, with no evidence of other neurological disease on neuroimaging and absence of preexisting vascular risk factors.We report on a 9-year-old girl with acute onset of persistent diplopia seventeen days after a mostly asymptomatic SARS-CoV-2 infection. The main symptom reported during the course of the disease was headache, but there was no meningismus. The diagnosis was confirmed using a molecular nasopharyngeal swab that detected the presence of SARS-CoV-2 RNA by real-time reverse transcriptase polymerase chain reaction (PCR). The test resulted negative after 10 days from the beginning of the infection.An intercurrent episode of fever (temperature 38.5 °C) lasting 72 hours was reported one week before the onset of the eye disorder. The patient’s parents noticed the sudden onset of convergent strabismus of the right eye while the child was using her mobile phone. She had no pain with eye movements or other neurologic symptoms such as weakness, ataxia, paresthesia and hyporeflexia or anosmia/ageusia.The first clinical examination at the Pediatric Department Child Neurology Unit revealed complete and isolated failure of abduction of the right eye. The horizontal diplopia, persisted in the primary gaze and was exacerbated by distant fixation Fig. . Ocular On ophthalmologic examination her best-corrected visual acuity was normal in both eyes, no afferent pupillary defect nor nystagmus was observed. The colour vision (Hishihara plates), slit lamp examination and visual field were normal in both eyes. Ocular motility examination revealed an incomitant esotropia (convergent squint) with limitation of abduction in the right eye and abnormal position of the head when turned to the right shoulder. Horizontal binocular vision with image separation was typically worse at long distance than at near distance, and worse for right gaze. The fundus of the eye was normal.Magnetic resonance imaging (MRI) of the brain was performed with contrast-enhanced multiplanar reconstruction study, revealing normal findings.Lumbar puncture showed a normal opening pressure and normal cerebro-spinal fluid (CSF) components. The search for the main neurotropic viruses including the analysis for SARS-CoV-2 RNA as well as for oligoclonal bands was negative.Routine blood tests were normal including complete blood count, coagulation, inflammatory markers, liver and kidney function.The patient was discharged after 48 hours of observation with no change in double vision.After the collegial discussion of the case no treatment was undertaken.At one week follow-up, the patient reported the persistence of diplopia with a fluctuating course. Patching of the good eye was advised and performed. At 2-month follow-up, the patient had improved binocular vision and reduced eye deviation.The aetiology of sixth nerve palsy can be congenital or acquired. The most common cause of acquired abducens palsy in children has been found to be neoplasm, unlike the more common vascular aetiology in adults , 17. TheRegarding SARS-CoV-2, while the amount of data published on central and peripheral neurological manifestations is continuously growing, few studies describe the specific neuro-ophthalmological manifestations . These iWe therefore decided to report the case of a young girl with diplopia caused by VI nerve palsy, investigating the possible etiopathogenetic relationship with primary SARS-CoV-2 infection.The neurotropism of SARS-CoV-2 , 26 is mDirect viral involvement through retrograde or anterograde transport mechanisms from peripheral nerves to the CNS .A route of hematogenous dissemination could provide entry into the CNS for SARS-CoV-2 by three mechanisms: 1) Transcellular migration that involves the binding of the virus to its receptors  on brain microvasculature endothelial cells and subsequent crossing of the endothelial cells via transcytosis , 28; 2) A systemic inflammatory response (including autoinflammatory and hyperinflammatory responses) associated with the deleterious role of the cytokine storm in disrupting the BBB .An immune-mediated para-infectious or post-infectious effect; it is believed that SARS-CoV-2 has auto-immunogenic effects mainly via molecular mimicry, leading to autoimmune neuropathies , as demonstrated in cases of GBS and its variants MSF .The proposed pathogenetic mechanisms to explain SARS-CoV-2 neuro-invasiveness and neuro-virulence include:In our case, the primary infection was asymptomatic and the neurological disorder appeared after about 2 weeks, suggesting a post-infectious immune-mediated mechanism underlying the neuropathy rather than direct viral infiltration.Additionally, besides diplopia, the neurological examination was completely negative (no strength deficit and good evoked reflexes were found) and the level of proteins in the CSF was normal, reducing the likelihood that it was a GBS variant. Even if in this case the anti-ganglioside antibodies have not been tested, the Brain MRI revealed normal findings.In light of these considerations, virus-induced immune-mediated neuropathy may be the most likely pathogenic mechanism.Further studies are needed on a larger population to verify the association between SARS-Cov-2 infection and neuro-ophthalmological disorders in order to clarify the exact etiopathogenesis.