The convergence of the HIV and SARS-CoV-2 pandemics is an emerging field of interest. In this review, we outline the central nervous system (CNS) effects of COVID-19 in the general population and how these effects may manifest in people with HIV (PWH). We discuss the hypothetical mechanisms through which SARS-CoV-2 could impact the CNS during both the acute and recovery phases of infection and the potential selective vulnerability of PWH to these effects as a result of epidemiologic, clinical, and biologic factors. Finally, we define key research questions and considerations for the investigation of CNS sequelae of COVID-19 in PWH. As the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic continues, millions of individuals worldwide remain at risk for coronavirus disease 2019 (COVID-19). The convergence between the HIV and SARS-CoV-2 pandemics is an emerging field of interest . AttentiHIV infection, even when well-controlled with antiretroviral therapy (ART), has long-term implications on health through mechanisms related to viral persistence, chronic inflammation, and incomplete immune reconstitution –11. The A wide range of neurologic disorders has been observed in acute SARS-CoV-2 infection. However, most studies have focused on hospitalized patients, and less is known about the prevalence and spectrum of neurologic symptoms in COVID-19 patients who do not require hospitalization. In a UK surveillance study of 125 case notifications for neurologic and psychiatric complications among hospitalized COVID-19 patients over a 3-week period, altered mental status and cerebrovascular disease constituted the majority of syndromes reported . NeuroloIt may be challenging to differentiate encephalopathy due to the direct or indirect effects of SARS-CoV-2 infection from the impact of critical illness, prolonged ICU hospitalization, and other medical issues  on the brain. While some degree of encephalopathy is prevalent in hospitalized patients with COVID-19, encephalitis, defined as inflammation of the brain parenchyma by neuroimaging or cerebrospinal fluid [CSF] analysis, occurs infrequently –31. In bMounting evidence points to elevated cerebrovascular risk during acute SARS-CoV-2 infection. In a study of 1916 patients who were hospitalized or sought emergency care at a single center in New York City over 2 months, 31 (1.6%) had an acute ischemic stroke compared with 3 of 1486 influenza patients  . SimilarFollowing acute illness, an unknown but likely substantial number of people experience post-acute sequelae of SARS-CoV-2 infection (PASC) . InitialPersistent cognitive changes are a predominant feature of CNS PASC. People can experience new difficulties staying focused (attention), holding onto and manipulating information (working memory), remembering recent events that improves with cues (executive function), and slowed thinking (processing speed). Collectively, these symptoms suggest fronto-executive brain network dysfunction. Cognitive difficulties now reported in several cohorts –41, inclWhile there was initial concern that PWH would exhibit increased vulnerability to SARS-CoV-2 infection based on greater susceptibility to other respiratory pathogens , studiesOverlap or synergy in potential mechanisms driving neurologic complications of COVID-19 and HIV may place PWH with COVID-19 at higher risk for CNS dysfunction and/or injury , a protein present in myelinated axons of neurons , 92. SimPost-infectious autoimmunity: Given the infrequent detection of SARS-CoV-2 neuroinvasion, some data suggest that CNS injury or dysfunction is caused by immune-mediated mechanisms that occur secondary to systemic infection. A recent study found that 5 of 7 hospitalized COVID-19 patients with a spectrum of neurologic issues displayed evidence of CNS autoantibodies [tibodies . Furthertibodies •, 94–96,Neurovascular injury: The long-term projections for brain health and recovery after SARS-CoV-2 infection may be influenced by mechanisms driving neurovascular risk during acute infection, including a dysfunctional immunologic and endothelial response triggering cerebral microvascular injury and thrombosis [rombosis . Furtherrombosis , 98, cerThe proposed mechanisms by which SARS-CoV-2 may lead to elevated cerebrovascular risk may be particularly relevant for PWH. PWH contend with a heightened risk of cerebrovascular injury, attributed in part to chronic systemic immune activation and inflammation , 9. CereSyndemic comorbidities: Syndemic comorbidities in PWH, including substance use and metabolic disorders, may increase risk for contracting SARS-CoV-2 infection and lead to more severe disease and worse clinical outcomes [outcomes ••, 102. outcomes , 104, evoutcomes . PWH whooutcomes , which ioutcomes , 107. Amoutcomes –110, areoutcomes , could aPreliminary data and expert opinion suggest that one or more of several mechanisms might contribute to the potential neurologic effects of COVID-19 in PWH:more vulnerable to CNS effects of SARS-CoV-2 infection than the general population?Are PWH more severe or more persistent in PWH compared to people without HIV?Are CNS manifestations of COVID-19 during the acute or recovery phase potentiated in PWH?Are certain pathogenic mechanisms that underlie the CNS effects of SARS-CoV-2 infection mitigate the impact of neurologic complications of SARS-CoV-2 infection in PWH?Which interventions, including SARS-CoV-2 preventative or therapeutic interventions or HIV-targeting interventions, could Understanding the epidemiology and pathogenesis of neurologic complications of SARS CoV-2 infection will inform our approach to investigating potential CNS effects of COVID-19 in PWH. The optimal study approach will depend on the specific research questions that are of greatest interest to PWH and their providers. Although there are many potential questions related to CNS effects of COVID-19 in PWH -derived data can be used to measure neurologic effects of COVID-19 among those hospitalized during the acute period, variability in access to care, provider assessments, symptom screening, and participant reporting will complicate the use of these data for non-hospitalized individuals and for all individuals during the post-acute period. In addition, the quality of measurements is relevant. Most existing studies have queried participant symptoms through self-report or interview. Few have included objective testing, which will require a combination of structured assessments, neurologic examination, and neuropsychological testing. In our experience, routine cognitive screening tests developed for the detection of dementia, such as the Montreal Cognitive Assessment and Mini-Mental State Examination, may not utilize sensitive enough thresholds to detect the cognitive inefficiencies observed following COVID-19, particularly as this population includes a younger age range of patients . CollectIn line with the importance of studying population-based cohorts that reflect the distribution of demographic and socioeconomic factors in communities affected by COVID-19, these considerations are also germane to the investigation of potential CNS effects of COVID-19. Some of these factors have been associated not only with risk of SARS-CoV-2 infection but also with differential outcomes during both the acute and recovery phases of COVID-19 , 114–116To understand the mechanisms driving neurologic involvement of SARS CoV-2 infection and interactions in PWH, we will need longitudinal data on systemic and CNS inflammation, neuronal injury, the presence of autoantibodies, and other biologic factors beyond the acute infection period. In addition to blood measurements, CSF analyses may be critical. For example, some patients with COVID-19 and neurologic complications have displayed differential inflammatory profiles in CSF compared with plasma, suggesting compartmentalized CNS immunologic responses may contribute to neurologic disease . AvailabThrough intense community advocacy, PWH were included in initial SARS-CoV-2 vaccine trials –136, altThe convergence of the dual HIV and SARS-CoV-2 pandemics has highlighted the urgent need for research on potential interactions between these two infections. Much of our knowledge of how viruses impact the brain and brain function derives directly from HIV research. Leveraging research infrastructure and scientific methods developed over decades of effort in the fight against HIV has allowed for rapid, hypothesis-driven study of the CNS effects of COVID-19. Studies investigating the determinants of CNS effects of SARS-CoV-2 infection in PWH and whether HIV infection potentiates the mechanisms underlying these effects will further illuminate our understanding of virally-mediated CNS disease and could potentially improve the quality of life of millions of PWH worldwide.


As previously reported in various cell types, TGFβ/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFβ/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFβ/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFβ and hypoxia to human ventricular cardiac fibroblasts  Heart disease and subsequent heart failure (HF) is the major cause of death worldwide . HF primThe HIPPO pathway is a growth suppressor signaling pathway that prevents organ overgrowth by inhibiting cell proliferation and inducing differentiation or apoptosis . The keyCardiomyocyte-specific gain- and loss-of-function studies demonstrated that Yap promotes embryonic cardiomyocyte proliferation and supports cardiac homeostasis in adult mice by protecting cardiomyocytes from apoptosis and preventing heart fibrosis ,10,11,12In the current study, we aimed to determine the expression pattern of Yap in cardiac tissues from patients with LV HF. We further examined the role of Yap in proliferation and pro-fibrotic activation of human adult cardiac fibroblasts. Our data show that Yap appears to predominantly accumulate in cardiac fibroblasts of HF patients and that LV tissues from these patients present with down-regulation of signaling of the Yap major upstream inhibitor LATS1. We further provide evidence that cardiac fibroblast Yap is upregulated under pathophysiological stimuli and identify its role in human cardiac fibroblast proliferation.HIPPO-Yap/Taz signaling plays an important role in cardiac growth of embryos as well as adult mouse cardiac cells and tissue remodeling post injury ,10,11,12in vitro studies. Failing hearts are subject to regional hypoxia [Analysis of cardiac samples from our HF patients revealed increased protein levels of connective tissue growth factor (CTGF), a regulator of cardiac extracellular matrix and fibrosis ,19 and a hypoxia , which i hypoxia ,26. Furt hypoxia  and to e hypoxia ,30,31,32in vitro in human primary adult cardiac fibroblasts  and phospho/total Akt ratio (Akt activation) were significantly higher in LV tissues from patients with HF compared to controls  ratio or protein level  Yap signaling is enhanced in LV from patients with non-ischemic HF, (ii) Yap is induced in human adult cardiac fibroblasts by the pro-proliferative pro-fibrotic TGFβ/hypoxia exposure and (iii) silencing Yap expression inhibits cardiac fibroblast proliferation potentially via down-regulation of Akt signaling but, (iv) without affecting mTORC1 signaling. This work provides a basis for future studies on the role of cardiac fibroblast signaling in the pathogenesis of HF, paving the way for development of new treatment avenues for this progressive disease.Yap and its upstream negative regulator HIPPO are confirmed modulators of cardiomyocyte growth and survival ,47 whileGiven the established pro-proliferative role of Yap in other cell types and confirmed link between cardiac fibroblast proliferation, extracellular matrix production and HF pathogenesis ,16,17, wWe found that depletion of endogenous Yap protects adult cardiac fibroblasts from TGFβ/hypoxia-induced proliferation. Of note, the pro-proliferative role of Yap had also been reported in pulmonary fibroblasts from fibrotic lungs , in agreYap is known to promote activation of Akt , which, Further, Yap plays a mediatory role in crosstalk between HIPPO and mTOR  and in mDeidentified snap frozen LV tissue specimens from non-failing (control) hearts from male Caucasian subjects and LV from gender-matched non-ischemic HF patients whom had undergone heart transplant were provided by Cleveland Clinic Human Heart Tissue Bank and University of Pittsburgh, Pittsburgh Heart, Lung and Blood, Vascular Medicine Institute in compliance with protocols approved by University of Pittsburgh and Cleveland Clinic institutional review boards.Immunofluorescence analysis was performed as described previously ,55. BrieSer473, (P)-AktThr308, -Lats, -P-Lats, -beta-tubulin, -CTGF, -S6, -P-S6, -P-P70S6K, -P-4E-BP1, -Raptor, -mTOR, -P-mTOR and -GAPDH rabbit, anti -Akt and -P-Erk mouse , anti-collagenI goat  and anti-tubulin rat  overnight at 4 °C. Membranes were then incubated with fluorescent  or HRP-linked  secondary antibodies at room temperature for 1 h. The images were scanned on an Odyssey system imager, developed on film or BioRad ChemiDoc Imaging system and relative band intensities were quantified by densitometry using ImageJ (NIH).Immunoblot analysis was performed as described previously ,55. Brie2) for indicated times. For gene silencing, Yap shRNA-producing or control adenovirus  was transduced into cells [Human adult ventricular fibroblasts were purchased from LONZA , plated, and expanded according to manufacturer protocol using fibroblast medium supplemented with FGM-3 Bullet Kit LONZA . Cells were exposed to TGFβ (10 ng/mL) and/or hypoxia  separately or in combination. Cell counts were performed using the automated Coulter Counter Z1 . A minimum of three repetitions per experimental condition were performed in each experiment.Cell growth assay was performed as described previously . Brieflyp-value of <0.05 was used as a criterion for statistical significance. Data are presented as means ± SEM.Statistical analyses were performed using GraphPad Prism . Comparisons between two groups and multiple groups were performed using Mann-Whitney and Kruskal-Wallis with Dunn’s test, respectively. A Our study provides evidence that Yap is up-regulated in human LV HF and supports pathological cardiac fibroblasts proliferation. Given that pharmacological inhibition of HIPPO and up-regulation of Yap are currently under consideration as an attractive option to induce cardiomyocyte regeneration in HF, our study suggests that pro-proliferative function of Yap in cardiac fibroblasts might be also considered as a potentially unfavorable outcome of HIPPO/Yap-targeted therapy. Further studies are needed to delineate the cell-specific roles of YAP and therefore educate potential future therapeutic strategies.


Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of  N-terminal proteoforms raised upon alternative translation.Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics , we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by  translation initiation in  Arabidopsis  and tomato (Solanum lycopersicum), translation (initiation) landscapes have been mapped by means of Ribo-seq   peptides in Arabidopsis cell suspension cultures have been described previously   equal or higher than a certain threshold, where:The mapped profiles from the initiating ribosomes, obtained after lactimidomycin (LTM) treatment, were found accumulated in AUG or near-cognate start codons using a ±1 nt window  read coverage compared to elongation (CHX) read coverage. The thresholds for TIS “minimum profile count” and RLTM −RCHX were optimized for database annotated  methionine-labeled translation products were generated using the TnT T3 RNA polymerase and 2 μl of [35S] methionine (10 μCi/ml) per reaction. After 1 h of incubation at 30°C, and to stop the translation reaction, 4 μl of the reaction mixture  was diluted 12.5-fold in 50 mM Tris (pH 8) and NuPAGE® LDS Sample Buffer , and the samples were heated for 10 min at 70°C. The samples were separated on 4–12% or 12% NuPAGE® Bis-Tris gradient gels  using MOPS Buffer. Subsequently, proteins were transferred into a PVDF membrane, air-dried, and exposed to a film suitable for radiographic detection .To inspect translation products, the mutagenized constructs served as templates for https://iomics.ugent.be/icelogoserver) using the precompiled Swiss-Prot composition of Arabidopsis as reference set. Nt peptide data stored in the Plant PTM Viewer  were cross-referenced to the Ribo-seq-called TIS data. Additionally, proteins were appointed a subcellular location based on SUBA4 consensus locations .All mass spectrometry proteomics data and search results of Arabidopsis  was apparent among the 393 TIS identified in so-called intergenic regions  protein sequences corresponding to all in silico translations matching Ribo-seq-called TIS. In total, we identified Nt peptides matching 6,493 unique protein N-termini supported by at least two peptide-to-spectrum matches  (in vitro) Nt acetylated (NTA) peptides, NTA patterns corresponded with the known specificities and NTA efficiencies of major NAT  enzymatic activities in eukaryotes  . Of thes ≤ 0.01)   . Moreove2 test adjusted p < 0.05). Taken together, these Nt peptide identifications and their modification status provide unambiguous complementary proteomics and Ribo-seq proof of the translation of Nt-truncated proteoforms from dTIS for the 68 genes  with the Ribo-seq-called dTIS identified in this study. Overall, Nt peptide evidence was also found for 42 out of 68 dTIS-indicative N-termini in various Arabidopsis studies, and 34 of these were identified as NTA (Arabidopsis tissues.Next to Nt peptides matching annotated TAIR10 N-termini (dbTIS), complementary Nt peptide was evidenced for 81 Ribo-seq-called aTIS . The maj68 genes . Next to68 genes . Next tod as NTA . An addid as NTA . Moreoved as NTA  and a tyd as NTA . Hence, in vitro coupled transcription and translation (TnT). First, we tested ISOPENTENYL DIPHOSPHATE ISOMERASE2 (IPP2), for which Ribo-seq LTM evidence suggests translation initiation from both the annotated dbTIS and dTIS possibly resulting in the expression of an IPP2 proteoform pair  resulted in the exclusive expression of the Nt proteoform translated from the non-mutated TIS (UBX DOMAIN-CONTAINING PROTEIN1 (PUX1) was performed, for which a dTIS matching AA position 22 was identified in our riboproteogenomics analysis   and in vitro (TnT). Besides, mutations of TIS further confirm downstream translation initiation, likely via leaky ribosome scanning, giving rise to the expression of Nt-truncated proteoforms and, thus, the possible (co-)expression of proteoform pairs from a single CDS.To further support alternative translation initiation at in-frame dTIS as a source for the generation of N-terminal proteoform(s) (pairs), we tested if the mutagenesis of annotated TAIR10 TIS (dbTIS) and/or Ribo-seq-called dTIS would result in altered proteoform expression by orm pair . In addiated TIS . Second,analysis . Also he(NAC014)  supporteN-acetyltransferase (GNAT) domain-containing proteins, which show diverse NAT substrate specificities , 426 (20%) N-termini were not directly indicative of translation initiation, as they did not match dbTIS or Ribo-seq-called dTIS . PlottinArabidopsis cell culture findings with in vivo degrees of NTA and to confirm the location of NTA neo-N-termini indicative of post-translational cTP-processing in the chloroplast, we additionally performed Nt COFRADIC analyses on proteomes of enriched chloroplasts and corresponding stromal protein fractions from Arabidopsis leaves. Both fractions were processed in two different ways. First, as performed for cell cultures   were also identified. Besides the 17 annotated protein N-termini of chloroplast-encoded genes, the remainder are from cytosolic origin and were likely identified as impurities in the chloroplast isolation procedure as also evidenced by their general greater cellular abundance. For all NTA peptides, the degrees of NTA were calculated  and plotIPP2   or, alteIPP2  exactly ition 54 , top. Moition 54 . Hence, Arabidopsis genome  of non-AUG start codons (Solanum lycopersicum (tomato) that similarly employed LTM for halting initiating ribosomes   represents a powerful technique to empirically determine translated regions in the genome, and has revealed yet an unexplored proteome complexity. Using inhibitors that halt ribosomes during translation initiation, TIS can be uncovered on a genome-wide scale  given the absence of preceding Ribo-seq signal and lack of peptide evidence in the Arabidopsis Peptide Atlas  confirmed translation initiation at 42 dTIS  or downstream processing .In the next phase, PROTEOFORMER   resulted in alternative transcripts, encoding cTP-containing proteoforms targeted to the chloroplast and Nt-truncated cytosolic proteoforms lacking a cTP , and to serve as a mechanism for dual protein localization to the mitochondria and cytosol , ThrRS (AT5G26830), and GlyRS (AT1G29880)  . In facton (TnT) . Furtheron (TnT) . Besideson (TnT)  were alron (TnT) . Similarendorfii .Arabidopsis peptides identified and a maximal deviation ≤ 5 AA residues of TargetP2.0 predicted cleavages, more than 1,000 unique N-terminal sorting signal processing sites  were identified (in vivo NTA (%) of annotated protein N-termini and stromal neo-N-termini  are typically identified in Nt proteomic experiments  can be found in the article/PW and PVD conceived the research. VJ and PVD conducted the experiments. PVD performed the proteomics. PW, EN, and PVD analyzed the data. PW, FV, and PVD wrote the article. All authors contributed to the article and approved the submitted version.This study was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (PROPHECY grant agreement no. 803972 to PVD), the Research Foundation-Flanders  and the Research Foundation-Flanders-Fonds de la Recherche Scientifique (Excellence of Science project no. 30829584 to FV).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli, whereas AZT displayed no selectivity for any antibiotic-resistant strain. Motility assays revealed that β-lactamase-producing Escherichia coli strains were less motile in nature and more strongly affected by CPX than a parental strain. Resistance against CPX was not observed in E. coli even after 25 days of growth, whereas AZT resistance was observed in less than 2 days. Moreover, CPX effectively killed AZT-resistant strains with different resistance mechanisms. Our findings indicate that CPX may be utilized as an alternative or supplement to AZT-based medications to treat opportunistic Gram-negative bacterial infections.Multidrug-resistant (MDR) Gram-negative bacteria are the top-priority pathogens to be eradicated. Drug repurposing  may be helpful to overcome the limitations of current antibiotics. Zidovudine , a licensed oral antiviral agent, is a leading repurposed drug against MDR Gram-negative bacterial infections. However, the rapid emergence of bacterial resistance due to long-term exposure, overuse, or misuse limits its application, making it necessary to develop new alternatives. In this study, we investigated the efficacy of ciclopirox (CPX) as an alternative to AZT. The minimum inhibitory concentrations of AZT and CPX against MDR Gram-negative bacteria were determined; CPX appeared more active against β-lactamase-producing  The emergence of multidrug-resistant (MDR) Gram-negative bacteria urgently require new strategies to treat infections caused by such bacteria. High costs and prolonged development time characterize new antibiotics. Drug repurposing, i.e., the identification of non-antibiotics for new therapeutic indications, is a promising alternative or adjuvant to treat MDR bacterial infections, obviating antibiotic-resistant biological pathways .Escherichia coli (E. coli)  or the gene-specific primers tdk-F and tdk-R  The chromosomal tdk gene was amplified as described above using gene-specific primers and used for sequencing. The presence of the mobilized colistin resistance (mcr-1) plasmid in either KS7001 or KS8001 was confirmed by colony PCR with pQE-60-specific primers  methyl}-guanine  would also reduce the antiviral potential of AZT [A recent report suggested that resistance to AZT under its own pressure could occur in an unmutated-Tdk strain that contained eight mutated genes, including  protein . Howeverdespread , how rapdepicted a. As shoKeio-tdk ; Table 1neration b,c. Howe) of AZT b,c. Thisl of AZT .E. coli is a thymidine kinase (Tdk) expressed from the tdk gene in the bacterial chromosome [tdk gene in AZT-R and ATCC 25922 cells was performed with tdk-specific primers  as that for the control strain, with retained bactericidal properties of CPX  and share target pathways with CPX. Second, the activation of CPX action against specific bacterial species is required. The formulation of CPX into biocompatible nanoparticles is a promising strategy for this purpose, since nanoparticles have been utilized to increase the selectivity pressure of antibiotics to target species. In addition, synthetic derivatives of CPX can be utilized to increase its specificity. This possibility was proved in a recent study, in which synthesized CPX derivatives effectively inhibited the growth of Cryptococcus neoformans [In summary, we showed that CPX is a potential alternative agent to AZT against Gram-negative bacterial infections, especially against oformans . It wouloformans , a noveloformans . With th


We present two scenarios where Retzius-sparing robotic-assisted radical prostatectomy (RS-RARP) provided additional versatility that was translated into a more desired clinical outcome.The Retizius-sparing (RS) approach to prostatectomy has several proposed advantages over the conventional retropubic approach. This includes reduced post-prostatectomy stress urinary incontinence rates and better preservation of potency.The first case was a 76-year-old renal transplant patient diagnosed with intermediate-risk prostate cancer. His well-functioning low-lying graft kidney in the left lower quadrant was in close relation to the prostate gland, precluding either conventional retropubic prostatectomy or radiotherapy as a treatment option. RS-RARP was performed for the patient, yielding satisfactory oncological and functional outcomes. The second case was a 61-year-old patient who had a rectal tumour with suspected prostate invasion on MRI. After neoadjuvant chemotherapy and radiotherapy, robotic en-bloc abdominal-perineal resection of the rectum, using a Retzius-sparing prostatectomy technique, was performed to save the patient from total exenteration and double stomata. A modified RS-RARP was adopted to keep the surgical plane between the rectum and prostate. Both procedures were uneventful.In conclusion, the Retzius-sparing approach is a versatile technique which helps the surgeon to conduct a prostatectomy when special anatomical considerations are at play.Supplementary file1 (MP4 260251 kb)Supplementary file2 (DOCX 25 kb)Below is the link to the electronic supplementary material.


Currently, there are two antifibrotics used to treat idiopathic pulmonary fibrosis (IPF): pirfenidone and nintedanib. Antifibrotics slow disease progression by reducing the annual decline of forced vital capacity (FVC), which possibly improves outcomes in IPF patients. During treatment, patients occasionally switch antifibrotic treatments. However, prognostic implication of changing antifibrotics has not yet been evaluated.This multi-center retrospective cohort study examined 262 consecutive IPF patients who received antifibrotic therapy. Antifibrotic agents were switched in 37 patients (14.1%). The prognoses were compared between the patient cohort that switched antifibrotics (Switch-IPF) and those without (Non-Switch-IPF) using propensity-score matched analyses.p < 0.0001). In propensity-score matched analyses that were adjusted to age, sex, FVC (%), history of acute exacerbation, and usage of long-term oxygen therapy, the Switch-IPF cohort had significantly longer survival times than the Non-Switch-IPF group . The second-line antifibrotic therapy showed similar survival probabilities than those in first-line antifibrotic therapy in multistate model analyses.The median period between the initiation of antifibrotic therapy and the drug switch was 25.8 (12.7–35.3) months. The most common reasons for the switch were disease progression (n = 17) followed by gastrointestinal disorders (n = 12). Of the 37 patients that switched antifibrotics, only eight patients disrupted switched antifibrotics by their adverse reactions. The overall prognosis of the Switch-IPF cohort was significantly better than the Non-Switch-IPF cohort (median periods: 67.2 vs. 27.1 months, Switching antifibrotics is feasible and may improve prognosis in patients with IPF. A further prospective study will be required to confirm clinical implication of switching the antifibrotics.The online version contains supplementary material available at 10.1186/s12890-021-01587-3. Switching antifibrotics were generally well-tolerated. The prognosis of IPF patients switching antifibrotics was significantly better than those not switching, suggesting that switching is feasible and may improve prognosis in patients with IPF.Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing form of interstitial lung disease (ILD) with unknown aetiology –3. IPF iThese two antifibrotics have distinct types of antifibrotic mechanisms  and diffThis retrospective study was conducted on 312 consecutive patients with ILD, receiving pirfenidone or nintedanib, at Hamamatsu University of School of Medicine, Seirei Hamamatsu Hospital, and Seirei Mikatahara Hospital. All patients were treated between February 2009 and March 2020. Fifty ILD patients were excluded from the study: 37 patients were diagnosed with non-IPF ILD, and 13 IPF patients had insufficient clinical data. Thus, this study enrolled 262 patients with IPF. The IPF patients switching antifibrotics (Switch-IPF) was defined as patients who received first-line antifibrotics more than one month and followed by second-line antifibrotic therapy. The IPF patients who received first-line antifibrotics less than one months or were not treated with second-line antifibrotics were defined as Non-Switch-IPF. Diagnosis of IPF was based on the ATS/ERS/Japanese Respiratory Society (JRS)/Latin American Thoracic Association (ALAT) criteria . The Mann–Whitney U test was used to compare the continuous variables. Fisher’s Exact test for independence was used to compare categorical variables. Overall survival time was measured from the start date of first-line antifibrotic therapy, unless otherwise specified. Propensity-matched analyses were employed to determine the impact of switching the antifibrotics on prognosis. Propensity score matching was performed using the following algorithm: 1:1 optional match with a ± 0.05 calliper and no replacement. Univariate and multivariate analyses were also performed using the Cox proportional hazards regression model. The variables of propensity score matching and multivariate Cox-regression analyses that were considered clinically important, and specific risk factors for mortality in IPF were selected , 18–20.As “switching antifibrotics” was a time-varying event, the values of “switching antifibrotics” was evaluated using time-dependent Cox regression analysis and Markov multistate model to describe the clinical transitions of second-line antifibrotic therapy –23. CumuAs “switching antifibrotics” was a time-varying event, we evaluated the values of “switching antifibrotics” by time-dependent Cox regression analysis and Markov multistate model to describe the clinical transitions of second-line antifibrotic therapy. The Markov multistate models have traditionally been used to study effect of transitory state of illness (or interventions such as transplantation) on prognoses to death –23. The p values < 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism Version 6 , JMP , and R software version 4.0.2 . All analyses were two-tailed and The clinical characteristics at the initiations of first-line antifibrotics of IPF patients with Switch-IPF) and Non-Switch-IPF are shown in Table Among Non-Switch-IPF cohort, 48 patients 21.3%) discontinued the antifibrotics during the observation period with a median exposure time of 7.2 months. The characteritics of Non-Switch-IPF Continued cases and Discontinued cases were also presented in Table .3% discoAmong the Switch-IPF patients, the most common cause of switching the antifibrotics was disease progression , followed by GI side effects n = 12)  months vs. 5.3 (0–15.8) months, respectively, p < 0.0001]. Patients who switched antifibrotics due to Non-PD, mostly due to adverse drug reactions (ADRs), were initiated second-line treatment delayed from after resolutions of ADRs.The detailed time course and reasons for switching antifibrotics are shown in Fig. The declines of FVC (%) and FVC (L) between the first and second-line treatments are shown in Fig. The clinical characteristics at the initiations of second-line antifibrotics were showed in Additional file During the observation period, 17 patients in the Switch-IPF cohort and 130 patients in the Non-Switch-IPF cohort died. The most common cause of death was chronic respiratory failure, followed by AE. The causes of death did not differ between the Switch-IPF and Non- Switch-IPF patients Table : Fig. S2p = 0.0219, Fig. As shown in Table Not all Non-Switch-IPF patients received antifibrotics for the entire duration of the study; 48 patients (21.3%) discontinued antifibrotic treatment during the observation period, whereas the rest patients (n = 177) were continued first-line antifibrotic therapy. We therefore compared the survival times of Switch-IPF patients with Non-Switch-IPF Discontinued cases and Non-Switch-IPF Continued cases, respectively. The Switch-IPF patients still exhibited significantly longer survival times than Non-Switch-IPF Discontinued cases or Non-Switch-IPF Continued cases  had a significantly longer survival than those not (Non-Switch-IPF), and this survival benefit remained significant in propensity-matched analysis adjusted by age, sex, FVC (%), AE history and LTOT usages. Further, second-line antifibrotic therapy showed similar survival probabilities than those in first-line antifibrotic therapy using a multistate model analyses. These results suggested that the switch of antifibrotics is feasible and may have a prognostic benefit in patients with IPF.Currently, the antifibrotic medications, pirfenidone and nintedanib, are available for the treatment of IPF. Nintedanib inhibits multiple tyrosine kinases, including the receptors for vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor. The underlying mechanism of action for pirfenidone is still unknown , but it The most common reason for the switch was disease progression (45.9%), followed by GI side effects (32.4%) as determined by physicians. Compared to patients in the Switch-IPF group who switched for other reasons (Switch-IPF due to non-PD), the patients who switched due to disease progression (Switch-IPF due to PD) had significantly longer exposure periods to the first-line antifibrotics as well as shorter intervals from the end of first-line therapy to the start of second-line therapy. This is likely because the Switch-IPF due to non-PD patients mostly discontinued first-line antifibrotics due to ADRs within a short exposure periods from the initiation of first-line antifibrotics. Regarding the feasibility of second-line antifibrotics, among the 37 Switch-IPF patients, only eight (21.6%) discontinued second-line antifibrotics. None of the Switch-IPF due to PD patients had severe ADRs that caused them to discontinue second-line antifibrotics. By contrast, eight out of 20 Switch-IPF due to non-PD patients discontinued second-line antifibrotics because of ADRs. In particular, three patients terminated both first and second-line antifibrotics due to GI side effects. These issues are consistent with chemotherapy-induced nausea and vomiting (CNVI). In the field of oncology, experienced CNVI once was identified as a futured risk factor of CNVI in further chemotherapy . These dAlthough the causes of death did not significantly differ between Switch-IPF and Non-Switch-IPF patients, the survival times of Switch-IPF patients were significantly longer than those of Non-Switch-IPF patients. However, the baseline characteristics of Switch-IPF patients were not identical to those of Non-Switch-IPF patients. For instance, Switch-IPF patients had preserved lung function, lower incidences of AE history, and lower frequencies of LTOT usage compared with Non-Switch-IPF patients. Thus, we employed propensity score-matched analysis and multivariate Cox proportional analysis to adjust for these baseline differences. The propensity analysis adjusted by age, sex, FVC (%), AE history, and LTOT usages revealed that Switch-IPF patients still demonstrated significantly longer survival times than Non-Switch-IPF patients. Furthermore, the multivariate Cox proportional analysis revealed that patients with IPF who switched antifibrotics were identified as a population with better prognoses. Because 48 Non-Switch-IPF patients (21.3%) discontinued antifibrotic treatment during the observation period, we compared the prognoses between the Switch-IPF patients with Non-Switch-IPF Discontinued cases and Non-Switch-IPF Discontinued cases, respectively. We found that Switch-IPF patients still demonstrated significantly longer survival times than Non-Switch-IPF Discontinued cases and Non-Switch-IPF Discontinued cases. Collectively, these results suggest that switching antifibrotics from one to another may improve the prognosis among patients discontinuing first-line antifibrotics due to ADRs or those showing disease progression despite treatment.The rationale for the longer survival times of the Switch-IPF patients compared to the Non-Switch IPF patients is not entirely clear. In the time-dependent Cox proportional analysis, “switching antifibrotics” itself did not show great impact to change prognosis of IPF, but efficacies of second-line therapy was not inferior to those of first-line therapy by the multistate model analyses Fig. , suggestThe present study had several limitations. Although the present study showed that patients with IPF who switched antifibrotics as a population with better prognosis, the prognostic implications of switching the antifibrotics were not completely clarified even evaluated using time dependent Cox proportional model and multistate model. Second, this study was retrospective and the sample size was not large enough to provide conclusive results. Furthermore, because pirfenidone was approved before nintedanib, most of the patients switched from pirfenidone to nintedanib rather than the other way around. The disease behavior, such as a decline in FVC (%) before and after antifibrotic therapy, was not evaluated. Additionally, the decision to switching antifibrotics either due to disease progression or ADRs is subjective, and there were no formulated protocols used by physicians. Collectively, these limitations may cause potential biases in this study. However, this study may provide novel clue about the sequential usage of two antifibrotics. These findings are important because only two antifibrotics are currently available, and neither drug completely halts disease progression. Thus, balanced and large-scale prospective studies are required to overcome these limitations.In conclusion, the present retrospective study showed that 37 out of the 262 IPF patients who had received antifibrotics switched drugs during the observation period, and the second-line antifibrotics were generally well-tolerated. Moreover, patients with antifibrotics were identified as a population with better clinical outcomes as assessed by propensity-matched and multivariate Cox proportional analyses. Additionally, the present study showed survival benefit of second-line antifibrotic therapy was not inferior to that of first-line therapy Despite several limitations, the results of our study suggest that switching antifibrotics may be beneficial for the clinical management of IPF, especially among IPF patients discontinuing first-line antifibrotics due to ADRs or disease progression.Additional file 1: Figure S1. Survivals of patients with IPF with or without switching antifibrotics stratified by GAP system: Switched cases vs. Non-switched cases. Survival of patients with IPF GAP stage I with or without antifibrotic switching (A). Survival of patients with IPF GAP stage I with or without antifibrotic switching (B). p values were determined by the log-rank test. Cumulative survival probabilities from initiation of antifibrotic therapy were calculated. An event was defined as death from any cause and patients who were lost or still alive were censored at the last follow-up date.Additional file 2: Figure S2. Survival of patients with IPF switching antifibrotics as analyzed by antifibrotic medication or cause of switching. Survival of patients with IPF switching antifibrotics by antifibrotic medication (A), and causes of switching (B). p values were determined by the log-rank test. Cumulative survival probabilities from initiation of antifibrotic therapy were calculated. An event was defined as death from any cause and patients who were lost or still alive were censored at the last follow-up date.Additional file 3:Supplementary Table 1. Reasons for discontinuation of antifibrotics in 48 patients with Non-Switched IPF. Supplementary Table 2. Timeline of switching antifibrotics. Supplementary Table 3. Differences between the clinical characteristics in first-line and second-line anti-fibrotic therapy. Supplementary Table 4. Propensity-matched 74 patients with IPF with or without switches of the antifibrotics.


The aim of this narrative review is to give an overview on current and emerging imaging methods and liquid biopsy for prediction and evaluation of response to PRRT. Current limitations and new perspectives, including artificial intelligence, are discussed.A literature review of PubMed/Medline was performed with representative keywords. The search included articles published online through August 31, 2020. All searches were restricted to English language manuscripts.Peptide radio receptor therapy (PRRT) is a prospectively evaluated and approved therapy option in neuroendocrine tumors (NETs). Different ligands targeting the somatostatin receptor (SSTR) are used as theranostic pairs for imaging NET and for PRRT. Response assessment in prospective trials often relies on the morphological RECIST 1.1 criteria, based on lesion size in CT or MRI. The role of SSTR-PET and quantitative uptake parameters and volumetric data is still not defined. Monoanalyte tumor marker chromogranin A has a limited value for response assessment after PRRT. New emerging liquid biopsy techniques are offering prediction of response to PRRT and prognostic value.New response criteria for NET patients undergoing PRRT will comprise multiparametric hybrid imaging and blood-based multianalyte markers. This represents tumor biology and heterogeneity. The incidence of neuroendocrine tumors (NETs) has increased over the years , with ga68Ga]Ga-DOTATATE, [68Ga]Ga-DOTATOC, and [68Ga]Ga-DOTANOC F-FDG-PET response criteria in solid tumors (PERCIST) are not directly transferrable to NET disease Ga -labeled SSA or other PET-tracers ([18F]F-FDG). Previously widely used scintigraphy with [111In]In or [99mTc]Tc-labeled SSA is more and more replaced by SSTR-PET. SSTR scintigraphy underestimates SSTR expression in small NET metastases Ga -labeled DOTA-agonists are discussed.Imaging SSTR by positron emission tomography is realized by the combination of a positron emitter, 68Ga, a chelator, DOTA, and an SSA. Different SSA, −TOC, −NOC, and −TATE, show different affinities to SSTR-subtypes. However, they all bind to SSTR2, the receptor subtype predominantly expressed on GEP-NETs . Recentl68Ga]Ga-DOTA-SSA PET is a predictor for the outcome of PRRT and might thus also have an impact on PFS [68Ga]Ga-DOTATATE PET at initial diagnosis as being predictive of PFS max) F-FDG-PET targets increased glycolytic metabolism and is generally recommended for imaging of NETs with high proliferation index (G3) and poorly differentiated neuroendocrine carcinomas. While per patient positivity may account for around 60% of G1 and G2 NETs, per lesion positivity is lower, due to disease heterogeneity. These values are typically not provided in the majority of studies. As a consequence, [18F]F-FDG-PET is not part of the routine clinical workup in well-differentiated tumors [18F]F-FDG-PET should be considered.In-pentretreotide; Octreoscan) [177Lu]Lu-scintigraphy after PRRT in GEP-NET patients as well as in other disease treated with PRRT F-FDG-PET but also applied to SSTR-PET, albeit data is still sparse. The aspect of whole-body SSTR-PET protocols facilitates fast, automatic, whole-body tumor burden assessment. By manual segmentation, whole-body tumor burden assessed by PET can already be correlated to survival . PET-MRIBefore reaching its full potential, wide application of AI requires standardized examinations. Training of sophisticated algorithms on large prospective trials with high-quality data, including relevant outcome information, is mandatory.Although SSTR-PET is used in everyday practice as a gatekeeper for patients before undergoing PRRT, data of this imaging method is not incorporated into frequently used response assessment criteria. RECIST 1.1 has well-described shortcomings, as only morphological information is included. First, structured PET response criteria PERCIST, however, are only available for [18F]F-FDG-PET. This underlines the need for response assessment criteria for SSTR-PET and other incoming theranostics. Recent advances in structured reporting systems for SSTR-PET might help to overcome current use of data from SSTR-PET in an empirical, non-standardized way. As results for the predictive and prognostic significance of quantitative uptake values from SSTR-PET are inconclusive, new parameters assessing tumor volume and heterogeneity should be included. Data on the predictive or prognostic role of quantitative SSTR-PET uptake values are in the majority of cases extracted from retrospective data sets. Instead, we need a fundamental understanding of change of conventional PET-derived parameters under therapy that can be generated from large prospective trials or by data-mining, including high-quality data from large centers. This pooling of retrospective data sets is currently hampered by the limited standardization of PRRT and SSTR-PET, including the use of different radioligands for imaging and therapy. To ensure repeatability of PET measurements, currently associated with relevant variability , harmoniCapturing the overall clinical status of the patient by providing an insight into molecular biology of the tumor, multi-analyte liquid biopsy overcomes certain disadvantages of tissue-based analysis. Hence, blood-based information remains the new frontier of longitudinal patient management offering real-time information. Multi-analyte biopsy might be included into routine clinical-based and imaging-based assessment before PRRT. Assessing the molecular biology and heterogeneity of the tumor by a multiparametric approach including all the aforementioned imaging and liquid biopsy options will improve response assessment in NET patients . TogetheNew prospectively evaluated criteria for response assessment based on hybrid imaging should overcome previously described shortcomings. Imaging-based criteria, associated with additional predictive and prognostic value, are warranted to complement blood-based assessment.Strategies for modern treatment in oncology are no longer only based on tissue biopsy followed by tumor marker measurements and assessment of tumor size. In the era of precision imaging and liquid biopsy, capturing the complexity of tumor biology in real time will become standard of care. Hybrid imaging, including modern SSTR-PET, will be included into response assessment criteria. AI offers new opportunities for the assessment of tumor heterogeneity from hybrid imaging data acquired in routine clinical workup. Classical monoanalyte tumor markers, such as CgA, have been demonstrated to be inadequate to assess the treatment response. These markers are surpassed by the blood-based multigene biomarker NETest, which demonstrated 98% accuracy for early PRRT monitoring. In addition, the multigene test PPQ demonstrated to be an accurate specific prognosticator for PRRT response. Future strategies for response assessment and outcome prediction of PRRT will comprise the integration of state-of-the-art hybrid imaging of current and new theranostics with blood-based molecular information capturing the complexity of metastatic NET in the individual patient.


Making clothing and fabric with nanoparticles or nanofibers allows the improvement of fabric properties without a significant increase in weight, thickness, or stiffness. For example, incorporating nano-whiskers into fabric used to make pants produces a lightweight water- and stain-repellent material. Selected companies involved in this fast-developing arena include Nano-Tex, Aspen Aerogel, BASF, and Nano-Horizons. In line with this theme, four representative articles ,2,3,4 haAs shown by Kan et al. , the pre2) compounds in the form of coating. Treated textiles displayed very promising self-marking properties together with antibacterial activity. The strong interaction between Ir complexes and silica nanoparticles enabled easy detection under UV light, both when used as coating for textiles and after release in the environment. The homogeneous distribution of such luminescent coating can be usefully exploited as a wear diagnostic tool. In addition, the antibacterial testing showed interesting light-induced antimicrobial properties against both Gram(+) (Deinococcus radiodurans) and Gram(−) (Escherichia coli) bacteria systems, probably due to production of reactive oxygen species (ROS). For useful application, a preliminary human dermal exposure assessment candidate such as Ir@SiO2 coating (as a safety by design solution) can be easily tracked for the whole life cycle, i.e., from production to release of nanoparticles.Blosi et al.  obtained2 nanoparticles sensitized with water-insoluble disperse blue 183 dye  was achieved. The synergetic mechanism of both metal-nonmetal co-doping and dye sensitization on the photocatalytic activities of TiO2 nanoparticles under visible light irradiation was investigated. The superior photocatalytic activity of the dye-sensitized PET-Ag-N-TiO2 composite photocatalyst was primarily due to the quick separation of photo-generated electron-hole pairs and efficient interfacial charge transfer. The results of photo-degradation of methyl orange (MO) dye solution (with trapping experiments as well as ESR measurements) showed that the holes, in comparison with •OH and •O2− radicals, were the main reactive radical species for the PET filaments coated with dye-sensitized Ag-N co-doped TiO2. PET substrate and water insoluble disperse blue dye greatly contributed to the superior photocatalytic activity of the dye-sensitized Ag-N co-doped TiO2 coated PET filaments. This not only generated more holes, but also inhibited the recombination of electron-hole pairs by the transfer of electrons to the dyed PET substrate and Ag nanoparticles. In comparison to the PET filaments coated with TiO2 nanoparticles, the PET filaments coated with Ag-N co-doped and dye-sensitized TiO2 nanoparticles exhibited greatly enhanced light absorption capacity, efficient separation of electron-hole pairs, and substantial photocatalytic activity in degradation of MO dye under visible-light irradiation. The resultant photocatalytic composite filaments were capable of repeatedly photo-degrading MO dye without losing initial photocatalytic activity significantly. The proposed photocatalytic composite structure can be taken as a novel approach to design textile materials based composite photocatalysts for the photo-degradation of organic pollutants.In work by Zhang et al. , the enh−4 W/sq. Furthermore, the surface resistance of the hybrid films appeared to decrease due to the interconnected network formed by the AgNPs. The results indicated that these films are promising as nano-conductors in electronic devices. Silver-based electrodes show demonstrated potential not only in biosensor ECG systems for monitoring cardiac activity, but also in functional biological applications. However, exploring the large-scale production of these films is dependent on commercial viability and uptake. The use of dry electrodes for ECG devices is an exciting application for wearable technology featuring nanohybrid films.As reported by Lin et al. , highly


Statins have become the commonest lipid-lowering agent worldwide and have significantly reduced morbidity and mortality associated with cardiovascular diseases. Overall, statins are very well tolerated. However, in clinical practice, a wide variety of skeletal myopathic effects have been observed, ranging from asymptomatic patients with high creatine phosphokinase (CPK) to fatal cases of acute rhabdomyolysis. Recent reports suggest that statins are associated with immune-mediated necrotizing myopathy (IMNM), a unique autoimmune myopathy. Unlike other drug reactions, this can occur months to years after initiation of statin. It is a distinctive autoimmune myopathy where symptoms persist or even progress after statin discontinuation and requires immunosuppressive therapy. The presence of anti-hydroxy-methyl-glutaryl coenzyme-A reductase (HMGCR) antibody in serum strengthens the diagnosis of statin-associated necrotizing myopathy. Here we present a case of statin-associated IMNM in a 43-year-old Caucasian female who had statin-induced progressive deterioration of proximal muscle weakness with poor response to high-dose steroids and required further immunosuppressive therapy. Statins, also called hydroxy-methyl-glutaryl-coenzyme-A reductase (HMGCR) inhibitors, are generally considered safe and are well-tolerated drugs. Statins may be responsible for a wide array of musculoskeletal manifestations, such as an asymptomatic rise in creatine phosphokinase (CPK), myalgias, myositis, and rhabdomyolysis . Statin Inflammatory myopathies are characterized by progressive proximal muscle weakness due to autoimmune-mediated damage to muscle tissue. Most well-known inflammatory myopathies associated with statins include polymyositis (PM), dermatomyositis (DM), and inclusion body myositis ,5. HowevThere are two subtypes of IMNM including anti-3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (anti-HMGCR) and anti-signal recognition particle (SRP) . StatinsA 43-year-old Caucasian female initially presented to her primary care physician (PCP) with the chief complaint of progressive weakness of symmetric upper and lower proximal limbs for 4-5 weeks. Symptoms started suddenly and progressively worsened, and she was also experiencing diffuse muscle aches. The patient was having difficulties climbing stairs, standing from a sitting position, and combing her hair. The patient had type 2 diabetes mellitus, hyperlipidemia, and migraine headaches. She had been on atorvastatin, 40 mg daily, for almost five years. Initial outpatient lab work showed an elevated CPK level of 8000 IU/L and the patient was referred to the ED by her PCP for further evaluation. Atorvastatin was discontinued. The patient was started on intravenous hydration for possible rhabdomyolysis. The patient’s symptoms did not improve after aggressive hydration in the hospital, and the CPK level remained persistently elevated above 7000 IU/L. An MRI of proximal lower extremities showed diffuse, symmetric proximal lower extremity muscle edema. MRI also did not show significant fatty atrophy of posterior thigh muscles and this was indicative of acute myopathy Figures  and 2.Clinical symptoms of significant muscle weakness, very high CPK level, and abnormal MRI findings initially raised suspicion for inflammatory myositis. To rule out inflammatory myositis, a muscle biopsy was performed. The patient was empirically started on 60 mg of prednisone daily for suspected inflammatory myositis pending biopsy result. The muscle biopsy report was suggestive of statin-induced myopathy. Moreover, the subsequent workup showed positive antinuclear antibodies (ANA), negative extractable nuclear antigens (ENAs), and very high HMGCR antibodies (titer > 200). The patient was diagnosed with statin-associated IMNM. She reported resolution of muscle achiness, but no improvement of weakness with corticosteroid treatment. Due to a lack of adequate response to high-dose corticosteroids for eight weeks, she was started on IVIG 2 gram per kilogram of body weight per month for three months. In outpatient follow-up, the patient reported resolution of all muscle symptoms, CPK had been normalized and the patient did not require any further immunosuppressive drugs.Statins have been reported to be associated with inflammatory myopathies like PM, DM, inclusion body myositis, and IMNM . Statin-Statin-induced myalgia is a self-limited condition and the patient's symptoms improve within weeks or months of discontinuing statin . HoweverStatin-associated IMNM is rare, which does not respond to the discontinuation of statin alone. Lack of improvement in muscle strength and persistently elevated CPK level after statin discontinuation necessitate the importance of further workup including a muscle biopsy. After the diagnosis is confirmed, patients with IMNM often require aggressive immunotherapy. Due to the increasingly widespread use of statins, clinicians should consider the possibility of statin-associated IMNM in patients whose myopathic symptoms and elevated CPK levels persist despite discontinuation of statins. Further studies are needed for a better understanding of the pathogenic mechanisms, risk factors for developing the disease, management, and long-term prognosis of this disease.


Since the first report of SARS-CoV-2 in China in 2019, there has been a huge debate about the origin. In this work, using a different method we aimed to strengthen the observation that no evidence of genetic manipulation has been found by (1) detecting classical restriction site (RS) sequence in human SARS-CoV-2 genomes and (2) comparing them with other recombinant SARS-CoV-like virus created for experimental purposes. Finally, we propose a novel approach consisting in the generation of a restriction endonucleases site map of SARS-CoV-2 and other related coronavirus genomes to be used as a fingerprint to trace the virus evolution. Coronaviridae causing symptoms primarily in the upper respiratory tracts which range from common cold to severe to fatal illnesses1. They have been associated with two major disease outbreaks, the severe acute respiratory syndrome  and the Middle East respiratory syndrome 2. In December 2019, a new coronavirus (SARS-CoV-2) started to cause viral pneumonia bringing to severe and fatal infection. Although SARS-CoV-2 belongs to the same lineage of CoVs that causes SARS, it is genetically different and it cluster apart exploiting phylogenetic trees3. Phylogenetic analysis demonstrated the highly similarity between human SARS-CoV-2 and the sequence isolated from the Bat-Cov-raTG134 (97.2% identity) and the Pangolin-SARS-CoV5 (80% identity), particularly in the receptor-binding-domain (RBD) of the S protein, important to mediate binding to human-receptor-angiotensin-converting-enzyme-2 (hACE2)6. The World Health Organization declared a coronavirus disease 2019 (COVID-19) pandemic in March 2020. Therefore, one of the major discussions around SARS-CoV-2 has been related to its origin with the assumption that SARS-CoV-2 could have been the result of genetic manipulations or spill-over from laboratories studying these viruses. In March 2020, Anderson and colleagues published a detailed analysis showing that SARS-CoV-2 does not derive from a laboratory construct7. Although other several coronavirus experts have discredited the hypothesis of a man-made coronavirus12, here we aim to present a different method based on the analysis of restriction site (RS) sequences in the genome of SARS-Cov-2 to reconstruct its origin and follow the new variants.Coronaviruses (CoVs) goes into the family 13. This resulted in the so-called infectious clone icSARS-CoV containing atypical markers of the wild-type (WT) virus. In particular, several Bgl1 RSs were introduced into the icSARS-CoV cDNA, which can be recognized since mutation are included in the newly formed cDNA. Figure Bgl1 RS used to produce icSARS-CoV.During the SARS-CoV epidemic outbreak in 2003, a method called reverse genetic to assemble a full-length cDNA of the SARS-CoV-Urbani strain, as a template for manipulation of the viral genome, was published to develop and test candidate vaccines and therapeuticsBgl1 RS ‘marker’ (GCCNNNN/NGGC). All the genomes did not contain these sites  to look for es Table , in partBy analysing the sequences of Bat-CoV-raTG13 and the hCoV-19-Pangolin, we observed that only one sequence from SARS-CoV-Urbani (GCCAGCGTGGT) was found in SARS-CoV-2Wu (Wuhan). This is expected since the first part of these two genomes show high similarities.14. The identity of MA-SARS-CoV compared with the original SARS-CoV-Urbani is 99.97% with only six distinct nucleotides, that cannot be used as markers of this recombinant virus since same mutations are naturally acquired by the WT-SARS-CoV, as demonstrated from the sequences of other isolated SARS-CoV14. Both the icSARS-CoV and the MA-SARS-CoV have become the most widely used recombinant viruses to study SARS-like viruses and no specific sequences were found in the human SARS-CoV-2Wu.Another recombinant SARS-CoV was produced in 2007 which derived from fifteen passages of the SARS-CoV-Urbani in BALB/c mouse lungs, therefore it was named Mouse-Adapted (MA)-SARS-CoV15. The full-length Bat-SCoV infectious clone, generated with the method described by Yount et al.13 include in the recombinant sequence specific markers such as the Bgl1 RSs. These RSs have specific nucleic base pairs in the “N” positions of the recombinant Bat-SCoV . We observed that these specific sequences were all absent  was generated starting from four Bat-SCoVs genomes HKU3–1 (DQ022305), HKU3–2 (DQ084200), HKU3–3 (DQ084199), and RP3 (DQ071615)9 was genent Fig. .16. Therefore, SL-CoVs were not able to infect hACE2 expressing cells, but only chimeric viruses expressing the spike protein of the SARS-CoV were able to bind the hACE2 which is the functional receptor of SARS-CoV. The authors identified a specific region responsible for the virus entrance into hACE2-expressing cells consisting of a minimal region of less than 200 amino acids. Interestingly, this group showed that chimeric spike proteins, whereby different regions of the SARS-CoV BJ01 (BJ01-S) spike were substituted into the spike of the bat SL-CoV (Rp3), and able to bind the hACE receptor. We generated in silico two of this chimeric spike (CS) sequences (the CS424-494 and the CS45-608), and then performed a multiple alignments to check similarities between other spikes identified after 2008, including the Bat-Cov-raTG13, the hCoV-19-Pangolin and the human SARS-CoV-2. The similarities of these two chimeric spikes are limited in the RBD of the spike  from horseshoe bat, which has a high similarity to SARS-CoV, differed in the N-terminus of the spike protein and particularly in the receptor binding RBS regionike Fig. . Thus, tMoreover, we performed a nucleotide blast sequence to find whether these recombinant spikes are found in the recently identified SARS-CoV-2 viruses. As shown in Figure 17 using the well establish reverse genetic approach13. According to this method, several Bgl1 RSs were included into the sequence  to two other identified WT bat coronaviruses, RsSHC014 and RS3367. This study suggested that direct bat-to-human infection is a possible scenario for some bat SL-CoVs. In 2015, Vineet et al.made a recombinant virus between the spike of the bat coronavirus SHC014 and the mouse-adapted SARS-CoV backbonece Table . MoreoveExploiting the RS sequences, which are approximately 6–8 base pairs of DNA, as specific markers, we propose an alternative way to trace the SARS-CoV-2 origin. This approach consists in the generation of a RS map of SARS-CoV-2 and the other four related coronavirus genomes. Using the Serial Cloner Restriction Enzyme Library, we generated the RS barcoding map based on the frequency of finding specific RS sequences in the genome. First, we generated a RS barcoding map which was used as genetic fingerprinting of the specific sequence analysed and which easily highlights sequence differences between the genomes. The pattern of the barcode’s reconstruction demonstrated high similarity between the coronavirus isolated from the Bat-Cov-raTG13 and the Pangolin, suggesting a natural evolution and adaptation of the virus. Different sequences of HIV, SARS-CoV and MERS-CoV were used as controls Fig. .Figure 218. Moreover, this approach can easily be used to discriminate between false negative and false positive which are the reasons for important additional socio-economic disruptions19.From the full restriction enzyme barcoding map, we identified in the spike (S) gene a sequence of 300 bp that can be used as a barcode to identify the virus and differentiate from others Fig. B and S7.Using the data to generate the full barcode map we performed principal component analysis (PCA) to determine whether the observed frequency of RSs is related to the hierarchical distance of the genomes analysed. The PCA plot shows a cluster formed by the Pangolin, the Bat-Cov-raTG13 and human SARS-CoV-2   were exploited to manipulate the transmissible gastroenteritis coronavirus (TGEV) PUR46-MAD virus26 and although nowadays other genetic manipulation mechanisms are known that allow no traces to be left, such as the use of the Crispr-cas system28, these remain more disadvantageous because they require higher technical capacity and higher costs and times. Furthermore, according to our knowledge, in the literature, there are no reports of SARS-CoV-2 coronavirus modifications through these more sophisticated techniques yet.Here, we analysed the peer-review literature of the SARS-related viruses generated in the laboratory over the years used to study the evolution of Coronaviruses and to generate drugs for their treatment. We have demonstrated through the analysis of RS, that SARS-CoV-2 does not contain peculiar RS or other markers that suggest a manipulation deriving from the recombinant viruses known in the literature. Indeed, the use of RS remains today the simplest, fastest and safest way to modify and study recombinant DNA. However, it should be mentioned that other methods have been used for generating recombinant clones, such as the one called transformation-associated recombination (TAR) cloning30 while Son and colleagues developed a simple method to discriminate SARS-CoV-2 from SARS-CoV using one-step RT-PCR followed by restriction fragment length polymorphism31. Although this method, based on only three restriction enzymes, succeeds in distinguishing between SARS-CoV2 and SARS-CoV, it may not be sensitive to discriminate any mutations outside that region within the virus genome and does not allow new variants to be tracked. Guan and colleagues developed an elegant study based on the identification of a barcode that allows the SARS-CoV-2 genome to be assigned to 5 different clades, however this process requires advanced bioinformatics skills32. Here instead we have shown that with our method it is sufficient to sequence a region of 300 bp to build a specific barcode to distinguish the genome of a virus, included the new variants  and to trace its evolution over time. This would allow us to have useful information quickly and economically during the classic tests performed on swabs.Finally, we used RS as markers to build a barcode map that could uniquely identify a particular virus. Recently, other authors used mathematical algorithms to find barcodes that identified a particular viral genome and build phylogenetic treesSARS-CoV-2 Wuhan-Hu-1, GenBank: MN908947.3; SARS-CoV-2-English, Gisaid: EPI_ISL_816718; SARS-CoV-2-African, Gisaid: EPI_ISL_678597; SARS-CoV-2-Brazilian, Gisaid: EPI_ISL_792680; SARS-CoV-2-19/Italy/VR (Gisaid accession id: EPI_ISL_422438|2020-03-25); SARS Urbani, GenBank: AY278741.1; SARS-CoV TW1, GenBank: AY291451.1; SARS-CoV-2 HKU, GenBank: JN854286.1; SARS-CoV-2 ZJ0, GenBank: DQ182595.1; SARS-CoV-2 Tai, GenBank: AY348314.1; HIV-1, GenBank: KY580639.1, MT222953.1, AF224507.1, MN703133.1, MH327766.1, KC156130.1; MERS-CoV, NCBI Reference Sequence: NC_019843.3; MERS-CoV, GenBank: KX034098.1, KT006149.2, KT225476.2, MW086533.1; Bat SARS-like Rs4231, GenBank: KY417146.1; Pangolin-CoV, GISAID accession numbers EPI_ISL_410721; Bat CoV RaTG13 GenBank: MN996532.1.All the genomic sequences and recombinant spikes sequences used in this study were generated following the materials and methods of the literature taken in considerations and saved in xdna format which is compatible with Serial Cloner. The files are available upon request to the authors.33, ClustalW34 and Jalview35.The sequences were aligned using Serial Cloner, Blastn suite36 was used to make Venn diagram.The restriction enzyme map barcode of each genome was obtained using Serial Cloner library. Using this software each genome was analysed in order to obtain the frequency of each restriction site to occur in that genome. The total frequencies of all the restriction sites present in the library were used to generate the barcode map. The InteractiVennThe genomic distance in bp between two or more restriction enzymes sites was calculated with serial cloner and then reported graphically using Prism GraphPad v8.PCA analyses was performed on the frequencies of the restriction enzymes sites on the different viruses’ genomes and plotted by ggbiplot R-studio. Codes available upon request to the authors.The heatmaps were generated in R-studio by using frequencies of the restriction enzymes sites on the different viruses’ genomes. The hierarchical clustering was performed using Pearson correlation as distance metric and Ward D clustering algorithm. Codes available upon request to the authors.The 300 bp region was determined analysing the area of major discrepancy (low identity) between genomes, in particular between related genomes. This area was identified inside the spike (S) region. To generate the barcode map of these 300 bp regions we used the same method used for the full-length genomes. Thus, we calculated the frequencies of the restriction sites to generate the heatmap.As informative sites we chose all those restriction sites that showed strong discrepancy in the cut-off frequency between the various genomes. Thus, to give an example, sites that had a high cut-off frequency in genome A compared to genome B, or sites unique to genome B that are repeatedly frequent in genome A (and vice versa). Then all non-informative sites, designated as those sites equally frequent across genomes, were discarded. In total we selected 104 informative sites here listed: "AatII" "AccBSI" "AcyI" "AfeI" "AloI" "Aor51HI" "AspA2I" "AsuNHI" "AvrII" "AxyI" "BarI" "BbvCI" "BcgI" "BlnI" "BmtI" "BplI" "BsaHI" "Bse21I" "BseYI" "BsiWI" "Bsp19I" "BspOI" "BsrBI" "BssNI" "BstACI" "Bsu36I" "BtgZI" "CchIII" "Cfr9I" "Ecl136II" "Eco32I" "Eco47III" "Eco53kI" "Eco81I" "EcoICRI" "EcoRV" "GdiII" "Hin1I" "Hsp92I" "MbiI" "MreI" "NcoI" "NheI" "NmeAIII" "Pfl23II" "PfoI" "Psp124BI" "PspLI" "PspOMII" "PsrI" "RpaBI" "SacI" "SauI" "SmaI" "SplI" "Sse232I" "SstI" "TspMI" "UcoMSI" "XmaI" "XmaJI" "ZraI" "AasI" "AccIII" "AgeI" "AsiGI" "BsePI" "BshTI" "Bsp13I" "BspEI" "BspMII" "BssHII" "CspAI" "DinI" "DrdI" "DseDI" "EciI" "Eco147I" "EgeI" "FspAI" "KasI" "Kpn2I" "KroI" "KspAI" "McaTI" "Mly113I" "MroI" "MroNI" "NaeI" "NarI" "NgoMIV" "PacI" "PasI" "PauI" "PceI" "PdiI" "PinAI" "PteI" "RceI" "SalI" "SfoI" "SseBI" "SspDI" "StuI". To generate the barcode map of the informative sites we used the method described for the full length and the 300 bp region.Supplementary Information.


Resting-state functional magnetic resonance imaging (rest-f-MRI) is a neuroimaging technique that has demonstrated its potential in providing new insights into brain physiology. rest-f-MRI can provide useful information in pre-surgical mapping aimed to balancing long-term survival by maximizing the extent of resection of brain neoplasms, while preserving the patient’s functional connectivity. Rest-fMRI may replace or can be complementary to task-driven fMRI (t-fMRI), particularly in patients unable to cooperate with the task paradigm, such as children or sedated, paretic, aphasic patients. Although rest-fMRI is still under standardization, this technique has been demonstrated to be feasible and valuable in the routine clinical setting for neurosurgical planning, along with intraoperative electrocortical mapping. In the literature, there is growing evidence that rest-fMRI can provide valuable information for the depiction of glioma-related functional brain network impairment. Accordingly, rest-fMRI could allow a tailored glioma surgery improving the surgeon’s ability to increase the extent of resection (EOR), and simultaneously minimize the risk of damage of eloquent brain structures and neuronal networks responsible for the integrity of executive functions. In this article, we present a review of the literature and illustrate the feasibility of rest-fMRI in the clinical setting for presurgical mapping of eloquent networks in patients affected by brain tumors, before and after tumor resection. In the setting of brain tumors, the primary goal of the surgery aims to maximize the tumor resection while preserving brain function ,2,3. ResRest-fMRI is a novel neuroimaging technique that explores functional connectivity networks associated with both normal and pathologic neurologic function . It workAlthough rest-fMRI is still under standardization, there is evidence that it can provide useful information about brain neuronal networks organization in the routine clinical setting. In particular, it can be used to in vivo map eloquent areas. Thus, rest-fMRI could be used for detecting glioma-related functional brain network alterations, improving the surgeon’s ability to increase the tumor resection and simultaneously minimize the risk of damage to brain neuronal networks responsible for executive functions integrity ,6.In this review, we illustrate the principles and the value of rest-fMRI in the clinical setting for presurgical mapping of neuronal networks in patients affected by brain tumors, and after tumor resection.Rest-fMRI is an emerging neuroimaging technique depending on the quantification of hemodynamic changes following the activation of brain areas. The increase in neuronal activity involves a greater demand of energy, and therefore oxygen, by neurons. Thus, neuronal activity induces a hemodynamic response that alters local brain concentrations of oxyhemoglobin and deoxyhemoglobin. This process produces time-dependent alterations in T2- and T2*-relaxation times, constituting the basic principles of the blood oxygen level-dependent (BOLD) contrast imaging . Since tThe human brain networks are active even during the resting or relaxing state. Furthermore, resting-state brain activity is far more significant than task-related activity, consuming 60–80% of the brain’s energy . These nThe proof of the existence of the RSNs is based upon the reproducibility of the networks in the single subject, the consistency of the networks between different subjects, and the correspondence of the cortical areas identified with different methods of study ,6,20,21.Brain MR imaging accurately demonstrates the morphological aspects of brain tumors with standard sequences, such as the three-dimensional (3D) magnetization-prepared rapid acquisition with gradient echo (MPRAGE) T1-weighted images obtained before and after gadolinium enhancement. Echo-planar imaging (EPI) sequence for rest-fMRI can be performed in a routine clinical setting, instructing the patient to stay still with their eyes closed while relaxing ,6. The rRest-fMRI is an emerging technique valuable for the exploration of brain physiology. Recent studies have explored the potential applications of rest-fMRI in various neurologic diseases  ,23,24,25Several studies reported high overlap between rest-fMRI and task-driven fMRI in various neurologic diseases when comparing the motor network, as well as high concordance with cortical stimulation mapping . Other sResting-state fMRI can reliably detect common functional connectivity networks in patients with glioma, and has the potential to anticipate network alterations after surgical resection. Alterations of RSNs can be analyzed at the level of the single subject and in group analysis, with a functional mapping demonstrating a good correspondence with cortical stimulation mapping . AlthougClinical presurgical rest-fMRI can be merged with standard anatomic sequences  and imported onto the neuronavigational system, thus can be used to guide intraoperative stimulation.Rest-fMRI evaluation of functional brain connectivity has demonstrated different RSNs, which depict specific functions and varied spatial location ,15,18 F. AnatomiDifferent post-processing and statistical approach have been described to assess the functional connectivity by rest-fMRI. There are mainly three widely used techniques for rests-fMRI connectivity analysis: the seed-based analysis, the independent component analysis (ICA), and the graph theory analysis. The seed-based analysis was the first method used to identify the resting state networks by the selection of a seed region of interest (ROI) to locate by computational and statistical analysis the linear correlation of the seed regions with all the other voxels of the entire brain ,40,41. TThe graph theory analysis is used to define a mathematical model of complex network functions within the human brain. With this method, the resting-state networks are represented by groups of nodes connected by edges ,6,45,46 https://www.fil.ion.ucl.ac.uk/spm/ (accessed on 1 September 2021)), FSL (Available online: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki (accessed on 1 September 2021)), Analysis of Functional NeuroImages (AFNI Available online: https://afni.nimh.nih.gov/ (accessed on 1 September 2021)), and CONN toolbox (Available online: https://web.conn-toolbox.org/ (accessed on 1 September 2021)). However, these software require a high knowledge and expertise to conduct postprocessing analysis as they lack standardization, thus requiring ad hoc adjustments or custom-made scripts to tailor the postprocessing pipeline.Therefore, the graph theory analysis shows the relation between the nodes and edges and describes these interactions through different graph parameters . Using these analysis methods for functional connectivity, different RSNs in the brain can be identified, such as the salience network, auditory network, basal ganglia network, higher visual network, visuospatial network, default mode network, language network, executive network, precuneus network, primary visual network, and sensory-motor network. Importantly, for rest-fMRI image processing, a variety of different post-processing software packages can be used, mainly freely available as open-sources, such as Statistical Parametric Mapping ) and FreeSurfer (Available online: https://surfer.nmr.mgh.harvard.edu/ (accessed on 1 September 2021)), and the Conn Toolbox (Available online: https://web.conn-toolbox.org/ (accessed on 1 September 2021)) running under MatLab ), using a 64-bit Intel-based High Performance Computer (HPC) with a 16 core CPU, 64 GB RAM, nVidia Tesla 100 graphic board, and 7 TB storage hard disk.In our clinical setting, we implemented a post-processing pipeline based on the opensource software fMRIPrep (Available online: https://brainmap.org/training/BrettTransform.html (accessed on 1 September 2021)) on the subjects, and a low pass filter to reduce signal fluctuations due to cardiac and respiratory pulsations. A 10% intensity threshold was applied to the raw images to remove spurious correlations outside of the brain. Atlas-based cortical parcellation before and after surgery was obtained with FreeSurfer ) after transforming the coordinates from the MNI atlas into the Talairach atlas (Available online: https://brainmap.org/training/BrettTransform.html (accessed on 1 September 2021)). The sensorimotor network (SMN), encompassing primary sensory and motor areas, was mapped using the atlas-based left BAs 1–3 (BA1-3L). The language network was mapped using either left Bas 44 and 45  for Broca’s area or left BAs 22, 39, and 40  for Wernicke’s area. Detailed post-processing steps used included each 3D MPRAGE T1w (T1-weighted) volume being corrected for intensity non-uniformity and skull-stripped. Brain surfaces were reconstructed with recon-all of FreeSurfer, and the brain mask estimated previously was refined with FreeSurfer-derived segmentation of the cortical gray-matter. Spatial normalization was performed through nonlinear registration using brain-extracted versions of both T1w volume and template. Brain tissue segmentation of cerebrospinal fluid (CSF), white-matter (WM) and gray-matter (GM) was performed on the brain-extracted 3D MPRAGE T1w with FSL (Available online: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL (accessed on on 1 September 2021)). EPI rest-fMRI images were slice time-corrected using AFNI (Available online: https://afni.nimh.nih.gov/ (accessed on 1 September 2021)) and motion-corrected using FSL. This was followed by co-registration to the corresponding 3D MPRAGE T1w images using boundary-based registration with six degrees of freedom, using FreeSurfer. Motion-correcting transformations, BOLD-to-T1w transformation, and T1w-to-template (MNI) warp were concatenated and applied in a single step using ANTs (Avatailable online: https://github.com/ANTsX/ANTs (accessed on 1 September 2021)).The preprocessing steps of the rest-fMRI steps included: rigid body motion correction, spatial smoothing , spatial normalization using the Montreal Neurological Institute (MNI) atlas . Indeed, functional network preservation should be considered when evaluating the EOR planning. In this setting, rest-fMRI can predict the impairment in the executive functions or other cognitive processes after surgical resection of a brain tumor, and can be valuable in identifying behavioral changes.Rest-fMRI can thus contribute to tailoring a personalized surgery for brain tumor resection improving the surgeon’s ability to increase the EOR, and simultaneously minimize the risk of impairment of critical functional networks responsible for specialized function .Rest-fMRI can also play a role in the postoperative evaluation of patients after tumor resection by estimate the potential alterations in functional connectivity and the onset of new neurologic deficits due to surgical resection. Moreover, in longitudinal studies, rest-fMRI allows assessing functional reorganization after surgical resection of brain tumors. Therefore, this advanced neuroimaging tool may be implemented in the routine clinical management of brain tumor patients.Despite further validation is currently necessary in a large population of patients to define the clinical value of task-driven fMRI and rest-fMRI for neurosurgical planning of brain tumors ,53,54,55There are still various problems affecting the performance and analysis of rest-fMRI in the clinical setting of brain tumors. The susceptibility artifacts and neurovascular uncoupling (NVU) issues, as well as head motion artifacts, which result in rest-fMRI image quality degradation. Susceptibility effects are more evident at 3T and higher field strength. They reflect the extent of magnetization of a substance in the presence of an external magnetic field. Field distortions and MR artifacts are commonly present around metal objects and implants, as they contain ferromagnetic materials which is perturbative to the external magnetic field. Susceptibility-induced signal loss from T2*-dephasing results in “geometric distortion” affecting rest-fMRI acquisition which is worse at high field strength ,6.Head motion and physiologic disturbance due to respiratory and cardiac cycles are sources of artifacts that heavily affect rest-fMRI. Head restraints used to reduce the effects of head motion and compensation of respiratory fluctuations and cardiac pulsations can achieve an improvement of these sources of artifacts. False-positive BOLD activation due to eye motion, random noise, partial volume effects, and physiological pulsations, along with post-processing statistical issues, may reduce the reliability of rest-fMRI studies ,6,58.To address these issues, retrospective motion and physiological noise correction methods can be used. Correction for the non-neural noise from white matter and cerebrospinal fluid (known as “nuisance regression”) can be utilized to improve the image quality of the rest-fMRI dataset. ,6,58. ImAnother limitation to this approach is the heterogeneity of lesion localization and spatial extent is altered anatomy due to a lesion mass effect that impacts cortical parcellation and seed selection . AutomatArtificial intelligence-based classification of resting-state connectivity is expected to provide more precise analysis results. This may ultimately allow integration of real-time connectivity maps into neurosurgical guidance.Rest-fMRI is an advanced neuroimaging technique able to provide useful information concerning different neuronal networks and their alterations related to the tumor and surgery. It provides an assessment for diagnosis, prognosis, and personalized treatment, which can be improved with the complementary information obtained by diffusion tractography  and stanThis approach has the potential for real-time pre-surgical mapping of eloquent cortex in patients with brain tumors. Future developments include parcellation of eloquent cortex based on combining rest-fMRI with diffusion tensor imaging (DTI)  by meansStandardization and validation of advanced imaging techniques and post-processing of fMRI will improve the risk–benefit assessment for each patient allowing neurosurgeons to better understand the clinical outcome of resection.