To determine whether there is a correlation between myelin oligodendrocyte glycoprotein (MOG) antibody-associated diseases and varicella zoster virus (VZV) infection. We provide a case report and performed a study to determine the frequency of MOG antibodies (MOG-IgG) in neurological VZV infections. Patients admitted to the Medical University of Innsbruck from 2008–2020 with a diagnosis of a neurological manifestation of VZV infection (n=59) were included in this study; patients with neuroborreliosis (n=34) served as control group. MOG-IgG was detected using live cell-based assays. In addition, we performed a literature review focusing on MOG and aquaporin-4 (AQP4) antibodies and their association with VZV infection. Our case presented with VZV-associated longitudinally extensive transverse myelitis and had MOG-IgG at a titer of 1:1280. In the study, we did not detect MOG-IgG in any other patient neither in the VZV group  nor in the neuroborreliosis group. In the review of the literature, 3 cases with MOG-IgG and additional 9 cases with AQP4 IgG associated disorders in association with a VZV infection were identified. MOG-IgG are rarely detected in patients with VZV infections associated with neurological diseases. Varicella zoster virus (VZV) is an exclusively human neurotropic herpes virus, presents with chickenpox (varicella) as inaugural infection, and remains latent in the dorsal root ganglions, cranial nerves, and the autonomic nervous system, and upon reactivation, results in rash and pain in one or more dermatomes, known as shingles (herpes zoster). This would occur often decades after the primary infection, particularly in susceptible immunocompromised patients, and older patients due to immunosenescence .Typical VZV neurological complications include postherpetic neuralgia, VZV vasculopathy, cranial nerve neuropathy, and radiculopathy . CentralHowever, until now, there has been no systematic analysis about the association of MOG-IgG and neurological manifestations of VZV infection. Here, we describe a patient with MOG-IgG positive VZV-associated longitudinally extensive transverse myelitis (LETM), perform a study to determine the MOG-IgG frequencies in patients with VZV infection and neurological involvement and present the results of a literature review.The retrospective study included 59 patients who were admitted to the Medical University of Innsbruck between 2008 and 2020 with the diagnosis of a neurological manifestation due to VZV infection and had an available serum sample of at least 500 µl. Diagnosis of VZV infection with neurological involvement  was based on the presence of typical dermatomal rash followed by neurological symptoms and supported by laboratory findings  as determined by polymerase chain reaction (PCR), or increased CSF VZV-IgG) , 20. CNSPatients with neuroborreliosis [previously published ] were inResults of routine diagnostic procedures, clinical, magnetic resonance imaging (MRI), and CSF data were collected. Routine CSF work-up comprised red blood cell (RBC) and white blood cell (WBC) count, CSF total protein concentration, CSF/serum albumin quotient, and CSF and serum IgG, IgM, and IgA concentrations. Intrathecal synthesis of IgG, IgM and IgA were calculated by the Auer and Hegen formula  and exprThe presence of MOG-IgG was determined by live cell-based immunofluorescence assay with HEK293 cells transfected with full-length human MOG , as previously described . ScreeniThe study was approved by the Ethics Committee of the Medical University of Innsbruck . Written informed consent was obtained from all patients. Authorization has been obtained for disclosure (consent to disclose) from the index case patient.2 tests were applied, as appropriate. Two-sided P-values <0.05 were considered statistically significant.Statistical analysis was performed using SPSS 26.0 . Non-parametric data were displayed as median and interquartile range. Categorical variables were reported as frequency and percentage. For group comparisons, Mann-Whitney-U and χWe conducted a literature search in MEDLINE and Google Scholar. Search terms were: VZV AND MOG or AQP4 or NMOSD or LETM; herpes zoster AND MOG or AQP4 or NMOSD or LETM. Abstracts that primarily did not deal with VZV infection and MOGAD or NMOSD or LETM were excluded. In addition, articles identified in reference lists of the individual papers were selected if considered appropriate.A 30-year-old, previously healthy man presented in 2019 with sensomotor paralytic syndrome (sensory level below T6), subsequent gait ataxia and neurogenic bladder disturbance requiring catheterization at our emergency department. Six days before the first neurological symptoms, the patient had developed herpes zoster infection (dermatome T6 right side) treated by his general practitioner with oral acyclovir . There was no history of constitutional symptoms or a recent vaccination. A MRI of the spinal cord showed a T2 hyperintense lesion extending from T1 to conus medullaris confined to gray matter Figure 1A total of 59 patients with neurological involvement due to VZV infection and 34 patients with neuroborreliosis were included into this study Figure 2All patients – those with VZV infection with CNS involvement and those with neuroborreliosis – were negative for serum MOG-IgG. One patient with VZV infection and radiculitis had a borderline MOG-IgG positive titer of 1:160. This result was not confirmed by using heavy chain–specific secondary antibodies and was therefore regarded as negative.We identified 2 case reports , 25 and Here, we present a case with MOG-IgG associated LETM triggered by VZV infection. In a subsequent retrospective study of 59 patients with VZV infection and neurological involvement; however, we did not found MOG-IgG in any patient .MOG-IgG are more often present in children than in adults and are associated with a variable clinical spectrum. Typical clinical presentation of MOGAD, particularly in children, is an acute disseminated encephalomyelitis (approximately 50%), whereas in adults myelitis (up to 30%) or optic neuritis (up to 50%) are more common , 26–28. The pathophysiological basis of the association between VZV infection and MOG or AQP4 antibody-associated associated LETM is unclear. In the majority of the reported cases, CSF VZV DNA PCR results were negative Table 3,A limitation of our study is the retrospective design and small number (n=15) of patients with myelitis, encephalomyelitis, or encephalitis. However, data specifically excluded a nonspecific bystander reaction, as patients with VZV infection without parenchymal involvement were negative for MOG-IgG (n=44). As CSF analysis of MOG-IgG improve the sensitivity by 7%, another limitation is that MOG-IgG was only tested in patient sera .Overall, we showed that the presence of MOG-IgG is a rare finding in patients with a VZV infection complicated by CNS demyelinating-inflammatory diseases. Nevertheless, due to therapeutic implications, antibody screening is a useful tool, particularly in patients with a higher pre-test probability, e.g. with LETM. Further prospective larger studies, including children, are required to analyze the frequency of neurological antibody-associated diseases triggered by VZV infection.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical University Innsbruck. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FDP conceptualized the study, collected data, case analysis, statistical analysis, drafted the manuscript, and revised the manuscript for intellectual content. PM collected data and revised the manuscript for intellectual content. KB collected data and revised the manuscript for intellectual content. MA collected data and revised the manuscript for intellectual content. AB collected data and revised the manuscript for intellectual content. TB case analysis and revised the manuscript for intellectual content. GB collected data and revised the manuscript for intellectual content. PR MRI analysis and revised the manuscript for intellectual content. KS collected data and revised the manuscript for intellectual content. AZ collected data and revised the manuscript for intellectual content. FDe case analysis, conceptualized the study, and revised the manuscript for intellectual content. MR conceptualized the study, and revised the manuscript for intellectual content. HH conceptualized the study, collected data, case analysis, statistical analysis, drafted the manuscript, and revised the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.This study was funded by a research grant from the Austrian Science Fund .FDP has participated in meetings sponsored by, received honoraria  or travel funding from Almirall, Bayer, Biogen, Celgene, Janssen, Merck, Novartis, Sanofi-Genzyme, Roche and Teva. Her institution has received research grants from Roche. KB has participated in meetings sponsored by and received travel funding from Roche. MA received speaker honoraria and/or travel grants from Biogen, Merck, Novartis and Sanofi. AB has participated in meetings sponsored by Merck and Biogen. TB has participated in meetings sponsored by and received honoraria  from pharmaceutical companies marketing treatments for multiple sclerosis: Almirall, Biogen, Biologix, Bionorica, Celgene/BMS, GSK, MedDay, Merck, Novartis, Roche, Sandoz, Sanofi/Genzyme, TG Pharmaceuticals, TEVA-ratiopharm and UCB. His institution has received financial support in the last 12 months by unrestricted research grants  and for participation in clinical trials in multiple sclerosis sponsored by Alexion, Bayer, Biogen, Merck, Novartis, Roche, Sanofi/Genzyme, and TEVA. GB has participated in meetings sponsored by, received speaker honoraria or travel funding from Biogen, Celgene, Lilly, Merck, Novartis, Roche, Sanofi-Genzyme and Teva, and received honoraria for consulting Biogen, Celgene, Roche and Teva. AZ has participated in meetings sponsored by, received speaking honoraria or travel funding from Biogen, Merck, Sanofi-Genzyme and Teva. FDe has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Almirall, Alexion, Biogen, Celgene, Genzyme-Sanofi, Merck, Novartis Pharma, Roche, and TEVA ratiopharm. His institution has received research grants from Biogen and Genzyme Sanofi. He is section editor of the MSARD Journal (Multiple Sclerosis and Related Disorders). MR was supported by a research support from Euroimmun and Roche. His institution receives payments for antibody assays  and for MOG and AQP4 antibody validation experiments organized by Euroimmun . HH has participated in meetings sponsored by, received speaker honoraria or travel funding from Bayer, Biogen, Merck, Novartis, Sanofi-Genzyme, Siemens, Teva, and received honoraria for acting as consultant for Biogen and Teva.The remaining 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.


DANG) to locate malignant gliomas and guide accurate excision. Both peptides/Cy7-SPIONs probes displayed excellent tumour-homing properties and barrier penetrating abilities in vitro, and both could mediate precise aggregation of the nanoprobes at gliomas sites in in vivo magnetic resonance imaging (MRI) and ex vivo near-infrared (NIR) fluorescence imaging. However, compared with ANG/Cy7-SPIONs probes, DANG/Cy7-SPIONs probes exhibited better enhanced MR imaging effects. Combining all these features together, this MRI/NIR fluorescence imaging dual-modal nanoprobes modified with retro-enantio isomers of the peptide have the potential to accurately display GBMs preoperatively for precise imaging and intraoperatively for real-time imaging.Glioblastoma (GBM), one of the most common primary intracranial malignant tumours, is very difficult to be completely excised by surgery due to its irregular shape. Here, we use an MRI/NIR fluorescence dual-modal imaging nanoprobe that includes superparamagnetic iron oxide nanoparticles (SPIONs) modified with indocyanine (Cy7) molecules and peptides (ANG or  At present, surgical operation combined with both radiotherapy and chemotherapy is still the most common treatment method for GBM.6 However, using the current enhanced MRI technology, over 10% of GBMs and 30% of undifferentiated astrocytic GBMs cannot be identified. The failure of glioma imaging is mainly attributed to the distinctive characteristics of this intracranial tumour.7 The blood-brain barrier (BBB) is an important transit barrier that controls the exchange of substances between the blood and the central nervous system (CNS), maintaining homeostasis of the CNS.8 Due to the tight connection between cerebrovascular endothelial cells, which hinders the transport of substances, it is difficult for probes to enter the lesion area in the brain. Similarly, the identification of GBMs with clinical MR contrast agents has limited performance due to their inability to penetrate physiological or pathological BBB. Furthermore, these contrast agents have poor specificity and a short residence time in vivo. Therefore, it is of great significance to develop tumour-targeting imaging probes for the preoperative diagnosis of glioma and accurate location of tumour margins.Magnetic resonance imaging (MRI) is one of the most important preoperative diagnostic methods for intracranial GBMs. To improve the sensitivity of contrast among different tissues, enhanced MRI with contrast agents, such as Gd-DTPA, is used to show a macroscopic region of the GBM.12 In this study, considering the characteristics of gliomas, we focused on MRI/optical imaging. MRI has a superior temporal and spatial resolution, excellent soft-tissue contrast resolution and no depth limit.13 However, its image acquisition time is long, so it is very susceptible to the influence of tissue movement, which produces motion artefacts.14 On the other side, optical imaging has the ability of continuous imaging but has poor image resolution and limited light penetration,15 so its application in intraoperative navigation is relatively limited. Fortunately, near-infrared (NIR) fluorescence imaging, attributed to the properties like reduced light scattering, higher signal-to-noise ratio, relatively higher penetration depth and image resolution,16 has been currently popular among the exploitation of intraoperative navigation. Practically, some NIR fluorescent dyes, such as indocyanine green (ICG) and methylene blue, have been approved by Food and Drug Administration for clinic use, indicating great clinical value of NIR fluorescence imaging.18 In some of the latest studies, a variety of NIR dyes after specific modifications could bring signals to specific lesion areas such as various tumours from different origins,20 and specific tissues,16 to guide the operators for further medical actions. Therefore, by combining these two imaging techniques, MRI and optical imaging, we can obtain both accurate preoperative diagnostic information and intraoperative pathological information to provide guidance for a more precise surgical resection of tumours.With the rapid development of multimodal imaging, the construction of imaging nanoprobes based on molecular biology, nanomaterials and molecular imaging increase the possibility for specific tissue imaging. Multimodal imaging, such as MRI/optical imaging, MRI/radionuclear imaging, and radionuclear imaging/optical imaging, etc., integrates their advantages, overcomes the limitations of a single imaging model and finally improves the clinical imaging specificity and sensitivity.23 Some particular molecules that cross the BBB can also function as transmembrane transporters by receptor-mediated transfection (RMT) or adsorption-mediated transfection (AMT).24 Therefore, the design of cross-BBB shuttle tools should achieve physical material delivery to the brain.The application of an endothelial cell-mediated endocytosis transport mechanism is an effective strategy for the delivery of probes into the brain. Most small-molecule compounds and polar molecules, such as glucose, amino acids, and short peptides, have specific vectors on endothelial cells.25 However, the peptide sequence is commonly linear in structure and is composed of L-amino acids, which makes it easy to be degraded by the protease in vivo and further reduces the targeting efficiency. Fortunately, we can improve the antiprotease degradation function of peptides by utilizing nonnatural amino acids, such as cyclic peptide chains, or the application of D-amino acids.28 As reported, the retro-enantio isomers of peptides show anti-enzyme degradation properties while preserving the topological characteristics of the sequence, becoming highly enzyme-resistant peptide analogues.31Ideal BBB shuttle carriers exhibit the following characteristics: (1) BBB receptors are highly expressed at the cavity site in cerebrovascular cells; (2) they are able to mediate transcytosis; and (3) they possess high flux and extensive substrate recognition capabilities. Peptides, as ligands, have obvious advantages, such as simple chemical synthesis, low immunogenicity, easy modification, and low influence on the function of delivery.34 Therefore, angiopep-2 (ANG) is a promising ligand to trigger transcytosis.34 It not only traverses the BBB but also targets glioma cells by recognizing LRP-1. To enhance the stability of ANG in vivo, we used the retro-enantio isomer of ANG (DANG) to overcome degradation by various enzymes in the blood and cells. Finally, we constructed a new dual-modal imaging probe for effective GBM imaging by combining superparamagnetic iron oxide nanoparticles (SPIONs) with the NIR fluorescent dye indocyanine (Cy 7) and targeting peptides (ANG/DANG).The aim of this study is to introduce targeting ligands with high affinity and stability for the construction of imaging nanoprobes. Low-density lipoprotein receptor protein 1 (LRP-1) is highly expressed on brain capillary endothelial cells and glioma cells.DANG were separated and identified by HPLC and MS. In HPLC analysis, ANG and DANG peptides reached their single distinctive peaks at 10.344 and 12.088 min, respectively, with their purity reaching 95%  , oxygen (O) and carbon (C) were analysed by energy dispersive spectroscopy (EDS)  imaging showed that the obtained PEG-DSPE-SPIONs had a cubic spinel crystal structure Fig. , which wDANG) and Cy7 dyes were conjugated to the PEG terminus through a condensation reaction. UV–vis spectroscopy was used to test whether the peptides had successfully attached to the surface of PEG-DSPE-SPIONs, and the UV–vis absorption spectra of the peptide solutions before and after the reaction were shown in Fig. DANG, respectively. The connection of Cy7 molecules and peptide-PEG-DSPE-SPIONs was detected by a multifunctional fluorescence microplate reader. After connecting the Cy7 molecule, the probes were excited by 720 nm excitation, and there was an obvious emission peak at 820 nm, which indicates successful connection of Cy7 molecules and peptide-PEG-DSPE-SPIONs. For 2 types of peptide-PEG-DSPE-SPIONs (ANG and DANG) containing 10 mg Fe3O4, the amounts of Cy7 molecules bound to the probes were 10.03 and 9.87 µg, respectively  of SPION cores is 78.24 emu/g  value, longitudinal relaxation (r1) value and r2 to r1 ratio are effective indexes that determine the imaging efficiency of MRI contrast agents. As shown in Fig. r2 values of Cy7-SPIONs, ANG/Cy7-SPIONs, and DANG/Cy7-SPIONs were 419.2 S−1 mM−1, 515.83 S−1 mM−1, and 536.28 S−1 mM−1, respectively; the corresponding r1 values were 2.1271 S−1 mM−1, 1.5537 S−1 mM−1 and 1.7444 S−1 mM−1  from healthy BALB/c mice injected with the peptide/Cy7-SPIONs suspension showed no noticeable abnormalities or tissue damage 1, 3 and 7 days after the mice were sacrificed, which indicated that the designed probes displayed nonvisible effects in vivo  measurement, the final in vitro BBB model TEER value was recorded in the following 10 days. As shown in Fig. −6 cm/s, which was significantly different from that of the blank group (Papp = 67.80 × 10−6 cm/s). These results indicated that the in vitro BBB model had a strong ability to limit permeability.Another test with NaFl showed that the NaFl Papp of the BBB model group was (11.24 ± 0.73) × 10DANG/Cy7-SPIONs probe solutions passing through the upper chamber were 158 ± 47.53, 624.6 ± 41.6, and 674 ± 62.22, respectively  between the peptide-modified groups and the control group, which indicated that the penetrating ability of the probes was significantly improved after peptide modification.The ability of probes to penetrate the in vitro BBB model was evaluated by measuring the fluorescence intensity of probes modified by peptides in the solution passing through the upper chamber in the Transwell and comparing the fluorescence intensity of probes without peptide modifications in the control group. 60 min after adding the same amount of the probes to the upper chamber, the fluorescence intensities of the Cy7-SPIONs, ANG/Cy7-SPIONs, and ely Fig. . ApparenWe conducted quantitative analysis using flow cytometry (FCM). As shown in Fig. T2WI and SWI .Ex vivo NIR fluorescence imaging was another method to confirm whether the probes we designed had successfully targeted the intracranial gliomas. The tumour size of tumour-bearing mice was confirmed by 7.0 T MRI. Then, NIR fluorescence imaging detected the aggregation of probes at glioma sites, and then the total fluorescence intensity at each site was calculated, which was effective for evaluating the average aggregation of probes in gliomas. Cy7-SPIONs probes had the lowest aggregation at glioma sites, while both ANG/Cy7-SPIONs and ive Fig. . The afoThe distribution in the brain was confirmed by scrutinizing slices of fixed brain tissue stained with Prussian blue, and the results showed that all probes aggregated at the margin of the tumours and that the probes modified by peptides had obviously higher aggregation than the unmodified probes  and retro-enantio angiopep-2 with PEG-DSPE-SPIONs. These nanoprobes show a dual-modal imaging capability that utilizes both MRI and NIR fluorescence imaging. Compared with previous methods, the clever synthesis method for these probes enables them to possess the key functions of glioma molecular probes, such as BBB penetration ability, glioma-targeted binding and a favourable safety profile.36 The introduction of Cy7 molecules helps facilitates the dual-modal imaging capability of the nanoprobes, and the peptides (ANG/DANG) allow the nanoprobes to penetrate the BBB and target glioma tissue.First, we used a well-designed method to synthesize the nanoprobes. In our study, we encased the SPIONs with DSPE-PEG to form PEG-DSPE-SPIONs, in which DSPE-PEG, because of its distinct hydrophilic-hydrophobic segment and self-assembly ability, could effectively mediate the formation of an iron oxide core, maintain its colloidal stability and monodispersity in an aqueous solution and prolong the in vivo circulation time of SPIONs.DANG peptide is a retro-enantio isomer of angiopep-2 (ANG), and it can also specifically bind to LRP-1 and then deliver the nanoprobes by receptor-mediated transcytosis. Compared with ANG, DANG has a weaker receptor affinity. However, thanks to its unnatural structure, it is resistant to protease degradation, hence acquiring a longer metabolism time in vivo to prolong the functional time of the nanoprobes, as proven by the former PET/CT assay. The results above are consistent with those in a previous report.37 The successful construction of the in vitro BBB model in our research allowed us to test the penetrability mediated by DANG, and the positive result shown in the aforementioned Transwell assay suggested excellent BBB penetrability of DANG/Cy7-SPIONs. In the cellular uptake assay, the performance of DANG/Cy7-SPIONs was similar to that of ANG/Cy7-SPIONs, while both performed significantly better than Cy7-SPIONs, indicating the ideal mediatory function of DANG for cellular internalization. All of these factors ensure the good performance of DANG-modified probes in the in vivo tests.The T2WI and SWI. T2WI is a basic magnetic resonance sequence and is widely used clinically.38 SWI uses the inhomogeneity of the magnetic field in the tissue to form an image, and is thus extremely sensitive to variations in the magnetic field.39 SPIONs probes exploit the characteristics of T2WI and SWI, and their aggregation in the tissue can strongly enhance the contrast of the image. In our research, DANG/Cy7-SPIONs probes were accurately targeted to gliomas and effectively enhanced their contrast to display a relatively dark signal on the image. This contrast enhancement effect was significantly better than that of either ANG/Cy7-SPIONs or Cy7-SPIONs probes, and it extended to the tumour edges, an ability that could not be achieved by the latter two probes. Consequently, DANG/Cy7-SPIONs probes can be used for precise imaging and provide guidance for precise surgical resection of glioma.Application for MRI is a core requirement for our probe. To optimize the performance of our probe, we used two MRI sequences in the test, DANG/Cy7-SPIONs probes acquire the capability of real-time fluorescence imaging and evolve into a dual-modal probe. In the corresponding experiments described in the previous sections, the inherent functions and superiority of the DANG/Cy7-SPIONs were shown. In vivo and ex vivo NIR fluorescence imaging successfully showed the location of the tumour, indicating that the DANG/Cy7-SPIONs probe can be used for intraoperative navigation. Furthermore, ex vivo NIR imaging of the isolated brain further reflected the function of intraoperative navigation. Consistent with the results of the MRI assay, the DANG/Cy7-SPIONs probe performed better and accumulated to higher levels in glioma margins than either the ANG/Cy7-SPIONs or Cy7-SPIONs probe, especially the latter, which showed almost no aggregation inside gliomas and exhibited an extremely fast metabolism. Specifically, DANG/Cy7-SPIONs could show the edge of the tumour when the tumour tissue is exposed, which helps to effectively remove the tumour tissue during the operation. Such a practice provides an important reference for the clinical translation of such probes in the future.Considering the advantages of NIR fluorescence imaging dyes in intraoperative navigation and fluorescence imaging, we introduce Cy7 dye to the probe, which help 40 Thus, the development of new contrast agents is one of the ways to improve biosafety. Previous report about biosafety of iron oxide nanoparticle (IONP) underlined that the biological safety of IONP was significantly better than that of GBCA, especially it would not cause nephrogenic systemic fibrosis or had a long retention in vital organs.41 Our findings supported such arguments, the cytotoxicity test indicated that the probe had no cytotoxicity. The results of histological examination of the tissue showed that the probe would not cause damages to organs and tissues, indicating a very good biosafety. Finally, the biodistribution of the probe suggested that the metabolism and distribution of the probe followed normal rules and that the probe would not remain in the vital organs of the body for too long, thus minimizing its impact on the body.Finally, the biosafety and biodistribution of the probe also need to be considered. Gadolinium-based contrast agents (GBCA) are commonly used clinical MRI contrast agents, but recent studies have shown that their biosafety cannot be guaranteed, for this type of contrast agent is associated with renal fibrosis and the nervous system deposition.DANG/Cy7-SPIONs nanoprobes possess good MRI and NIR fluorescence imaging efficiency simultaneously, which is valuable for preoperative diagnosis and intraoperatively locating gliomas. Consequently, we believe that they have the potential to stand out among next-generation glioma-targeting contrast agents.The current contrast agents used for diagnosis are generally restricted by their limited functions; thus, next-generation integrated contrast agents are in demand. Our group provides a novel approach to overcome this dilemma. The designed glioma-targeting 2.U87-MG (human GBM cells), HUVECs and bEnd.3  cells were obtained from the Institute of Basic Medical Science Chinese Academy of Medical Sciences . All types of cells were cultured in high-glucose DMEM (HyClone) containing 1% penicillin/streptomycin (HyClone) at 37 °C with 5% COµg/mL). With limited dilution, the individual clones of the transfected cells were screened for detecting the luciferase activity by an IVIS spectrum  for 4 weeks.Transfection and stable cell line generation: The luciferase plasmid provided by the State Key Laboratory of Biotherapy  was transfected into U87-MG cells using lentiviral vectors. Then, the transfected cells were selected and cultured using puromycin  and kept under SPF conditions. All animal experiments were performed with the approval of the Animal Care and Use Committee of Sichuan University.DANG peptides (Ac-yeetkfnnrkgrsggyfft-OH) were synthesized through a standard solid-phase synthesis method, which used active ester chemistry to couple the Boc-protected amino acid with the deprotected resin. The crude products were purified by high-performance liquid chromatography (HPLC) and identified by mass spectrometry (MS).Peptides: The ANG peptides (Ac-TFFYGGSRGKRNNFKTEEY-OH) and g, 10 min) and the precipitation was distributed to hexane. Finally, after the obtained SPION solution was centrifuged at 3000 × g to extract the aggregates, the DSPE-PEG 2000 AMINE molecule encased the SPIONs by hydrophobic action.PEG-DSPE-SPIONs: SPIONs were synthesized by using a hydrothermal method. First, 2 mmol of acetone iron, 10 mmol 1,2-diol, 6 mmol oleic acid, 6 mmol oil amide and 20 mL benzyl ether were mixed in a round-bottom flask, and the air in the reaction system was expelled by argon. Then, the mixture was heated to a constant temperature at 200 °C for 2 h and was heated to reflux at 300 °C for 1 h, and the mixture turned black. After the black mixture cooled to room temperature, ethanol was added to the system for precipitation. Then, the fluid was removed by centrifugation : Cy7 was provided by Professor Ke’s team .µL, 1 mg/mL) were added to the solution, and the solution was oscillated at 4 °C in the dark overnight. At the end of the incubation, the peptide/Cy7-SPIONs probes were obtained by removing the unreacted dye molecules via an ultrafiltration tube.Peptide/Cy7-SPIONs: PEG-DSPE-SPIONs were pretreated in 0.02 M borate buffer at pH 8 and filtered through a 10 kDa ultrafiltration tube. Moreover, peptides (2 mg) were dissolved in 0.02 M MES buffer at pH 5.5. To activate the carboxyl group of the peptides, 1 mg EDC molecules and 0.5 mg NHS molecules were added into the solution and shaken at 180 rpm with a constant temperature at 25 °C for 25 min. After activation, the solution was centrifuged twice in 2 KDa ultrafiltration tubes to remove the unreacted EDC/NHS . Then, the activated peptide and PEG-DSPE-SPION solutions were blended together and reacted in a shaker for 2 h. When the reaction ended, the unreacted peptides were expelled by a 30 kD ultrafiltration tube. Cy7-NHS molecules . 68Ga were chelated after pH adjustment (pH = 4) with sodium acetate. The reaction mixture was heated to 95 °C for 10 min, and the completeness of the reaction was checked by radio-liquid chromatography. The 68Ga compounds (68Ga-DOTA-peptides) were processed by solid-phase extraction before PET.2-PEG-DSPE-SPIONs probe was measured by SAED. Moreover, the particle size distribution of various nanoparticles was measured by DLS .The morphology of the SPIONs probes was visualized by transmission electron microscopy (TEM). The NH2-PEG-DSPE-SPIONs probe at 300 K were acquired by vibrating sample magnetometry  by using an applied field from 0 to 2 T.Hysteresis curves of the NH2-PEG-DSPE-SPIONs were detected by an ultraviolet spectrophotometer . The amount of peptide remaining in the solution collected after the reaction was determined by HPLC  and then compared with the total peptide content involved in the reaction to obtain the coupling efficiency. The introduction of fluorescent molecules was detected by a microplate reader .The successful introduction of the peptide structure can be confirmed by comparing the decreased spectra at 280 nm before and after coupling. The spectral changes in peptide/Cy7-SPIONs and NHT-2weighted MRI was conducted on a 7.0 T MRI system, and the r2 value was determined by the formula 1/T2 = 1/T20 + r2 CFe .The magnetic resonance relaxation data were acquired by using a 7.0 T MRI scanner . The probes were dissolved at gradient concentrations in PBS containing 1% agarose and were placed in a series of tubes. 4 cells in each well and cultured at 37 °C with 5% CO2. After 24 h, probe-based medium with gradient Fe concentrations of 0.000, 3.125, 6.250, 12.500, 25.000 and 50.000 µg/mL was added to the plate, and the cells were cultured for another 24 h. Then, the cells were washed with PBS and incubated in 100 µL of CCK-8 solution (10 µL of stock solution and 90 µL of DMEM) for another 2 h. Finally, the absorbance was measured at a wavelength of 450 nm.The cytotoxicity of the probes was analysed with a CCK-8 kit. U87-MG and human umbilical vein endothelial cells (HUVECs) were inoculated in 96-well plates with 1 × 10DANG were labelled separately with 68Ga-DOTA. First, 5 × 105 cells were plated in six-well plates overnight prior to use. Then, the cells were incubated with 74 kBq of 68Ga-DOTA-DANG or 68Ga-DOTA-ANG for 1 h. In the competitive group, a 20-fold molar concentration of nonlabelled peptide was added 1 h prior to adding 68Ga- labelled peptide. The medium was removed after incubation. Then, the cells were washed three times with PBS and lysed with 0.1 M NaOH. All cell lysates were collected, and the radioactive particles were counted with a Wizard 2470 gamma counter .For in vitro evaluation of the affinity of peptides for cells, ANG and 68Ga-DOTA-DANG or 68Ga-DOTA-ANG via the tail vein, and imaged at 1 h post injection. PET data were acquired for 10 min and reconstructed with a three-dimensional ordered-subset expectation-maximization (3D-OSEM) algorithm with a Monte Carlo-based accurate detector model. CT acquisition was performed with 50 kV, 1 mA X-ray output and a total acquisition time of 140 s.For in vivo evaluation of peptide stability, PET/CT imaging experiments were performed on an IRIS small animal PET/CT imaging system . Each animal was injected with 3.7 MBq of DANG/Cy7-SPIONs probe solutions were mixed with equal volumes of human serum and preincubated in a shaker at 37 °C for 3 h.Preincubation of probes: To investigate the stability of probes in vitro, ANG/Cy7-SPIONs and Cellular uptake analysis: Quantitative analyses of the cellular uptake of probes were conducted by flow cytometry .2. When the cells grew in good condition, they were distributed to a 12-well plate at a density of 5 × 105 cells per well. After cell adhesion, two types of 50 µg Fe3O4/mL peptide/Cy7-SPIONs solutions and two types of 50 µg Fe3O4/mL preincubated peptide/Cy7-SPIONs solutions were added to the medium to coculture with the cells for another 4 h, respectively. Afterward, the cells were washed with PBS and collected in 200 µL PBS for FCM assays in which the signal channel was switched to APC-A750, and the acquired results were analysed by FlowJo.The cellular uptake efficiency of probes and preincubated probes was measured by FCM. U87-MG cells and bEnd.3 cells were cultured in high-glucose DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO42To evaluate the penetrating ability of the probes, we constructed an in vitro BBB model and tested permeability with a method modified from a previous literature.2. Moreover, a layer of a collagen solution was smeared on the inner side of the polycarbonate Transwell  and dried naturally on a clean bench for 30 min. Then, bEnd.3 cells were inoculated into the upper chamber of 24-well transwells units at a density of 5 × 104 cells per well. Two days later, U87-MG cells were inoculated into the lower chamber at a density of 1 × 105 cells per well and cocultured with bEnd.3 cells for one week.Construction of the in vitro BBB model: U87-MG cells and bEnd.3 cells were initially cultured in high-glucose DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% COµL medium and 900 µL medium were added to the upper chamber and the lower chamber respectively, then the fluid level in each chamber was recorded. After 4 h, the fluid level was checked to determine whether there were some changes.In vitro BBB permeability test: The first test was the leak test, 200 ® ERS-2 Electrical Resistance System . After U87-MG cells were inoculated into the lower chamber, TEER measurements were performed once a day at the same timepoint of cell seeding for 7 consecutive days. In addition, the resistance of the blank culture transwell unit was recorded once daily. Then, the final in vitro BBB model TEER value in Ω·cm2 was attained by subtracting the TEER value of the blank culture transwell unit from the TEER value of the in vitro BBB model transwell unit. Results were evaluated as referenced in a previous review.43TEER measurements were implemented by using a MillicellIn addition, immunofluorescence was used to test in vitro BBB permeability. Endothelial cells (bEnd.3 cells) from the in vitro BBB model were fixed with a 4% paraformaldehyde solution for 30 min at room temperature and washed twice with PBS. After fixation, the cells were incubated with 3% BSA in PBS for 1 h to block the nonspecific binding of antibodies. Then, bEnd.3 cells were incubated with rabbit polyclonal antibodies against ZO-1  at 37 °C for 1 h. After being washed with PBS three times and incubated with FITC 488-conjugated anti-rabbit IgG (Invitrogen) at 37 °C for 1 h, the stained cells were re-washed with PBS three times. Subsequently, the cells were further stained with DAPI for 10 min and observed by confocal microscopy .44 First, NaFl was resolved in PBS to produce a series of standard solutions with NaFl concentrations of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 µg/mL. The absorbance of standard solutions was obtained by a multidetection monochrometer microplate reader, and subsequently, a NaFl concentration-absorbance curve was made. With the help of the curve, we measured the in vitro BBB permeability with NaFl. Then, 200 µL NaFl solution (10 µg/mL) and 900 µL PBS were added to the upper chamber and the lower chamber, respectively, to continue culturing the cells at 37 °C with 5% CO2. A 100 µL aliquot of solution from the lower chamber was extracted to a 96-well plate after 30, 60 and 90 min while maintaining 900 µL of PBS in the lower chamber. Then, a multidetection monochrometer microplate reader (485/535 nm) was used to measure the amount of NaFl in the upper chamber. The apparent permeability coefficient (Papp) was calculated as follows:Another test taken as an example from a previous study using sodium fluorescein (NaFl) was also introduced to evaluate in vitro BBB permeability.Q/dt × 1/(A × C0)Papp (cm/s) = ddQ/dt: the transfer rate of NaFl from the upper chamber to the lower chamber.2).A: membrane surface area .µL of PBS with 10.0 µg/mL Cy7-SPIONs (control) solution and 200 µL of PBS with two types of 10.0 µg/mL peptide/Cy7-SPIONs solutions were added to the upper chamber, and 900 µL PBS was added to the lower chamber. After a 60-minute incubation, 100 µL of solution from the lower chamber was extracted and distributed to a black 96-well plate in which a multifunctional fluorescence microplate reader  was utilized to examine the fluorescence from the probes to determine how many probes had penetrated the in vitro BBB model.After successfully constructing the in vitro BBB model, PBS was used to rinse the Transwell, 200 2.45 As the cells grew well, trypsin was used to digest the cells so that the initial cell assembly broke into fragments for the following in situ glioma inoculation. Then, the mice received 5 × 104Luc-U87-MG cells per mouse by injection of a 5 µL single-cell suspension via surgery. Three weeks after the surgery, an IVIS imaging system was used to observe and confirm the success of the model.An in situ glioma model was established in BALB/c nude mice with weights of ~20 g and ages of 5–6 weeks. Luc-U87-MG cells were first cultured in high-glucose DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% COT2-weighted imaging (T2WI) and susceptibility-weighted imaging (SWI) MRI. The parameters are listed as follows:In vivo MRI studies were based on tumour-bearing nude mice in which in situ glioma models were successfully established. It was carried out on a 7.0 T magnetic resonance instrument  by acquiring brain section images via T2WI: TE = 33 ms; TR = 2520 ms; slice thickness (SL) = 0.6 mm; field of view (Fov) read = 20 mm; matrix size = 256 × 256; NEX: 3.SWI: TE = 11 ms; TR = 581 ms; slice thickness (SL) = 0.6 mm; field of view (Fov) read = 20 mm; matrix size = 256 × 256; NEX: 2.DANG/Cy7-SPIONs probes (10 mg Fe/kg dose) were injected into the other two groups.To investigate the performance of different peptide SPIONs probes in penetrating the BBB and stability in vivo, the mice were divided into 3 groups. The first group was the control group, in which Cy7-SPIONs probes (10 mg Fe/kg dose) without peptides were injected into the mice, while ANG/Cy7-SPIONs and T2WI and SWI were obtained for the first time. Then, T2WI and SWI images were obtained at 0 min, 2 h, 4 h and 24 h after the injection of the probes.The mice in each group were first anaesthetized, and images of DANG/Cy7-SPIONs probes. One hour later, mice were anaesthetized for craniotomy. Then, the brain tissue where the glioma was located was exposed, and the mice were imaged with an IVIS imaging system  for in vivo intraoperative imaging of the gliomas.Tumour-bearing nude mice were divided into three groups and injected with Cy7-SPIONs, ANG/Cy7-SPIONs or After finishing the in vivo imaging experiments, the isolated brains of mice were fixed for 2 days and completely dehydrated with a 30% sucrose solution three times. Then, the fixed brains were embedded with optimal cutting temperature (OCT) compound in a cryostat to obtain frozen slices. The slices were stained with DAPI and finally placed in an automated quantitative pathology imaging system  to complete the imaging.3O4/kg Cy7-SPIONs, ANG/Cy7-SPIONs or DANG/Cy7-SPIONs. One hour later, the mice were sacrificed by cardiac perfusion with saline and 4% polyformaldehyde. Then, the brains and main organs were extracted for glioma imaging, and the probe distribution in organs was observed via an IVIS imaging system .NIR fluorescence imaging of intracranial glioma required craniotomy, so the in vitro imaging method was chosen. The tumour-bearing mice were divided into three groups and received 10 mg FeThe distribution of probes in the brain was confirmed with the following process. After finishing the imaging experiments, the isolated brains of mice were fixed for 2 days and completely dehydrated with a 30% sucrose solution three times. Then, the fixed brains were embedded with OCT compound in a cryostat to obtain frozen slices. Prussian blue was used to stain the slices for histological examination through an optical microscope.3O4/kg Cy7-SPIONs, ANG/Cy7-SPIONs or DANG/Cy7-SPIONs solutions). All mice were sacrificed 24 h after the injection, and the main organs were extracted. For the histological examination, the organs were embedded in paraffin, sectioned (4 µm thick), stained with haematoxylin and eosin (H&E) and finally observed under an optical microscope. For the quantitative distribution of probes in mice, the main organs were cut up and digested in 1 mL hydrogen peroxide and 2 mL nitric acid for 2 days. The mixture was then placed in an oil bath at 120 °C until no precipitation remained. After it fully evaporated to dryness, 5 mL of 2% nitric acid was added to measure the volume. Processed by ultrasound for 20 min, 4 mL of solution was transferred to an Eppendorf (EP) tube, and the iron content was measured by inductively coupled plasma-mass spectrometry .The distribution of probes in other organs was confirmed with the following process. A number of healthy BALB/c mice without tumour implantation were divided into four groups, one of which was the control group (injected with 0.9% NaCl solution), while the other five were the experimental groups (injected with 10 mg Fep < 0.05 was considered statistically significant. The data are presented as the means ± SEMs. ANOVA and Student’s two-tailed t-tests were used to analyse the data.For all the data, Supplementary Materials for Retro-enantio isomer of angiopep-2 assists nanoprobes across the blood-brain barrier for targeted magnetic resonance/fluorescence imaging of glioblastoma


We reported the S. aureus (n=315) and CoNS (n=100) clinical isolates expressing various resistance phenotypes. The isolates were collected in 2002-2019 from medical centers located in the United States , Europe , Asia-Pacific region , and Latin America . These isolates originated mostly from the year 2019 (n=323).The isolates were susceptibility tested by the CLSI broth microdilution method. MIC interpretations were based on CLSI and EUCAST criteria where available.LSVT-1701 and comparators were tested against 415 S. aureus and CoNS isolates with MIC90 values of 2 mg/L for all S. aureus, methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and CoNS (Table). The highest LSVT-1701 MIC values were 4 and 8 mg/L among S. aureus and CoNS, respectively. LSVT-1701 retained potent activity against S. aureus isolates showing resistance or decreased susceptibility to oxacillin, vancomycin, teicoplanin, telavancin, linezolid, daptomycin, ceftaroline, or lefamulin; MIC50 values ranged from 0.5 to 1 mg/L and MIC90 values ranged from 1 to 4 mg/L among S. aureus resistant subsets.LSVT-1701 was highly active against Summary of LSVT-1701 activity against S. aureus, CoNS and resistant subsetsin vitro activity against contemporary clinical isolates of S. aureus and CoNS collected from medical centers worldwide and against resistant S. aureus isolates with uncommon resistance phenotypes. The results of this study support further clinical development of LSVT-1701 to treat staphylococcal infections.LSVT-1701 demonstrated potent David Huang, MD, PhD, Lysovant (Consultant) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Paul R Rhomberg, Cidara Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) Katyna Borroto-Esoda, PhD, Lysovant (Consultant) Eric Gaukel, BS, Lysovant (Employee)


Since its launch in 2006, Twitter has become a commonly used platform for sharing medical information, especially in the field of oncology. However, its role and impact on young oncologists’ education remain unclear. Moreover, COVID-19 and congress virtualization is likely to have modified Twitter use by the medical society.We conducted a national survey (27 questions) in France among medical oncology, hematology, and radiation therapy young doctors to help better understand the role played by Twitter on their medical education. One hundred eighty-three young oncologists participated in our survey. A majority does not use Twitter (72.1%), mostly to reduce their time spent on social media. Participants using Twitter (27.9%) often use it more than once a week, mostly by scrolling on their news feed. Interestingly, they rarely express their own opinion on Twitter: a majority of them (75.5%) tweet less than once a month while the rest of them mostly retweet others’ tweets. They mainly follow English-speaking experts, scientific societies, and medical journals. Pharmaceutical laboratories’ accounts are of less significance. Overall Twitter usage seems increasing since COVID-19 pandemic and the consequent digitalization of congresses. No statistical difference was observed between the baseline characteristics of Twitter users and non-users.This survey shows that Twitter is a relevant mean of continuous medical education used by around a third of French young oncologists, especially since COVID-19 pandemic and the virtualization of congresses. This media should be considered and evaluated for its educational advantages or potential biases.The online version contains supplementary material available at 10.1007/s13187-021-02119-7. For the last two decades, Internet has deeply modified our way to practice and learn medicine. Social networks have become ubiquitous, and with them, the conventional education means have been overturned. More and more students now use digital media for their formation, including medical education .Twitter is a microblogging network that was created in 2006, where users can share contents within a 140-character limit. It has quickly grown into one of the most used social networks, with around 330 million users in 2019, around half of which declare a daily use [Recently, COVID-19 pandemic has pushed the international medical community to attend online congresses, and medical content has increased on the platform . It is tTwitter has also become a customary tool for medical students: it seems complementary to academic courses , 7, and Moreover, even if the proportion of medical practitioners using this network seems rising, only a few express themselves, whereas a large majority retweet medical influencers’ content . YoungerWe conducted a national survey in France to help better understand the role of Twitter in the continuous medical education of young oncologists and its possible evolution due to COVID-19 pandemic and congress digitalization.We developed a questionnaire aiming at understanding the role of Twitter in the continuous formation of French young oncologists, which was validated by a working group comprised of resident and senior clinicians.Twenty-seven questions were asked, concerning medical curriculum (4 questions), the use of Twitter (2 questions), the characterization of Twitter non-users (5 questions), the characterization of Twitter users (10 questions), and the possible evolution due to COVID-19 and digitalization of congresses (6 questions) (see Supplementary 1).The survey was available online for 35 days, from September 14 to October 19, 2020. Residents and young oncologists (less than 40 years old) were invited via e-mail, website, and social networks (except Twitter) through the National Association of Medical Oncology Residents (AERIO), the Society of Young Radiation Oncologists (SFJRO), and the National Association of Hematology Residents (AIH) to participate in this nationwide prospective survey. Two reminder e-mails were sent to increase the number of responses. The questionnaire was accessible from a computer, tablet, or smartphone using Google Forms.2 test was used for large samples (n ≥ 60) and Fisher’s exact test was used for small samples (n ≤ 60). For each test, statistical significance was set at a two-sided p value of < 0.05.Categorical variables are described as frequencies (percentage). Subgroup analysis was carried out according to hospital status , medical specialty , and online congress attendee since the beginning of COVID-19 pandemic (yes/no). The chin = 93, 51.1%) followed by medical oncologists . Hospital status was quite well balanced, with a predominance of older residents   completed the whole questionnaire. Radiation oncologists were the most represented in this population  declare using Twitter as a medical media , the main reason was to reduce time spent on social medias , followed by the fear not to learn as much on Twitter as via other means  or that the invested time would be too important compared to the educational benefice  created their Twitter account during the residency and 4 out of 18 post-residency oncologists created their account after the residency.n = 31, 60.8%, Fig. n = 39), whereas the remaining 23.5% (n = 12) only retweet medical influencers’ contents. Besides, they had few followers , compared to the number of accounts followed .Twitter users seemed to use it intensively, with a daily use by 19 participants (37.3%), and more than once per week in a majority of cases (n = 41) followed scientific societies, and 78.4% (n = 40) medical journals. Literature monitoring accounts (such as @OncoAlert) were used by 24 participants (47.1%). Interestingly, pharmaceutical laboratories play a much poorer role with only 13.1% of participants (n = 7) following such accounts. 82.4% of participants mainly followed English-speaking accounts.All Twitter users followed world experts, 80.4% (n = 45), whereas 11.8% (n = 6) prefer exploring an account’s content, and the ideas and articles related to it. According to the users, Twitter’s main interest was to guarantee a fast and easy access to the opinion of world-class experts on medical news , more than the rapidity of access to medical information .Twitter users mainly spend their time on this media scrolling to read news  estimated that they used more Twitter than 6 months before and 43.1% (n = 22) estimated that it was stable. In 29.4% (n = 15) of cases, congress virtualization due to COVID-19 pandemic pushed the user to use Twitter more than they used to. Live tweets are regarded as a necessary complement to oral presentations by 81% (n = 30) of Twitter users.Among Twitter users, 41.2%  .On the other hand, close to a third of them use Twitter as a medical education tool and are clearly active: more than 40% connect every day. Twitter is for them a full part of their training, completing (and in some cases replacing) academic teaching. They prefer following expert accounts rather than journals or pharmaceutical companies. An explanation would be that they are willing to fully understand medical actuality through key opinion leaders, rather than just being informed. This need became stronger with the global pandemic and suspension of physical congresses. Students watching online talks alone are seeking for experts’ comments. In our study, more than 80% of them considered Twitter an essential complement to meetings, which is consistent with the rising use of Twitter during international congresses . HoweverIn our study, more than 40% of Twitter users increased their use of this social media since the beginning of COVID-19 pandemic. This underlines the challenge of digitalization of medical information, due to social distancing. Twitter plays an important role in such a process and can help users follow medical actuality when they cannot physically attend courses or congresses.In this survey, we failed to identify significant differences between these two profiles that determine their use of medical Twitter. First, it should be noted that the goal of our questionnaire was to focus on Twitter use, more than on the characteristics of users. Moreover, reasons for using or not using Twitter seemed unrelated to medical specialty or hospital status as they are mainly related to personal beliefs. We can therefore assume such a difference does not exist. In both cases, residents lack guidance for a wise use of Twitter as a professional tool, some of them thus deciding to avoid it, the others to abstain posting original content. An American study already demonstrated that only few physicians, known as “superusers,” mastering the codes of their specialty and social media, are responsible for the majority of tweets in oncology .Like Attai et al.  showed, COVID-19 pandemic has underlined the importance of medical media in the medical education of French residents, which contrasts with the lack of formation they receive on their use. To help residents optimize their time on social media, some societies have written recommendations , 23. TheIn conclusion, close to a third of young French oncologists are using Twitter as a tool in order to learn mostly from key opinion leaders. This phenomenon increased with COVID-19 pandemic and the access to many online talks without experts’ live commentaries. However, guidance is needed to make the most from the time spent on this platform and provide a clearer perspective to a majority of physicians regarding the value of Twitter and potentially encourage its use in order to avoid the “superusers” bias.Supplementary file1 (DOCX 13 KB)Below is the link to the electronic supplementary material.


With a particular focus on atherosclerosis imaging, we highlight recent approaches to report on the activity of cathepsins (K and B), matrix metalloproteinases (MMP-2 and MMP-9), thrombin, heme oxygenase-1 (HO-1) and myeloperoxidase (MPO).Enzyme-activated probes enable complex biological processes to be studied in real-time. A wide range of enzymes are modulated in diseases, including cancer, inflammatory diseases and cardiovascular disease, and have the potential to act as vital diagnostic and prognostic biomarkers to monitor and report on disease progression. In this perspective article, we discuss suitable design characteristics of enzyme-activated fluorescent probes for  This perspective provides an overview of the design requirements and fluorogenic mechanisms of enzyme-activatable probes in optical imaging, focusing particularly on detecting enzymatic targets overexpressed in vulnerable atherosclerotic plaque. Additionally, such probes have the advantage of visualising direct molecular processes and abnormalities, compared to Molecular Resonance Imaging (MRI) and Ultrasound (US) which provide much more detailed anatomical information.Optical Imaging (OI) is a powerful, highly sensitive and cost effective non-invasive diagnostic technique routinely used in chemical biology.2,3 pH fluctuations4 and biologically relevant enzymes5–7 in real-time. In particular, enzymes represent important biomarkers for diagnosis and monitoring the progression of a number of chronic diseases such as Parkinson's disease,8 arthritis9 and many types of cancer.10 Therefore, enzyme-activated fluorescent probes are an important area in this field.Over the years, a wide range of activity-based probes have been designed and developed to detect a number of metal ions,11 atherosclerosis is a chronic and often asymptomatic condition that is characterised by the narrowing of arteries following a steady build-up of plaque over a prolonged period of time.12 Atherosclerotic plaque broadly takes two phenotypes, named stable and vulnerable. One of the main challenges in this field is differentiating between these two dissimilar forms, particularly in the planning of medical interventions.One growing area of interest is utilising enzyme-activated fluorescent probes for the OI of atherosclerosis. Acting as a prerequisite to Cardiovascular disease (CVD) and stroke – two leading causes of death worldwide,13,14 Therefore, atherosclerosis is accelerated by IPH, and often results in the onset of ischemic stroke and myocardial infarction.15 In contrast, stable plaques display the opposite features and are significantly less prone to rupture.For example, vulnerable plaque is characterised by a high burden of inflammatory macrophages and a thin fibrous cap that is prone to rupture. One of the key processes in disruption is intraplaque hemorrhage (IPH), a bleed within the plaque itself, and strongly associated with more complete plaque disruption causing thrombosis and blockage of the lumen.In this perspective, we summarise the design aims and activation mechanisms that may guide the future development of enzyme-activated probes for disease diagnosis and prognosis. In particular, we focus on recently developed enzyme-activatable fluorescent probes for imaging atherosclerosis, and future directions that could be taken in this important area of fluorescence medical imaging. Chemical probes for other biological targets secreted by macrophages, such as cytokines, will not be discussed.2.Enzyme-activated probes are highly advantageous for OI applications, allowing molecular processes to be detected in real-time. Probes of this nature typically contain an enzyme-specific functionality and a fluorescent reporter moiety to provide the signal output. During the design and development process, a number of different factors must be considered, including the photophysical properties of the fluorophore and biological considerations of the probe prior to, and following activation. Moreover, these design requirements for enzyme-activatable fluorescent probes are inherently generalisable so would also apply to other medical OI probes in various biochemical contexts.2.1via a ‘turn-on/off’ mechanism. However, in some instances, the comparison of two or more wavelengths enables a ratiometric measurement. Such a ratiometric response is highly advantageous, due to enablement of self-calibration, eliminating artefacts resulting from the degree of cellular uptake or intracellular quenching during imaging.16 This facilitates much more precise assessment.As with all fluorescent probes used in OI applications, the signal-transduction and photophysical properties of enzyme-activated fluorogenic probes must be carefully considered. The fluorescence readout observed following activation is most commonly intensity-based 17 allowing an increased depth of tissue penetration. To this end, a range of fluorophores with a NIR-1 (700–900 nm)18 and NIR-2 (1000–1700 nm)19 spectral window have been developed in recent years for in vivo imaging applications. Additionally, probes that enable two-photon excitation are also extremely sought-after, due to the use of the lower energy excitation of two individual NIR photons.20In a biological setting a low-energy excitation (long wavelength) is extremely desirable for two reasons. The first is reduced absorbance by endogenous biomolecules such as hemoglobin,21 Finally, the fluorescent reporter must also be photostable with a high quantum yield (ϕ) and large extinction coefficient (ε).It is also important to consider the relative energy difference between the absorbance and emission of light of a particular fluorophore – termed the Stokes’ shift. Fluorophores that display a large Stokes’ shift are preferred and prevent the self-absorption of emitted light by molecules in the ground state.22 Additionally, the probe, and the products generated following enzyme activation, must be non-cytotoxic in order to prevent intracellular damage during in vivo imaging applications.In addition to the photophysical properties of the fluorophore, a number of biological factors must also be considered. The probe must respond selectively to the necessary enzymatic target in a complex biological environment at physiological pH.23 The unique properties of functionalised nanoparticles are often used to this end, as a strategy for the delivery of small molecule-based probes. One such approach, used in several probes designed for activation by proteases, is the use of a peptide-appended pegylated graft co-polymer to aid delivery through enhanced circulation of the agent.24–26 Additionally, functionalised liposomal nanoparticles can encapsulate an OI probe for delivery to a specific vascular site.27 In a very interesting approach, the endogenous protective nanoparticle High-Density Lipoprotein (HDL) has been reverse engineered as a potential carrier for therapeutic and imaging reagents.28,29 Furthermore, inorganic nanoparticles  and solid lipid nanoparticles34 have been developed as a carrier for an array of imaging techniques.In many instances, it is also desirable to incorporate biological targeting functionalities into probe design to ensure localisation in a specific region of the body or cellular compartment.35,36 An example by Urano and co-workers uses the specificity provided by antibodies to their cellular targets, in the application of activatable probe–antibody conjugates to cancer cells.37A further approach, used to good effect in the application of lipophilic cation-functionalised probes to facilitate mitochondrial localisation, includes a small targeting group on the probe scaffold itself.38The nature of the fluorophore itself can strongly influence the pharmacokinetic profile of the imaging agent, particularly in the case of extended systems for signal generation in high molecular weight probes. Therefore, an optimal balance between the desired NIR properties of a probe, and the undesirable perturbation it induces in the pharmacokinetics of a delivery system needs to be established. As such, strategies to improve localisation of an OI probe at the site of interest, through additional targeting moieties, encapsulation, or extended circulation times, are of great contemporary interest.2.2e.g. a specific, short peptide sequence) (e.g. an ester/thioester)  , or a fuioester) .Additionally, another common class of enzyme-activated fluorogenic probes involve the use of a self-immolative linker that masks the fluorescence of a fluorophore prior to enzymatic activation in the cellular environment. In this case, following enzyme activation, a chemical trigger group initiates a cascade of chemical reactions, initiating an increase in a fluorescence output .42,43Of course, other activation mechanisms are entirely possible too, but are not within the scope of this perspective. We point to two informative recent reviews in this area for a more detailed literature evaluation of a number of disease-relevant enzymes, particularly in oncology.44–46 Following selective enzymatic scission, a change in fluorescence response is observed, often due to the perturbation of Fluorescence Resonance Energy Transfer (FRET)††FRET is a distance–dependent interaction that involves the donation of energy from a donor to an acceptor fluorophore proving that there is a good spectral overlap, and there are in close proximity. between two fluorophores  acceptor acting as a fluorescent quencher . Due to this dual-signal approach, Cou-DEVD-TPET was used successfully to self-validate caspase-3 activity in live fluorescence imaging of HeLa and MDA-MB-231 cells.39Liu and co-workers used this approach to design a dual-signal ‘turn-on’ probe for caspase-3 – an enzyme responsible for the initiation and activation of cell apoptosis. Probe quencher .39 Follo‡PET is a non-radiative decay process that quenches luminescence. It is characterised by an electron transfer from the highest occupied molecular orbital (HOMO) of a receptor (electron donor) to the HOMO of a lumiphore in its excited state. prior to activation.47,48 A self-quenching mechanism can also be utilised to good effect with two fluorophores in close proximity, quenching the fluorescence prior to activation. Probes with these design features are collectively named enzyme “break-apart” probes, with enzymatic cleavage accompanied by a fluorogenic response.It is also important to note that in some cases, fluorescence quenching can occur by photoinduced electron transfer (PET)‡49–51 A similar functional group cleavage activation mechanism is observed for aminopeptidases, and this approach was used by Peng and Yoon to develop a CD13/aminopeptidases (APN) N-activatable fluorescent probe for tracking metastatic cancer  and cancerous cells (Hep-G2), an experiment designed to simulate the environment of a tumour.52 To achieve the mixed cultivation, cell planting and culture was achieved in a step-wise manner, within the same confocal dish over a 48 h period. A fluorescence signal of 655–755 nm was detected in the mixed culture and attributed to enzyme activation of YH-APN in Hep-G2 cells only. Furthermore, in the study, cancerous tissues were successfully identified by in situ spraying, outlining a high tumour-to-normal tissue ratio. Lesions smaller than 1 mm could be imaged precisely, superior to other diagnostic techniques used in clinical practice such as MRI and US. Such approaches, therefore, show a great deal of promise for use in image-guided surgery in the future.40Probes for other types of hydrolase enzymes, such as esterases and thioesterases, contain enzyme-cleavable ester and thioester functional groups respectively.c cancer . Probe Y41 All probes in this study contained a self-immolative benzyl thioether linker, that resulted in a bathochromic-shift in the absorbance and emission wavelength maxima following activation by the enzymatic target. For example, following detection of NTR , probe NTR-InD displayed a significant decrease in fluorescence at 612 nm (λex = 535 nm) and a 12-fold ‘turn-on’ in NIR fluorescence at 900 nm (λex = 730 nm). A highly favourable Stokes’ shift of 170 nm was displayed. Such a significant bathochromic-shift in fluorescence following enzyme activation enabled NTR-InD to be analysed in A549 cells by two-channel ratiometric fluorescence imaging. Additionally, promising results were displayed in a murine in vivo model where a time-dependent NIR fluorescence was displayed specifically in the tumour.41Finally, ‘turn-on’ fluorescent probes incorporating a self-immolative linker are increasingly being developed to detect disease-relevant enzymes. For example, in 2019 Zhao and co-workers developed a series of BODIPY-based activity probes for nitroreductase (NTR) and NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) for imaging hypoxia in cancer cells .41 All p3.31,53,54 OI,55 Photoacoustic Imaging (PAI)56 and Positron Emission Tomography (PET)57,58 have been developed, and summarised in an informative and recent review by Gao and co-workers.59Over the years an array of invasive and non-invasive medical imaging techniques has been used to detect the pathogenesis of atherosclerosis. Non-invasive imaging modalities utilising an assortment of biological targeting moieties for use in MRI,activatable fluorescent probes for enzymatic biomarkers associated with the diagnosis and pathogenesis of atherosclerosis. The primary challenge in this field is accurately differentiating between stable and vulnerable plaque, the latter of which is significantly more prone to rupture, due in part to the high burden of inflammatory macrophages.Here, in contrast, we summarise recent efforts to develop 60,61 Consequently, the development of chemical probes to monitor the enzymatic activity of macrophages is a popular approach to image plaque instability in atherosclerotic lesions.62 These include recent approaches to report on the activity of proteases , and thrombin), heme oxygenase-1 (HO-1) and myeloperoxidase (MPO). Due to commonality of molecular disease mechanisms, the enzymatic biomarkers developed for atherosclerosis may also be useful markers of other diseases, characterised by perturbation of the same enzyme activity.42,43,61Macrophages are responsible for the secretion of enzymes, reactive oxygen species (ROS) and cytokines as the primary response of a host to disease.3.1e.g. cathepsins, MMPs and thrombin) are a class of enzymes that are primarily responsible for the digestion of the extracellular matrix (ECM), the weakening of the fibrous cap, and subsequently leading to plaque destabilisation and rupture from this vulnerable state.63,64 Additionally, proteases play a major role in vascular remodelling, and over time can lead to the development of more advanced atherosclerotic plaques, and possible thrombus formation.As described in section 2, the design of enzyme-activatable probes for proteases predominantly involves the incorporation of a specific enzyme-cleavable peptide sequence as a linker between two fluorophores. Proteases  specific activation in a murine model of atherosclerosis.26 CatK-initiated cleavage results in the separation of the fluorophores and amplification of the fluorescent signal intensity. A 3.5-fold increase in signal intensity was seen compared to a peptide-based control probe, and a 13-fold increase compared to PBS (measuring autofluorescence). The structure of the probe is depicted in Cathepsins are a member of the cathepsin family of cysteine proteases, and two members of this family, cathepsin K and B, have been shown to be upregulated in atherosclerotic lesions.24,25 Indeed this probe, ProSense750/VM110, was used in the first demonstration of real-time intravascular Near-Infrared Fluorescence (NIRF) catheter-based imaging, by Jaffer and co-workers in 2008.66 Ultimately, two forms of commercially-available cathepsin-activated NIRF probes have been developed, based on the scaffold outlined above; ProSense 750/VM110, activated by cathepsins B, L and S, and ProSense680, activated by cathepsins B, L, S, K, V and D.67Earlier studies have also investigated a cathepsin B (CatB)-activated analogue in a murine atherosclerosis model.27 Rather, phosphatidylserine (PS)-functionalised liposomes were used as a macrophage-specific delivery mechanism. In this study, indocyanine green  that can be selectively cleaved by cathepsin B. Initially, the NIR emission of peptide-ICG2 was quenched 15-fold compared to ICG, assumed by the authors to be due to π-stacking between the two ICG moieties -functionalised liposome to target macrophages that had infiltrated vulnerable atherosclerotic plaque.69 For NIR-1 dyes, encapsulation in an nanocarrier platform can be highly advantageous as it dramatically increases the circulation time of the probe, allowing substantial accumulation in a biological target of choice. It is also thought that the stability of ICG and other cyanine dyes can increase considerably following encapsulation.70For ex vivo fluorescence imaging of the aortae of a murine imaging model (Apolipoprotein E knockout (ApoE−/−)) confirmed that the CatB-induced fluorescence signal observed was located in atherosclerotic plaque macrophages. Analogous results have been displayed in work by Kim and co-workers.71 In the latter, a weaker fluorescence intensity was displayed in mouse aortas with a high-fat diet following addition of atorvastatin, a known statin medication widely characterised to prevent cardiovascular disease by reducing plaque.71 Furthermore, it was suggested by the authors that a weaker fluorescence intensity correlates with more stable atherosclerotic plaque, an outcome that could not be predicted from a visual inspection.71 However, without any internal calibration or ratiometric response, this must be expressed with some caution as intensity-based probes can have systematic errors such as differing cell uptake, potentially affecting the intensity of the fluorescence output observed. One major limitation of self-quenching fluorophores is this inherent difficulty in obtaining ratiometric measurements. Such a response is achieved more readily when a donor–acceptor FRET quenching mechanism is employed.NIR Taken together these results are still very encouraging and demonstrate that OI probes for cathepsins are excellent candidates for imaging plaque vulnerability.3.1.272–74 Whilst MMP activity probes have been used to image and measure in atherosclerotic plaques, these are more limited.Matrix metalloproteinases (MMPs) are a family of 23 zinc-dependent proteinases associated with various physiological and pathological process. Consequently, a large number of enzyme-activatable probes have been developed to detect MMP activity in fluorescence cancer imaging.via a GGPRQITAG peptide sequence on a pegylated polylysine backbone.75 The NIR probe was based on work by Huang and co-workers,76 and demonstrated a 200-fold increase in fluorescence intensity following cleavage by MMP-2 and MMP-9.75Ex vivo fluorescence imaging of atherosclerotic aortas of a ApoE−/− murine model showed an MMP-2/MMP-9 specific increase in NIR fluorescence , the non-ratiometric behaviour of these probes would limit precision of in vivo imaging applications.In a notable example, Deguchi and co-workers developed a NIR Cy5.5-based self-quenching fluorogenic probe incorporating two Cy5.5 fluorophores linked rescence . Further3.1.377 directing researchers to invent a number of probes to detect and report on its activity. For example, Tung and co-workers have developed a thrombin-activated molecular NIR probe based upon a similar co-polymer scaffold to that employed in the group's other published works for imaging macrophage protease activity.78Thrombin is another serine protease with a significant role in a number of normal and pathological biological processes, including the development of vascular thrombosis. The enzyme is implicated in the pathophysiology of atherosclerosis in several ways,in vitro, using both exogenous thrombin in human blood  or following addition of CatD or CatB . Additioan blood  and in a3.2The vast majority of probes described in section 3.1 make use of a short, cleavable peptide sequence to facilitate enzymatic cleavage at sites of interest. However, a focus on different enzyme targets can facilitate activatable NIR probes based on alternative molecular scaffolds. Such scaffolds have the potential to be synthetically more accessible than high molecular weight bioconjugates based on co-polymers, peptides or proteins.80 HO-1 is induced during the IPH of vulnerable plaque, and has somewhat been neglected as a biomarker in atherosclerosis imaging to date.Heme oxygenase-1 (HO-1) plays a crucial role in vascular health, playing a protective role in hemorrhage and hematoma resolution.meso-position, from which it releases carbon monoxide (CO)  are well-known to display antioxidant and antiapoptotic properties.83,84HO-1 catalytically degrades ‘free’ (or unbound) heme, with regioselectivity for the α-ide (CO) .81 Whils85 Probe Fe–L1  lysates overexpressing human HO-1 (hHO-1) in a proof-of-concept study. Following addition and incubation with NADPH, a 6-fold increase in coumarin fluorescence intensity versus the control was reported. Mass spectrometry analysis (LC-MS and MALDI-MS) determined that porphyrin cleavage was regiospecific at the α-position, forming compound 1, and perturbing the efficient FRET process from the coumarin donor to the porphyrin acceptor moiety  region of the electromagnetic spectrum. However, high energy excitation is undesirable in OI due to a poor biological tissue penetration and can trigger intracellular damage as a consequence of the formation of ROS. Work in our research group is currently on-going to develop analogues of Fe–L1 with red-shifted absorbance and emission wavelengths for live-fluorescence imaging applications. For this purpose, probes with a high FRET efficiency remain essential, enabling a high signal-to-noise ratio and maximising the ‘turn-on’ increase in fluorescence observed following detection of HO-1 activity.Currently, 3.32O2) and a chloride anion (Cl−) as substrates to form hypochlorous acid (HOCl).88 At physiological pH, approximately half of all HOCl exists as a hypochlorite anion (OCl−), an extremely strong natural oxidant capable of causing tissue damage during inflammation.Myeloperoxidase (MPO) is a member of the heme peroxidase family that catalyses the formation of a number of reactive oxidant species including hypochlorous acid and peroxynitrite. MPO utilises hydrogen peroxide , with a high specificity over other reactive oxygen and nitrogen species  fluorophore in the structure of the probe. Such an approach is advantageous enabling the MB fluorophore to be in close proximity to the MPO active site, reducing complications arising from the short half-life of HOCl. Following formation of HOCl, the hydrazine bond in FD-301 is cleaved and a ‘turn-on’ increase in MB fluorescence is observed.92More recently, Yi and Wei developed a NIR emissive ‘turn-on’ probe to detect basal MPO activity, FD-301 .92 4-Amiin vivo. In this case, FD-301 was studied in vitro and in vivo in murine models for arthritis and ulcerative colitis.92 However, using a similar probe design and imaging approach in the future could accurately detect the overexpression of MPO in macrophages found in human atherosclerotic lesions.The study by Yi and Wei shows promising data for the selective detection and quantification of HOCl and, by implication MPO activity 4.Using recent approaches from the literature, we have summarised the desirable design considerations and activation mechanisms for enzyme-activatable probes in OI applications. With a particular focus on atherosclerosis imaging, and the detection of vulnerable atherosclerotic plague, an array of relevant enzymatic biomarkers has been evaluated, encompassing proteases , HO-1 and MPO.Differentiating between vulnerable and stable plaque remains the greatest challenge in this field. Whilst approaches described here are promising, they predominantly involve a “turn-on” activation mechanism, rather than a ratiometric behaviour. It remains key that new probes are developed in the future to ensure vascular diseases are detected earlier and more efficiently, before the onset of severe complications. The enzymes that are overexpressed in atherosclerosis also play key roles in a range of other chronic disorders, marked by inflammation and wound healing. Conversely, much information could be gained by repurposing enzyme-activated probes developed for other diseases, such as cancer. Such a strategy would improve the hugely challenging synthetic complexity which is often characteristic of enzyme-activatable probes. Ultimately, however, the development of new fluorogenic biomarkers for atherosclerosis is really required to significantly increase our understanding of this disease.67 In any case, the development of new fluorescent probes for less invasive in vivo imaging applications is also highly desirable. New NIR probes with high quantum yields, high stability, a high Stokes’ shift and low toxicity are required to this end. Furthermore, probes utilising two-photon excitation are also becoming increasingly desirable to facilitate greater depth penetration.In particular, enzyme-activated fluorescent probes to image atherosclerotic lesions offer extremely promising applications in catheter-based OI. However, further clinical application of such probes is still dependent on the availability of a clinically approved catheter.There are several challenges in developing OI agents applicable to atherosclerosis. These include a major trade-off between accessibility and wavelength. Longer wavelengths are better for tissue penetration and minimise tissue damage. For example, UV-excitable probes would be unacceptable. Yet, broadly, the longer the wavelength, the larger the dye molecule. Large fluorophores present challenges with entry in and out of the cells and tissue and may well present significant issues with steric hindrance of the enzyme site. Toxicity is a further consideration, particularly given the tissue is itself diseased. Appropriate instrumentation and route of delivery still remain challenges, although minimally invasive intracoronary approaches are now commonplace.94 as well as a superior tissue penetration over OI.95 PAI is being validated at preclinical studies, where it allows relatively high-resolution imaging of vessels. Additionally, the technique is being incorporated into multimodality coronary imaging. Enzyme-activatable PAI probes have been developed for MMP-2,56,96 and will form a vital part of imaging toolkit to improve the diagnosis of vascular disease moving forward.Other techniques, not discussed in detail over the course of this perspective, such as Photoacoustic Imaging (PAI) and luciferase-induced Bioluminescence Imaging (BLI), are appealing alternatives to OI. For example, PAI combines the advantages of OI and US imaging modalities, and possesses a higher intravascular resolution compared to MRI and CT,Luciferase-based imaging is already widely used in preclinical application, albeit predominantly based on genetic modifications, of a sort that would be challenging to translate clinically. For example, it has been widely used to image gene activation, based on insertion of artificial constructs comprised of gene regulatory sequence and an exogenous luciferase gene from firefly or jellyfish. A further evolution of this approach has been Bioluminescence Resonance Energy Transfer (BRET) which has primarily been used in the imaging of cell signalling.97,98 and is likely to be studied further in the near future to develop the next generation of chemical probes for atherosclerosis imaging.Dual-modal imaging techniques are highly advantageous and will be further explored in the future. Such approaches have the potential to utilise an enzyme-activatable fluorophore to directly report on molecular abnormalities, in combination with a modality to report in detail on anatomical abnormalities. The combination of OI with MRI, for example, is one possibility due to the high spatial resolution and depth penetration that is characteristic of MRI, but both significantly lacking in OI. Research to this end is currently of major interest,It is hoped that this perspective will provide an insight into the enzymatic targets for OI imaging of vulnerable atherosclerotic plaques, representing an exciting and emerging field within medical imaging. There are a number of directions to explore in the future, that could improve the diagnosis of vascular disease.The authors have applied for IP protection for a novel class of HO-1 fluorescence probes.