Formation of higher-order supramolecular complexes has emerged as a common principle underlying activity of a number of immune and regulated cell-death signalling pathways in animals, plants and fungi. Some of these signalosomes employ functional amyloid motifs in their assembly process. The description of such systems in fungi finds its origin in earlier studies on a fungal prion termed [Het-s], originally identified as a non-Mendelian cytoplasmic infectious element. Janine Beisson has been a key contributor to such early studies. Recent work on this and related systems offers a more integrated view framing this prion in a broader picture including related signalling systems described in animals. We propose here an auto-commentary centred on three recent studies on amyloid signalling in microbes. Collectively, these studies increase our understanding of fold conservation in functional amyloids and the structural basis of seeding, highlight the relation of fungal amyloid motifs to mammalian RHIM (RIP homotypic interaction motif) and expand the concept of Nod-like receptor-based amyloid signalosomes to the prokaryote reign. In this paper, she proposed that this invasive [Het-s*] to [Het-s] transformation results ‘from multiplication in the [Het-s*] strains of particles brought in by the [Het-s] strain’ (‘résulte de la multiplication dans la souche ssde particules apportées par la souche s’) . After a few years of practising experimental biology, one is likely to have faced at least once – when exposed to biological unorthodoxy – the choice between engaging the risky path of additional experimentation and autoclaving the whole business. In retrospect, one can only marvel at the insight and dedication of these scientists who simply would not let the ‘petit s’ genetic oddity rest unquestioned and who thus established the solid theoretical and experimental foundations, on which recent studies of such systems are based on. We will here very rapidly describe the biology of the [Het-s] prion and the path leading from the [Het-s] prion to the characterization of amyloid signalling in fungi. We then discuss three recent papers on [Het-s]-related systems that collectively expand the evolutionary span of amyloid signalling and attempt to integrate these systems into the broader picture of regulated cell death in animal, plant and prokaryotic organisms.On February 24uche s’) . Janine P. anserina exist as two genotypes termed het-s and het-S and the strains of these genotypes are incompatible, meaning that cell fusions between them result in regulated cell death [het-s-encoded protein is in its [Het-s] prion form. When in the soluble state-designated [Het-s*], the protein is inactive in incompatibility. [Het-s*] strains eventually all acquire the [Het-s]-prion state and as mentioned above, cytoplasmic contact between a prion-infected [Het-s*] strain induces rapid and invasive transformation of the [Het-s*] to [Het-s] phenotype. The HET-s protein displays a C-terminal prion-forming domain (PFD or HET-s (218–289)) comprising two 21 amino acid long pseudo-repeats (R1 and R2) adopting a specific β-solenoid amyloid fold, in which each repeat occupies one rung of β-strands  prion, and a general pattern of hydrophobic-polar residues , representing a cell-death trigger for the full-length HELLF protein. The sequence identity between the pseudo-repeats of the two distant homologs was found to be as low as 17%, nevertheless it was found that HELLF(209–277) formed amyloid fibres with a β-solenoid fold virtually identical to the backbone structure of HET-s. This discovery establishes that specific amyloids folds can be maintained well into the twilight zone of sequence identity and that (as established for globular and membrane proteins) very little sequence conservation is required to maintain structural relatedness. In spite of the very strong similarity of the amyloid backbones of the β-solenoids formed by HET-s and HELLF, no cross-seeding was observed between [Φ] and [Het-s] in vivo. These findings demonstrate that amyloid backbone similarity does not determine alone the cross-seeding ability of prion amyloids and suggested that key side-chain residues can impact the cross-seeding between amyloidogenic sequences. The latter statement finds additional support in the observation that a designed HELLF-derived variant (termed HEC), produced by substitutions of HRAM5-specific residues in HELLF with HRAM1-specific residues from HET-s, breaches the cross-seeding barrier in vivo with [Het-s]. Structural examination of HEC showed that the chimeric PFD adopts a HELLF/HET-s-like amyloid fold in vitro, highlighting the importance of the side-chains of the HRAM-specific residues for the breach of the cross-seeding barrier between HELLF and HET-s. The designed synthetic prion HEC shows some level of structural promiscuity both in vivo and in vitro and this type of plasticity  might be critical for the breaching of the seeding barriers. The characterization of HELLF shows that the HRAM families have diversified into signalling domains with distinct signalling specificities integrated into independent cell death pathways. The establishment of the HRAMs as a structurally unified superfamily provides a powerful experimental set-up for future investigations, because determinants specifying the amyloid signalling (ability to seed and cross-seed) must be reflected in the catalogues of natural variation found for these domains, which share a common β-solenoid fold.This hypothesis has been recently explored with the functional and structural characterization of a protein named HELLF, endogenous to anserina . The helPodospora anserina. The hellp gene occurs in a two-gene cluster immediately adjacent to a gene encoding an NLR termed PNT1 displaying an N-terminal PP-motif, a central nucleotide-binding and oligomerisation domain of the NB-ARC type and a C-terminal TPR domain (tetratricopeptide repeats) [In addition to HELLF and HET-S, HELLP constitutes a third amyloid-activated cell-death execution protein of repeats) . HELLP frepeats) . While, bona fide hits oras sequences analogous to animal or viral RHIM motifs. This observation primed a bioinformatic quest for related sequences in prokaryotic genomes [bacterial amyloid signalling sequences). These BASS motifs occur in proteins encoded by adjacent or overlapping genes at the N-terminus of NLRs and at the C-terminus of a protein domain termed BELL . This small domain predicted to form an α-helical bundle with a hydrophobic transmembrane helix shares some sequence features with the fungal HeLo and HeLo-related domains and homologous domains involved in the control of regulated cell death in animals and plants. However there is no direct detectable sequence homology between the BELL and HeLo domains, therefore functional and structural characterization of these domains are required to precisedefine its relation to HeLo. It is currently hypothesized that BELL functions analogously as a membrane-targeting cell death-inducing domain. Interestingly as described in fungi where amyloid signalling motifs occur most frequently but not exclusively in HeLo-related domains , the BASS-motifs do not occur only in association with BELL domains but also less frequently in association with other domains that have been implicated in host defence such as CHAT protease domains (Caspase HetF Associated with Tprs) or bacterial TIR domains (Toll/interleukin-1 receptor). The classification as 10 families is likely to represent a conservative estimate as a cut-off for the number of occurrences has been applied in the motif identification process. A lower cut-off or future expansion in genome coverage might increase the number of BASS families. Based on the current estimates however, there appears to be a greater diversity of amyloid signalling motifs in bacteria than in fungi, which in turn display more motif families than metazoans. This distribution is not surprising considering the differences in global phylogenetic diversity in these lineages and should probably not be taken as an indication that this form of signalling has experienced greater evolutionary success in multicellular bacteria than in fungi or metazoans.It is important to note that the fungal amyloid NLR-signalosomes  appear as far as it can be estimated from current genomic coverage limited to filamentous fungi ,32. Geno genomes . SpecifiThe present suggestion of the existence of fungal-like amyloid NLR signalosomes points at a more general aspect of NLR function and evolution in prokaryotes and fungi. There is experimental support for the role of NLRs in fungi in the control of regulated cell death and host defence  . If thesP. anserina [We discuss here recent findings concerning the molecular mechanisms of NLR-associated amyloid signal transduction  and the anserina , throughanserina , to the anserina . Paralleanserina ,37.Functional amyloids in bacteria have been foremost described as constituents of biofilms . Yet, ouper se) begins to be well defined, both the downstream events (how exactly do HeLo-related proteins bring about membrane damage?) and foremost the upstream activation steps (what are the ligands?) remain for the time being, at best, highly hypothetical. Then, an important knowledge gap resides in the definition of the actual structure of the NLR amyloid signalosomes, notably regarding the actual subunit stoichiometry of these complexes. In addition, what is unclear at present is whether the execution domains (such as HeLo) remain associated to the NLR moiety as a heterotypic amyloid assembly or whether the PFD regions receive the structural template information and then detach from the NLR amyloid hub. Thus, while some structural aspects of amyloid signalling in fungi have been explored in atomic detail [Nectria), occurs as natural prions. Only the latter two behave as infectious entities and cytoplasmic genetic elements transmitted between organisms. This is the very property that ignited in early studies the interest of pioneering scientists such as Janine Beisson.Aside from these structural and phylogenetic aspects, a major blind spot in these studies is the question of the biological role of the identified NLR signalling cascades. This is true for the microbial NLRs involving amyloid motifs but equally for the microbial NLRs in general. In particular, with the exception of a handful of NLRs involved in allorecognition in fungi, for none of these systems are the triggering cues known . While tc detail ,29, the


The flexibility imparted by these hinges may explain how multiple spikes act in concert to engage onto the flat surface of a host cell.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein enables viral entry into host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor and is a major target for neutralizing antibodies. About 20 to 40 spikes decorate the surface of virions. Turoňová Science, this issue p. 203 Flexible hinges shielded by glycans in the coronavirus spike protein may allow scanning of the host cell surface. The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo–electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines. The spike surface protein (S) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to initiate infection . We acquired a large-scale cryo–electron tomography dataset that consists of 266 tilt series covering >1000 viruses. Visual inspection of the tomographic reconstructions revealed a very-high-quality data set in which individual protein domains were clearly visible .. However, the isolate contained the Asp614→Gly (D614G) allele .S was mostly present in the prefusion conformation . PostfusSubtomogram averaging with NovaSTA . Classif. We will henceforth refer to this part of the stalk domain as the “upper leg.” Right-handed trimeric coiled coils were long thought to be absent from the structural proteome  and adopts an unusual right-handed coiled coil, consistent with a recent single-particle structure of the S head  simulations helped us to pinpoint the molecular origins of the flexibility seen in the tomograms. We performed a 2.5-μs-long all-atom MD simulation of a 4.1 million atom system containing four glycosylated S proteins anchored into a patch of viral membrane and embedded in aqueous solvent . In the Structures of S seen along the MD trajectory fit well into the tomographic density of S proteins protruding from the viral surface . In partAs a result of hinge bending, the stalk is diluted out in subtomogram averages focused on the head , and 4B.The predicted N-glycosylation sites, many already annotated in single-particle EM maps . ClusterN-glycosylation is also predicted on the knee (N1158HT and N1173AS) and the ankle (N1194ES) in regions not previously resolved by single-particle EM . We obseThe two primary structural analysis techniques combined in this study are complementary. Our MD simulations revealed three flexible hinges  within the stalk, consistent with the tomographic data. One might speculate that the high degree of conformational freedom of S on the viral surface is important for the mechanical robustness of the virus or may facilitate motions that interfere with antibody access to the stalk. It might also allow S to engage the relatively flat surface of host cells with higher avidity . Future .A particularly unusual feature masked at the edge of the resolved density of single-particle structures but well resolved in the subtomogram averages is the short right-handed coiled coil at the top of the prefusion stalk. Because this feature is lost in the postfusion structure as resolved for SARS-CoV (Overall, the observed distribution of S on the surface of the virion and its conformers is highly consistent with the findings of other studies (The fully closed prefusion conformation of S was abundant in situ. This finding emphasizes that the highly engineered, recombinant versions of S locked into this conformation . A lack By using cryo–electron tomography of intact viruses, we were able to resolve functionally important parts of S, including its connection to the viral membrane and its glycan coat, which were masked in studies of recombinant detergent-solubilized protein. Beyond S, our large-scale tomographic dataset contains rich, high-resolution structural information on SARS-CoV-2 particles in their native context. The in situ structures of several key viral components—including the nucleocapsid and the M protein that is highly enriched in the membrane—remain enigmatic. Our data might thus be explored to resolve such features in the future. Furthermore, high-resolution structural models can be fitted directly into the tomographic reconstructions, emphasizing the high quality of the data. This strategy might thus help us to build structural models of entire virions.


Pre-procedural contrast-enhanced CT and MRI imaging is typically acquired prior to deep venous recanalization procedures for post-thrombotic syndrome. This technical note reports the utility of live-overlay of augmented centerlines extracted from pre-procedural CT and MRI imaging in facilitating fluoroscopic-guided recanalization of post-thrombotic venous lesions.Six patients with pre-procedural CT or MR venography data were incorporated into a commercially available 3D overlay software  during venous disease interventions for post-thrombotic venous lesions. Procedures were performed on the GE Discovery IGS 740 fluoroscopy system. After manual determination of the vasculature from preprocedural CT or MR, centerlines were created representing the location and trajectory of the vessels. Steps showcasing the creation of centerlines and their representation during overlay with real-time fluoroscopic guidance in these cases are outlined. Time required to cross the post-thrombotic and occlusive venous segments were reviewed.All iliocaval recanalization procedures were successfully performed utilizing vessel centerline 3D overlay. In one case where occlusion extended to the femoral vein, mis-registration was identified over the femoral anatomy due to a complex leg rotation compared to pre-procedural imaging. No procedural complications related to utilization of software were noted. Average crossing time for occlusions was 3.4 min (range 1.6–5.2).3D overlay with vessel tracking from pre-procedural CT and MRI imaging is technically feasible and assists in catheter navigation for post-thrombotic venous segments. While results from these preliminary experiences support the continued use of this technology, further prospective and comparative evaluation of this technique is warranted to assess for added value in technical success, reductions in procedure time or reductions in radiation exposure. Recanalization of chronic deep venous obstructions or post-thrombotic venous segments can require extensive fluoroscopic time, contrast use, and repeat interventions  to December 2017, six consecutive patients with post-thrombotic iliocaval venous lesions were identified. Low-sample size stemmed from software implementation during hours of operation in which support staff was available in the event troubleshooting was required. Five patients had pre-procedural contrast-enhanced CTs while one patient had pre-procedural contrast enhanced MRI. Patients were treated on the same angiography unit . Before the start of each procedure, cross-sectional images from the most recent pre-operative scans were loaded from PACS to a designated workstation . Each dataset was prepared with one segmented volume to be used for initial registration and vessel centerline creation for the vessels of interest.The technique for centerline creation, vessel tracking, and fluoroscopic overlay is shown in Fig. In cases of multivessel recanalization, the method was repeated in similar fashion on the other vessel(s). With pre-operative CT datasets, bones were used for initial registration to live fluoroscopy and were automatically segmented by the software. Pre-operative MRI datasets required manual segmentation of the femoral heads or other structure of interest to allow for registration to live fluoroscopy Fig. . In pati0 apart (Fig. Once the patient was on the angiography table, and before the procedure started, the segmented anatomy and the vessel centerlines from the pre-operative scan were loaded into the overlay software and registered to the patient by the technologist using two fluoroscopic images 60art Fig. h-i. The art Fig. j-k. RefiTable While the benefits of fluoroscopic 3D overlay created from procedural digital subtraction angiography and cone-beam CT acquisitions are well demonstrated, the utility of incorporating peri-operatively acquired imaging data during vascular interventions remains limited (Gorges et al. Contrast-enhanced CT and MR imaging was sufficient for the provider to identify the course of the post-thrombotic venous segments and allowing for creation of a centerline. The software automatically calculates trajectory based on luminal opacification of the proximal and distal ends in the contrast enhanced cross-sectional examination with registration of the non-opacified portions of the vessel performed by bridging segments with manual clicks by either the operator or technologist. Such automatic registration does not correlate well with vessel wall thickness due to poor wall visibility relative to intraluminal post-thrombotic changes limiting the software’s ability to display a theoretical outline of the vessel.Currently, the implemented centerline technology relies on physician determination of the affected post-thrombotic venous channel. In the future, it may be possible to utilize this technology with non-contrast imaging based on landmark selection and triangulation with a selected vascular tract, though this was not formally interrogated in this study. Further automated segmentation of anatomical landmarks from imaging datasets for registration with fluoroscopic images may also be considered in the future.The use of augmented centerlines represents a promising road mapping tool regardless of the position of the C-arm. Displaying vessel centerlines allows for guidance through long segments of poor vessel opacification of contrast due to post-thrombotic changes. In the future, an augmented reality environment could be overlaid to the patient using more advanced visualization tools such as virtual reality glasses.Limitations of this technical note include a small retrospective patient cohort and lack of a control group showing differential time to recanalization or contrast volume use. Given the superior spatial resolution and standardization of dose parameters of CT compared to MRI, CT is preferred in the identification, extrapolation, and standardization of centerlines from occluded and atretic vascular channels over MR. However, in patients with prior MR imaging for other indications, these can be alternative options to develop guidance overlay centerlines. In one patient with pre-existing right external iliac to femoral vein stents and a left iliocaval post-thrombotic occlusion, a crossing time of 1.6 min was recorded, which is lower than others in this cohort. Presumably, the patient’s preexisting stents provided a more accurate landmark than the adjacent pelvic osseous structures when affixing 3D centerlines to the live images.In conclusion, 3D overlay with vessel tracking from pre-procedural CT and MR imaging data for catheter navigation during venous recanalization interventions is feasible. Procedure time to cross the occlusion was on average 3.4 min in this particular cohort, noting that reducing crossing times subsequently reduces procedure length in these often time-intensive procedures. Specifically, through a combination of the centerline creation and overlay process, intravascular ultrasound incorporation, and understanding of catheter and wire locations based on fluoroscopic landmarks, the potential for reductions in procedural contrast accumulation and dose exposure without compromising safety exists and would be fodder for future investigation in a prospective nature.


Laryngocarcinoma (LC), in most cases a squamous cell carcinoma, accounts for 1 ~ 5% of the incidence of all tumors. At present, laryngocarcinoma is mainly managed with the integration of surgery and radio- and chemo-therapies. The current development trend of treatment is to improve the local control rate of tumor and the quality of life of patients. Intraoperative radiation therapy (IORT) is a radiotherapy that delivers single high dose irradiation at a close range to the tumor bed during the surgical operation process. It has particular radiobiological advantages in protecting normal surrounding tissues by directly applying the irradiation dose to the high-risk tumor bed area.Two forms of IORT, i.e., high dose rate (HDR) brachytherapy and external beam radiotherapy , had been studied before the treatment of head and neck tumors (including laryngocarcinoma). However, no relevant assessment had been carried out on 50KV low-energy X-ray. We are convinced by certain arguments that the application of low-energy X-ray for intraoperative local radiotherapy of laryngocarcinoma can not only achieve the therapeutic effect of IORT but also reduce the incidence of high-energy irradiation related toxic and side effects. The purpose of this study is to observe the safety and short-term efficacy of IORT when used in conjunction with standard of care for the treatment of local advanced laryngocarcinoma (LAL).In consideration of the applications of precise targeted IORT in oncosurgery and in line with the application range and reference clinical medical guidances approved by SFDA , we have preliminarily planned the tumors suitable for IORT, determined the members of MDT in our hospital, improved the MDT diagnosis and treatment processes for the tumors, established the standards, indications and contraindications for the application of IORT, determined the indicators to be observed after the treatment of tumors with surgical operations plus IORT, and carried out follow-up visits and statistical analysis.This is a single-arm, prospective Phase II clinical trial of the treatment of LAL patients with IORT + EBRT. The study subjects are followed up for statistics and information of their acute/chronic toxic reactions and local control rate, DFS, and OS etc. The safety and short-term efficacy of the application of IORT as SIB for the treatment of LAL. The sample size of the study is 125 subjects.The safety and efficacy of IORT for the treatment of head and neck cancers have been proven in studies by multiple institutions (1–3). The purpose of this study is to investigate the maximum safe dose and short-term efficacy of IORT for providing a theoretical basis for clinical trials.Clinicaltrials.gov, NCT04278638. Registered 18 February 2020 - prospectively registered, https://clinicaltrials.gov/ct2/show/NCT04278638Trial registration:  The incidence of laryngocarcinoma accounts for 1–5% of the incidence of all tumors. According to Global Cancer Statistics 2018, there were nearly 200,000 new laryngocarcinoma patients and approximately 100,000 cases of laryngocarcinoma-related deaths in 2018 worldwide . HoweverIORT is a technique for intraoperative delivery of single high dose irradiation at a close range to the tumor bed, residual lesions, lymphatic drainage areas and potential invaded areas after tumor resection . The simNowadays IORT has been extensively used for the treatment of breast cancer, pancreatic cancer, head and neck tumors, thoracic and abdominal tumors and tumors at other body sites , 10. TheIn spite of the apparent advantages of IORT in the treatment of head and neck tumors, the application of IORT for the treatment of local advanced head & neck tumors is still in exploratory stage. In particular, there is a shortage of studies evaluating the efficacy of IORT in the treatment of LAL. Judged from the results reported in literature, the probability of complications after IORT of local advanced head and neck tumors is clinically acceptable. This study aims to investigate the safety and short-term treatment results of IORT as postoperative SIB radiotherapy of LAL and further detail and verify the assessment of toxicity and efficacy of IORT for the treatment of LAL. This study verifies the following hypothesis: compared with the current standard of care, IORT can improve local control, as observed in the patients in the current study.Clinical trial stage: Phase II.Design: single-center, single-arm, prospective clinical study.www.clinicaltrials.gov (NCT04278638).This is a single-center, single-arm, prospective clinical trial intended to investigate the safety and short-term therapeutic effect of IORT as a postoperative simultaneous integrated boost (SIB) radiotherapy for the treatment of LAL. The trial has been registered at 1. Feasibility and safety of IORT irradiation dose for the treatment of LAL;2. 2-year local recurrence rate;3. Acute and chronic toxicity, 2-year DFS, PFS and OS.Primary endpoint: local recurrence rate 2 years after the radiotherapy.Secondary endpoints: DFS and OS after the radiotherapy.Safety indicators: acute and chronic toxicological reactions  after IORT SIB, wound healing time, wound infection, wound rupture, pharyngeal fistula, radiation-induced pain, etc.Subjects of this study are patients with histologically confirmed LAL who have received total- or hemi-laryngectomy and postoperative radiotherapy and signed informed consent form at Tianjin First Central Hospital. Subjects in the treatment group are enrolled from 1 January 2019 to 1 July 2020.Adults aged over 18;Pathologically diagnosed with LAL (T2N1–3 / T3N0–3 / T4N0–3);Without remote metastasisResectable tumor marginWithout involvement of any important organ/tissue With surgical indications of total- or hemi-laryngectomy;Survival time expectation ≥3 months;With insight for signing informed consent forms for treatment and study.With concomitant involvement of important surrounding tissues , which may give rise to serious complications such as life-threatening angiorhagia, post-chemotherapy esophageal ulcer or stenosis that is hard to heal;With remote metastasis;With multiple primary cancers;With surgical tumor bed that is unsuitable for IORT;Pregnant or planned to pregnant;With contraindication(s) of follow-up visit CT/MRI examinations;Unable to receive treatment and/or follow-up visits as scheduled;With cachexia, organ function decompensation;Participating in other clinical trial.Examinations of the study subjects include physical examination, laboratory tests , laryngendoscopy, imaging examinations (CT or MRI examination of larynx) and, if necessary, histological examination and positron emission tomography (PET).The primary purpose of this study is to investigate the safety and efficacy of IORT as SIB therapy that directly acts on surgical tumor bed in place of conventional EBRT for the treatment of LAL; the primary study endpoint is the local recurrence rate 2 years after the treatment of LAL with surgery in combination with IORT. This is a single-center, single-arm clinical study. In our center, the 2-year local recurrence rate of LAL treated with conventional therapeutic regimen is approximately 20%; the 2-year local recurrence rate of laryngocarcinoma treated with surgery in combination with IORT reported in literature is approximately 15–70% , 25. In 1. Screening patients with LAL;2. Signing informed consent forms for surgery and IORT;3. Hemilaryngectomy/total laryngectomy under general anesthesia + IORT;4. Carrying out follow-up visits every day during the hospitalization after the surgery and every 1–3 months after discharge (Table Therapeutic regimens:1) Resectable range of tumor.a) Total laryngectomy; b) Tumor bed IORT; c) EBRT.2) Range of tumor resectable and laryngeal function retainable.a) Hemilaryngectomy; b) Tumor bed IORT; c) EBRT.Name: IORT radiosurgical treatment system.Trade Name: INTRABEAM®PRS500 System.Manufacturer: Carl AG, Germany.Laryngocarcinoma under general anesthesiaA spherical applicator of properly selected diameter is placed by the surgeon under direct vision and measurement to the postoperative tumor bed areaHemi laryngectomy + use of spherical applicator at tumor bed.Total laryngectomy + use of plate applicator at tumor bedAn appropriate irradiation dose is selected by the radiotherapist in order to protect normal surrounding tissues Fig.  26]..26].Surgical operation (surgery under anesthesia) ≤3 h.Connect a mobile 50 KV X-ray radiation source (Intrabeam) to the mechanical arm and maintain the source’s stability throughout the treatment process Fig. . Before The study subjects shall be followed up for 2 years after the treatment with surgery in combination with IORT for examination and assessment of the study subjects’ surgical wound and the first follow-up visit shall be done 1 month after the treatment. The frequency of regular follow-up visits shall be changed to once every 3 months from the third month onwards and once every 6 months from the second year onwards. The follow-up of the study subjects shall include physical examination, wound examination, and regular CT or MRI examination.Local recurrence: CT or MRI reexamination shows typical radiographic features of local recurrence.DFS: From subject’s enrollment for treatment until his/her first local recurrence or remote metastasis or other non-study-related death.OS: From subject’s enrollment for treatment until his/her death.Assessment of IORT-related toxic and side effects✓ Wound healing time✓ Infection✓ Pharyngeal fistula✓ RT-related mucosal reactions✓ Fibrosis of mucosa✓ RT-related toxic and side reactions of laryngeal nerveThis study implemented strict quality control in every aspect of the baseline and follow-up surveys, including the development of a unified questionnaire, the study protocol operation manual, etc.; all personnel in the baseline and follow-up surveys underwent strict training and technical examination before participating in the study; the field survey form was checked and verified by quality control personnel, and all data were double-entry.The primary endpoint of this study is local recurrence rate. The comparison of the local recurrence rate between subjects of this study and subjects treated with conventional therapeutic regimen shall be done with Chi-square test. The comparison of time to local recurrence shall be done with Kaplan-Meier survival curve and log-rank test. During the comparison of local recurrence rate, confounding variables shall be controlled with proportional hazards model.Figure 3Head and neck tumors are generally treated with an integrated treatment modality by a multi-disciplinary team (MDT). The precise application of a variety of therapies is an important component of individualized treatment. Postoperative prophylactic irradiation after LAL surgery can reduce the disease’s recurrence rate from 20 to 50% to 18–19%. This study aims to investigate whether the addition of low-energy X-ray source IORT to the standard of care of LAL for instantaneous SIB featuring high precision, high dose, and high biological effect can be effectively used in place of EBRT in order to reduce the local recurrence rate of LAL.The treatment idea of IORT is in line with the concept evolution from radical surgery to risk assessment based minimally invasive modified surgery in oncosurgery. Compared with conventional EBRT, IORT can precisely set the irradiation field for direct destruction of unresectable and/or residual tumor tissues with mild low-energy X-ray (50 kV) irradiation of tumor bed at shallow irradiation depth. The technique can increase the effective irradiation dose on local tumor bed without causing significant damage to normal tissues by virtue of its ability to evade the irradiation of dose-limiting sensitive tissues partially or completely. In other words, a single intraoperative large dose irradiation can destroy the micro-environment of tumor more effectively than conventional radiotherapy.IORT and conventional EBRT can mutually make up for each other’s shortcomings: the shortcoming of conventional radiotherapy, i.e., its inability to deliver high irradiation dose to target area due to the restriction of sensitive surrounding tissues/organs, can be addressed by IORT; the shortcomings of IORT, i.e., the non-uniform irradiation and limited irradiation unit dose during its application in target areas of irregular shapes, can be remedied by EBRT. Therefore, the combination of IORT and postoperative conventional irradiation can achieve ideal results featuring high irradiation dose in tumor target areas and complete irradiation dose coverage in areas at risk.Considering the huge potential of IORT for improving local control, we are looking forward to use this study to evaluate the effect of IORT in the treatment of LAL.


Patients infected with SARS-CoV-2 may be asymptomatic or present with symptoms ranging from mild clinical manifestations: such as fever, cough, and sore throat to moderate and severe form of the disease such as pneumonia and acute respiratory distress syndrome (ARDS). In some patients, SARS-CoV-2 can affect the heart and cause myocardial injury which is evidenced either by electrocardiographic (ECG) changes or by a rise in serum troponin level. Patients with myocardial involvement are generally at risk of developing severe illness and tend to have a poor outcome. We hereby present a case of a hypertensive male patient with undiagnosed, asymptomatic COVID-19, who underwent an emergency urologic procedure for ureteric calculi. He eventually sustained a postoperative myocardial injury resulting in his demise. This case highlights the importance of detailed preoperative assessment and anticipation of complications during this global pandemic.Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first identified on 8 Patients with COVID-19 who have underlying cardiovascular disease and diabetes are at added risk of poor outcomes. Myocardial injury has been considered as an essential pathogenic feature of COVID-19 however the precise mechanism is not completely clear. It has been hypothesized that it generally results either from direct damage of myocytes or systemic inflammation resulting in an interferon-mediated immune response. Additionally, the cytokine storm has also been considered to trigger stable coronary artery plaque which subsequently results in myocardial injury [SARS-CoV-2 has spread across the world in a very short period, posing an extraordinary challenge. According to the situation report published by the World Health Organization, as of April 30A 60-year-old male with a history of hypertension presented with an acute, severe, left lumbar pain, radiating to the groin, associated with nausea and dull pain on micturition. He denied any history of fever or hematuria. He reported upper respiratory tract symptoms, including cough and chest pain for 10 days before admission which had slowly resolved. The patient denied having been in close contact with a COVID-19 patient. On examination, he was alert and oriented with stable vital signs. An abdominal examination revealed a tender left lumbar region. The rest of the physical examination was unremarkable. Computed tomography of the kidneys, ureters, and bladder revealed an obstructive left ureteric calculus at the vesicoureteric junction (VUJ) measuring 4.7mm . Blood wOn workup, it was found that the patient had an abnormal ECG with ST-segment depressions and T wave inversion on the anterior (V1- V4) and inferior leads (III & aVF) , when coThe COVID-19 pandemic is one of the greatest international health care challenges we face in this modern time causing global turmoil with both health and economic challenges. So far, the focus of COVID-19 has primarily been the respiratory system. Extrapulmonary manifestation is still somewhat unfamiliar and variable, nevertheless, recent studies have recognized the role of cardiovascular disease as a risk factor of the severity of the infection . Cardiovet al.[et al.[Diagnosis of myocarditis is a diagnostic dilemma it is practically a clinical concept and very few cases have been confirmed radiologically. In a case published by Inciardi et al. the diaget al.. In terml.[et al. revealedThis case also highlights the need for detailed and standardized pre-operative assessment during the current COVID-19 pandemic, especially for asymptomatic patients. Comprehensive assessments will not only help to control disease transmission amongst staff but also prepare medics better for possible anticipation of complications. Since patients affected by SARS-CoV-2 can present with no or varying symptoms, it is logical to label or manage everyone as COVID-19 suspect until results are confirmed. It is becoming more obvious that many cases are pre-symptomatic or asymptomatic and the prevalence of myocardial injury in various populations is not yet known, as Troponin and ECGs are generally reserved for hospitalized patients.


Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue- or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein–protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein–protein interactions and offer considerations for future work in expanding genetic code expansion methods. Heterogeneous environments exist within living cells and this ever-changing environment affects the expression, dynamics, and state of proteins. However, the results and importance of the internal cellular changes are still unclear. Here, we introduce recent progress to observe the internal conditions in living cells using genetic code expansion (GCE).in vitro and in vivo for purposes such as protein purification, determining cellular localization, and visualizing and identifying interacting proteins alanine (3) in Escherichia coli  from E. coli  (8) at Phe codons in C. elegans by a mutant C. elegans phenylalanyl-tRNA synthetase (Light-activated BONCAT is a modified version of BONCAT where photocaged Aha (7) can only be incorporated into proteins upon uncaging by light exposure. The amount of protein labeling in HeLa cells could be controlled by varying the light intensity and proteins in specific regions can be labeled by aiming the light source, allowing spatial and temporal control of Aha incorporation . Howevernthetase . NeverthE. coli by recoding the UAG stop codon  (9) was site-specifically incorporated in the outer membrane protein eCPX (enhanced circularly permuted outer membrane protein OmpX) of op codon . By takiop codon .To visualize the spatial localization of newly synthesized proteins, BONCAT was modified to visualize proteins and termed fluorescence non-canonical amino acid tagging (FUNCAT) . Fixed cIn an effort to further improve upon the specificity of FUNCAT-PLA, SCROL (Stop-Codon-Read-thrOugh-Label) was developed to study expression of a specific protein of interest  Figure . A cassein vivo for protein visualization under different cellular conditions is bioorthogonal dye. Tetrazine dyes (11) have been extensively characterized for their use in labeling trans-cyclooct-2-ene (TCO∗) modified ncAAs (12) via click chemistry in a variety of mammalian cells for live cell imaging (An important component of labeling ncAAs  imaging . An attr imaging . A simil imaging . Interes imaging . The easE. coli (E. coli strain (ATMW1) where the endogenous tryptophanyl–tRNA synthetase/tRNATrp (TrpRS/tRNATrp) pair was replaced by the TrpRS/tRNATrp from Saccharomyces cerevisiae, rendering the E. coli TrpRS/tRNATrp pair available for ncAA insertion  Development of more sensitive detection techniques would enable a greater understanding of localization for more proteins. Although FUNCAT coupled to PLA allows amplification of a signal for a protein of interest  Although aaRS/tRNA pairs are tested to ensure their orthogonality within a system, cross-reactions may still occur. Developing more specific aaRS/tRNA pairs that minimally cross-react with endogenous aaRSs and tRNAs would lessen the impact on cellular metabolism and allow a more “natural” representation of the protein(s) of interest due to minimal disturbance of the intracellular environment. In addition, there is a need to produce more aaRS/tRNA pairs that are orthogonal in a greater variety of organisms  to studyinterest , a high interest , the SCRIn conclusion, GCE has become a versatile tool that can be modified to suit individual needs and incorporated into existing techniques. In the future, it is expected that more tools will be developed for expansion of GCE to other organisms and to improve on existing GCE methods for understanding heterogeneous environments within living cells.CZC and KA wrote the manuscript. DS edited it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study included a group of 15 patients with non-lesional ETLE who underwent a dynamic FDG PET study using a fully-integrated PET/MRI system (Siemens Biograph). FDG tracer uptake images were converted to MRGlc (μmol/100 g/min) maps using an image derived input function that was extracted based on the combined analysis of PET and MRI data. In addition, the same protocol was applied to a group of healthy controls, yielding a normative database. Abnormality maps for ETLE patients were created with respect to the normative database, defining significant hypo- or hyper-metabolic regions that exceeded ±2 SD of normal regional mean MRGlc values. Abnormality maps derived from MRGlc images of ETLE patients contributed to the localization of hypo-metabolic areas against visual readings in 53% and increased the confidence in the original clinical readings in 33% of all cases. Moreover, quantification allowed identification of hyper-metabolic areas that are associated with frequently spiking cortex, rarely acknowledged in clinical readings. Overall, besides providing some confirmatory information to visual readings, quantitative PET imaging demonstrated only a moderate impact on clinical management of patients with complex pathology that leads to epileptic seizures, failing to provide new decisive information that would have changed classification of patients from being rejected to being considered for surgical intervention.The purpose of this study was to establish a non-invasive clinical PET/MR protocol using [ Surgical interventions in patients with intractable epilepsy are planned based on a variety of diagnostic measures, including electrophysiological testing and neuroimaging procedures –3. The r18F]fluoro-2-deoxy-D-glucose (FDG) PET provides valuable imaging clues with respect to the location of electrophysiologically confirmed seizure onset zones , absolute metabolic rates have been rarely used in clinical routine to characterize abnormal brain areas in ETLE patients. The reason for this omission to fully embrace the potential of PET imaging in clinical applications is the requirement of arterial blood sampling, an invasive procedure not performed routinely. However, with the advent of hybrid imaging, quantification of MRGlc can be achieved non-invasively based on the simultaneous acquisition of both high-resolution anatomical as well as high-sensitive molecular functional data, yielding an image-derived arterial input function from an integrative analysis of PET and MR image data . ConsequIn the present study, we obtained regional MRGlc values from a series of patients with drug-resistant non-lesional epilepsy who were previously rejected as surgical candidates based on a comprehensive clinical work-up. The objectives of the study were as follows: (i) to establish a non-invasive clinical protocol that provides the physician with regional MRGlc values in epilepsy patients; (ii) to clarify the contribution of absolute quantification to clinical readings; and most importantly (iii) to determine whether quantitative PET imaging is able to provide sufficient new information that would allow re-classification of patients from being rejected to being considered for surgical interventions.In this prospective study we included 15 patients with drug-resistant non-lesional ETLE who were previously rejected for surgical intervention based on an extensive pre-surgical work-up at the Epilepsy Monitoring Unit of the Department of Neurology which included prolonged Video-EEG monitoring, a high-resolution 3T MRI scan using a predefined clinical epilepsy protocol (see below), formal neuropsychological testing and (in selected patients) interictal and ictal SPECT, as well as visual PET and functional MRI for lateralization of language functions.Video-EEG monitoring was recorded for an average of 5 days. The EEG was recorded according to the international 10–20 System with bilaterally placed additional sphenoidal electrodes. The absolute spike frequency over the entire recording time and its location was assessed by visual analysis. The clinical seizure semiology was evaluated with respect to the prediction of symptomatogenic area(s) and the ictal EEG patterns were determined over the time course by location and morphology. Two board-certified epileptologists (EP and SA-W) with 20 years of experience in epileptology reviewed all clinical information and Video-EEG monitoring results.MRI studies were assessed with respect to epileptogenic lesions including focal cortical dysplasia, hippocampal/mesial temporal sclerosis and epileptogenic tumors. Imaging was performed on a 3T MR system  with an 8-channel head coil. All studies employed a high-resolution epilepsy protocol that included the following sequences: a 3D volumetric spoiled gradient-echo T1-weighted  and a sagittal 3D fluid attenuation inversion recovery (FLAIR) MR sequence  with multi-planar reconstructions. Moreover, a T2-turbo-spin-echo MR sequence , an IR  and a FLAIR sequence  were acquired in a coronal plane perpendicular to the hippocampus. A board-certified neuroradiologist (GK) with 10 years of experience in epilepsy imaging, who was blinded to all other clinical information, reviewed all patients' MR scans and assessed them as being non-lesional.Based on the results of pre-surgical evaluation a suspected epileptogenic area was delineated and allocated to the corresponding brain region. If a clear assignment to a brain region/hemisphere/lobe was not possible, the seizure area was defined as non-localizable. The final decision regarding the procedure  was made individually for each patient based on case presentation and discussion at the institutional multidisciplinary epilepsy conference.Patients rejected as surgical candidates underwent subsequently a brain examination on a fully-integrated PET/MRI system in the Department of Nuclear Medicine, Medical University of Vienna, Austria, between September 9, 2017 and July 1, 2018.The study was approved by the Ethics Committee of the Medical University of Vienna and was performed in accordance with the Declaration of Helsinki (1964) including current revisions, the Austrian Drug Law and the GCP guidelines of the European Commission. Written informed consent was obtained from all subjects prior to the examinations.3 voxel size, TE = 3.6 ms, TR = 21 ms, 6 min duration). Subsequently, FDG (5.18 MBq/kg) was injected through the venous line as a slow bolus (40 s) and a 60 min list mode PET data acquisition was initiated. During PET list mode data acquisition, a second set of T1-w and T2-w sequences were acquired in order to assess potential disease progression relative to the initial MR scan as well as for the calculation of a pseudo-CT attenuation correction map  as described previously . In shortion map . List moWe have previously developed a methodology that enables the acquisition of a non-invasive image-derive input function (IDIF) based on simultaneously acquired structural and functional data obtained using a fully integrated PET/MR system . This mem), a standard deviation (MRGlcSD) as well as the corresponding coefficient of variation (COV = SD/mean) image, constituting a normative database. Given the observed physiological variation (combined within and between subjects) in MRGlc values determined in the control group, the normal COV across the brain is in the range of 10–20% (m). Regions of significant hypo- or hyper-metabolism were defined as areas in the patient's abnormality map that exceeded ±2 SD (as defined in the MRGlcSD image). Finally, these hypo/hyper-metabolic areas were projected back into the patient's native space and displayed onto the patient's own structural T1-w images. Quantitative readings (qPET) were then performed by viewing the patient's MRGlc images side-by-side with the hypo/hyper-metabolic areas overlaid onto the corresponding T1-w images. Quantitative analysis was performed by two collaborators  who were blinded to all clinical information about the patients.In order to assess abnormal MRGlc areas in epilepsy patients, the recently described normative database of MRGlc images was used . The datf 10–20% . The samIn a clinical setting, the added value of a test is assessed based on whether patient management is affected. Our main objective was to determine whether fully quantitative analysis of FDG PET data based on advanced PET/MR scanner methodology is able to provide added value to the established clinical epilepsy work-up which might change the classification of patients with complex epilepsy pathology from being rejected as surgical candidates to being considered for further testing. Given the fact that our study focused on the added value associated with quantitative PET imaging in relation to the established clinical evaluation procedure of ETLE patient in the absence of surgical outcome data , our results are necessarily descriptive. Thus, our primary outcome measure was the percentage of patients that was re-classified from being rejected to being considered for further testing. As a secondary outcome measure we also assessed the percentage of cases in which MRGlc maps may have generated additional information about possible underlying disease mechanisms or might have increased confidence in the original readings. Specifically, the added value provided by the abnormality maps is rooted in the sensitization of the reading physician to subtle abnormalities that might have not been appreciated during the initial clinical reading or in the confirmation of abnormalities that were initially detected but were considered marginal. To quantify the agreement between visual FDG PET/CT readings and MRGlc maps, we calculated the percentage of anatomical locations that were determined as abnormal by both methods against the total number of anatomical locations determined as abnormal by either method. Accordingly, an agreement score of 1.0 reflects perfect agreement  whereas an agreement score of 0 indicates no overlap between the two methods . All values are expressed as mean ± SD.Our dataset included complete clinical information and results of PET imaging for 15 patients with drug-resistant non-lesional ETLE. There were 7 females and 8 males with a median age of 26.5 years (IQR = 9.5 years). Clinical details about patients, including age at seizure onset, duration of disease, seizure frequency, results of Video-EEG monitoring, structural MRI and time to PET/MRI before last seizure are presented in With respect to the primary outcome measure, quantitative PET imaging did not provide sufficient added information to reverse the original clinical decision, which was to reject patients as surgical candidates. Overall, qPET demonstrated at least partial confirmation of prior clinical impression in 8/15 (53%) of cases, whereas in the remaining cases qPET and clinical readings either did not agree on the location of abnormality (4/15 = 27%) or both methods failed to detect any abnormalities (3/15 = 20%). Furthermore, in 5/15 (33%) cases, quantitative assessment yielded additional information about a potentially underlying disease mechanism . To demonstrate the performance of quantitative analysis, Several other cases deserve a closer inspection. In patient #4 visual analysis suggested a right-lateralized seizure onset in the temporo-parietal region, whereas qPET indicated bilateral seizure onset . In this particular patient the consensus decision during epilepsy conference was a unilateral right-sided seizure onset as indicated in the first column in An advantage of absolute quantification is the assessment of extensive WM abnormalities that are difficult to appreciate during clinical readings. One of our patients (#3) showed seizures originating independently from the left and right hemisphere. Both the clinical PET readings as well as quantitative assessment identified a hypo-metabolic area in the left temporo-parietal cortex . HoweverIn order to determine to what extent results of quantitative analysis deviated from visual readings of FDG PET/CT images, the average agreement score between the two approaches was determined. The average agreement score was found to be 0.47 ± 0.35, which included 3 cases with perfect overlap  and 3 cases with no overlap .Overall, in 12/15 (80%) of cases the two methods were correspondent and as a result qPET analysis either increased confidence in, or expanded upon, the original visual readings. In the remaining 3 cases, results obtained from the two methods differed as follows: in patient #2 qPET indicated bilateral focal hyper-metabolic areas at the R central and L precentral location which were not reported in the original PET readings, but were visually confirmed after detection by quantitative analysis and re-reading of semi-quantitative PET images. Moreover, in patient #5 qPET indicated bilateral hypo-metabolic areas in WM which were not reported by visual assessment and is not surprising, as abnormalities in the WM remain usually unreported during visual analysis. Finally, in patient # 12 visual assessment identified hypometabolic areas in the L parietal and L temporal lobe which were not confirmed by qPET analysis.Our work demonstrates moderately improved characterization of glucose metabolic abnormalities in patients with non-lesional ETLE epilepsy based on absolute quantification of MRGlc in a clinical setting. We report here four main findings: quantitative assessment of MRGlc: (1) does not provide sufficient additional information that could justify re-classification of patients who have been rejected as surgical candidates based on established clinical work-up; (2) contributes to increased confidence in the original visual readings in ~50% of cases; (3) identifies hyper-metabolic areas that are rarely acknowledged in clinical readings but might reflect dynamic changes in brain networks that are directly associated with seizure generation and propagation; and (4) sets a limit to the sensitivity of absolute quantification to detect hypo- or hyper-metabolic areas in epilepsy patients that is caused by the relatively large physiological variation (10–20%) of MRGlc values in the normal population.Although our findings indicate that the inclusion of quantitative MRGlc maps into clinical management of ETLE patients does not have the capability of changing classification from being rejected to being considered as surgical candidates, it can provide added confidence with respect to the location of suspected seizure onset areas, especially in cortical regions that are subject to extensive gyral folding pattern. Visual readings of FDG PET images are heavily influenced by the assessment of tracer uptake asymmetries between homotopic cortical territories that are sensitive to physiological variation in hemispheric cortical folding. Despite the fact that overlay of high-resolution MR images onto FDG PET images might shed light on whether the asymmetrical FDG tracer activity is due to an unbalanced gyral folding pattern, high-resolution MR images are not always available and even if available, might not provide a conclusive answer. As a result, expert readers find themselves often in a situation to make a judgment call whether a particular asymmetry is “real” or whether it is merely an artifact due to physiological variability in cortical shape. Moreover, in the absence of an absolute scale, it is unclear whether the observed asymmetries represent abnormal decreases of tracer concentration in one hemisphere or alternatively abnormal increases in the contralateral hemisphere, especially in complex pathologies characterized by more extensive abnormalities where visual comparison against areas that are assumed to be not involved in the epileptogenic network (such as the brainstem or cerebellum) is difficult. Visual PET readings therefore tend to be conservative in order to keep the level of false positive findings at bay, accepting the unavoidable higher false negative rate. Accordingly, the role of quantitative PET analysis is to provide added information with respect to the identification of “suspicious” territories that warrant a closer inspection by the clinical reader. This approach results in greater confidence of those abnormalities that have been identified independently by both methods and sensitizes the clinical team to scrutinize areas that were not identified by both methods. In addition, this approach also encourages both identification and reporting of hyper-metabolic areas that might represent frequently spiking cortex prone to epileptiform activity.Of special interest is the identification of abnormalities by quantitative analysis in two areas of the brain—the thalamus and white matter—that are difficult to evaluate by visual analysis alone. In our study, abnormalities in these areas were only detected by the quantitative analysis and the relevance of these findings remains unclear, although recent studies are beginning to address the contribution of these structures to ETLE pathologies . These sFinally, our analysis included several cases where qPET and clinical readings did not agree on the location of abnormality. The two cases  is a meaningful approach. The same applies also for the control group, as one of the main outcomes of our paper is the realization of high physiological variability of MRGlc (both across time and across subjects) in the normal population, setting a limit to the sensitivity associated with absolute quantification.In a selected group of patients with non-lesional ETL epilepsy who were originally rejected as surgical candidates, quantitative PET imaging failed to demonstrate sufficient added information to reverse the original clinical decision. However, absolute MRGlc values did contribute to the detection of subtle hypo- or hyper-metabolic brain areas that are often missed in clinical readings in ETLE patients, thereby increasing confidence in visual PET readings. Moreover, the high normal physiological variability of glucose metabolic rates limits the sensitivity of quantitative analysis. Taken together, our investigation documents both the potential as well as the limitations of absolute quantification based on advanced simultaneous PET/MR imaging for the management of non-lesional ETL epilepsy cases in clinical routine.The datasets generated for this study are available on request to the corresponding author.The study was approved by the Ethics Committee of the Medical University of Vienna and was performed in accordance with the Declaration of Helsinki (1964) including current revisions, the Austrian Drug Law and the GCP guidelines of the European Commission. Written informed consent was obtained from all subjects prior to the examinations.TT-W and EP: design and conceptualized study, analyzed the data, study supervision, and drafted the manuscript for intellectual content. OM: design and conceptualized study, analyzed the data, and drafted the manuscript for intellectual content. LS: analyzed the data and drafted the manuscript for intellectual content. SA-W: analyzed the data with respect to clinical assessment of patients. TB: design and conceptualized study, study supervision, and drafted the manuscript for intellectual content. MH and RL: drafted the manuscript for intellectual content. AH: provided expertise with MR navigator sequences. GK: provided expertise with assessment of MRI in epilepsy patients. E-MK: provided expertise with respect to clinical assessment of epilepsy patients. MM: design and conceptualized study. MP: provided expertise with regard to ethical issues and analyzed the data. IR: design and conceptualized study with respect to methodological aspects of PET/MR imaging. LR: provided expertise with respect to quantitative analysis of multimodality data. WW: design and conceptualized study and drafted the manuscript for intellectual content.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.


At the beginning of this century, due to well-established Brazilian recycling processes, geosynthetics’ manufacturers started to use recycled poly(ethylene) terephthalate (PET) yarns/filaments (from PET bottles) in geotextile production. Despite the fact that recycled products cannot act as reinforcement functions, geosynthetics are constantly under sustained tensile load and experiences evolutions of the axial strain (creep behaviour). Thus, this study aims to assess the influence of the structure of (needle-punched) non-woven geotextiles manufactured using recycled PET yarns on their creep behaviour. Two geotextiles with different fibre/filament production processes were investigated (short-staple fibres—GTXnwS—and continuous filaments—GTXnwC). Unconfined in-isolated conventional and accelerated  creep tests were performed at 5%, 10%, 20%, 40% and 60% of geotextiles’ ultimate tensile strength. The geotextiles investigated provided similar creep behaviour to geotextiles manufactured with virgin PET material. The standard deviation of the axial strain tends to increase as the load level applied increase. The structure of the GTXnwS harms its tensile –strain behaviour, promoting axial deformation under sustained loads, at least 50% higher than GTXnwC for the same load level applied. The influence of the load level and geotextile structure in the initial axial strain is pointed out. Long-term predictions based on creep tests performed using the stepped isothermal method have proven to be conservative and they must be restricted for quality control of the investigated geotextiles. Thus, tThe creep behaviour of geosynthetic materials is mainly affected by the geosynthetics’ manufacturing process (type), the characteristics of its former polymer and the load level applied. Structured geosynthetics  have a smaller creep tendency  than the non-structured ones (non-woven geotextiles) ,25,26,27e (PET)) ,35,36,37e (PET)) , a non-le (PET))  and, at e (PET)) .Especially for the present study, one must bear in mind the temperature effects in the geosynthetics’ creep behaviour. As the test temperature rises, an increase in creep strains and a decrease in the elastic stiffness occurs ,49,50,51perature ,52,53 anperature  are poinThe most important impact of the temperature on the creep behaviour of polymeric materials is its use to accelerate creep tests at a reference temperature and obtain the long-term creep behaviour of the material tested ,55. The −1.mol−1); The value of the shift factor . However, Bueno  and Thomr, Bueno  proved tThe Brazilian expertise in PET bottles’ recycling processes provides recycled materials (yarns) with high molecular weight. According to NBR 16757-1 , geosyntFurthermore, the study of non-woven geotextiles with recycled PET yarns is still incipient, and little information are present in the literature . Since the non-woven geotextiles’ structural characteristic dominate its axial deformation , this stThis study evaluated the performance of two needle-punched non-woven geotextiles manufactured with recycled poly(ethylene) terephthalate (PET) yarns. The recycled PET yarns adopted by the Brazilian manufacturers, initiated at the beginning of this century, results from the PET bottle recycling process commonly used as liquid containers by the industry . The first one is manufactured within continuous filaments (GTXnwC) and the other one within the short-staple fibres (GTXnwS). The difference in the fibre production process identifies the difference in the geotextile creep behaviour related to its manufacturing process.The manufacturers supplied each geotextile in 5.0 m long rolls . The roll’s lateral edges (300 mm) were discarded. Geotextile specimens were obtained to perform characterization and creep tests. The creep tests’ specimens were obtained randomly in the roll’s longitudinal direction and are 500 mm × 200 mm .The properties of both geotextiles used in this study, the relevant standard testing following the Brazilian Association of Technical Standards ,77 and tThe test program consists of assessing the creep behaviour (or long-term response) of specimens obtained from two geotextiles (GTXnwC and GTXnwS) using an in-isolate (unconfined) creep test in conventional and accelerated conditions. Unconfined in-isolation creep tests (ASTM D 5262-07 ), hencef 4595-05  at room For the conventional creep test, three specimens were tested for each load level applied in the metal hack illustrated in The close-range photogrammetry method adopted in this study is based on the Bueno  photograTo perform the accelerated creep test, a temperature-controlled chamber was constructed c and attIn the abovementioned equipment, accelerated creep tests were conducted using four isothermal steps (two hours long per step). The test was initiated at room temperature of (25.0 ± 1.0) °C and, after two hours from the first isothermal step, a first temperature jump of 15 °C was achieved in 60 s. Another three two-hour long isothermal steps were performed at the temperatures of 40, 50 and 60 °C within temperature jumps of 10 °C (achieved in 60 s). This test plan is similar to those adopted by Jeon et al. , ZornberAs expected, the creep behaviour of the geotextiles, for conventional and accelerated tests, exhibits an initial axial strain , there is variability in the axial strain at a specific time , the mean creep behaviour obtained from the accelerated creep test shows reasonable agreement compared to the mean creep behaviour obtained from the conventional test . To compThe accelerated creep tests in GTXnwC resulted in mean axial strain values 15% lower than the ones obtained using the conventional creep test for all load levels investigated, except for the 5% of the eep test a. In thieep test b, which The mean values of axial strain obtained from the accelerated creep test show values with reasonable agreements  when subjected to the sustained tensile load. The lower creep modulus of the GTXnwS than GTXnwC is responsible for this excess of the creep strain developed. Moreover, GTXnwC exhibited a smaller variability in the axial strain resulting from its manufacturing process. Despite the lower values of the creep strain rate exhibited by the accelerated creep tests compared with conventional creep tests, they provide good predictions of the 1000-h creep strains of the geotextiles tested. Longer predictions have shown conservative creep strains that must be considered by the geotechnical engineers.The investigated non-woven geotextiles showed creep strains with similar behaviour and order of magnitude compared to other geotextiles manufactured with virgin PET yarns/filaments. The variability of the non-woven geotextiles’ creep behavior tends to increase as the applied load level increases and also stems from the structural response of each sample (specimen) to the applied load level;Despite the existence of this variability, up to the limit of the conventional test period , the mean creep behaviour obtained from the accelerated creep tests show reasonable agreement compared to the mean creep behaviour obtained from the conventional test for the load levels higher than 5% of The representation of a mean creep behaviour of the geotextiles using three and four specimens for conventional and accelerated creep tests, respectively, provides values of the coefficient of determination  than the creep strains developed by the GTXnwC. The lower creep modulus of the GTXnwS attached to the higher structural variability resulting from the manufacturing process is responsible for this significant difference in the geotextiles´ creep behaviour.This study reported the results of a test program including a series of unconfined in-isolated creep tests performed at room temperature and unconfined in-isolated accelerated creep tests using the stepped isothermal method (SIM) conducted in two non-woven needle-punched geotextiles manufactured with recycled poly(ethylene) terephthalate (PET) yarns/filaments. The geotextiles have a different filament/yarn process and structure, one with continuous filaments (GTXnwC) and the other with short-staple fibres (GTXnwS). Based on the creep behaviour of the geotextiles tested in this study, the following conclusions are highlighted:Based on the conclusions obtained from this study, one must be aware of the evolution of axial strain when non-woven needle-punched geotextiles manufactured with recycled poly(ethylene) terephthalate are under sustained tensile in geotechnical works and consider it in design. The adoption of non-woven geotextiles with continuous filaments (GTXnwC) instead of the ones with staple fibres (GTXnwS) helps to avoid problems caused by the geotextiles’ creep behaviour. Furthermore, long-term predictions based on creep tests performed using the stepped isothermal method have proven to be conservative and it must be restricted for the quality control of the investigated geotextiles.


The severe acute respiratory syndrome coronavirus-2, formerly known as 2019 novel coronavirus, is a pandemic public health threat. This beta coronavirus potentially infects the alveolarcells of the lung leading to pneumonia. The disease may progress into acute respiratory distress syndrome especially in elderly patients with comorbidities. Therefore, it is of interestto design and develop candidates for treatment, therapy and prevention. The spike glycoprotein of the virus known to potentially interact with angiotensin converting enzyme 2 as a cellentry receptor is a suitable candidate for further consideration as vaccine and treatment candidate. Hence, we screened the spike protein of coronavirus-2 for potential B-cell and T-cellepitopes for further deliberation. Thus, we document several peptides on the spike protein with predicted high antigenicity, low allergenicity and good stability against selected proteases.The linear B-cell epitope with sequence 'GFNCYFPLQSYGF' is of particular interest in this context towards the design and development of short peptide vaccine candidates for combat and careagainst the virus. In late December 2019, multiple cases of viral pneumonia were reported near a local seafood market in the Chinese city of Wuhan .This viA concise overview for prediction pathway of this screening study can be summarized in a flowchart as seen in The amino acids sequence of SARS-CoV-2 spike protein crystal with code (6VXX) was downloaded as FASTA file from protein data bank ,18. ForThe continuous B-cells epitopes were predicted by using antigen sequence properties tool provided by IEDB website . The oneFor the prediction of discontinuous B-cells epitopes, we have used DiscoTope version 2.0 tool which can be accessed through IEDB website .This stTo predict peptides with intrinsic potential of being T-cells epitopes presented by MHC-I molecules, we have used a combined predictor tool available in IEDB website. This predictortool can generate a final score for the affinity of the peptide towards proteasomal degradation, transporter associated with antigen processing (TAP), and also MHC-I molecules binding. We haveWe have used MHC-II binding prediction tool, available in IEDB epitope analysis resource, to scan chain A sequence for any peptide that can be presented by MHC-II molecules. For thiTo evaluate the stability of sequence-based epitopes, we have used PeptideCutter tool to predict vulnerability of these peptides against a number of proteases .The seqWe have used Docktope tool to study interaction pattern between peptides and MHC-I molecules . The molThe summary of physicochemical properties in  indicateWe document the linear B-cell epitope 'GFNCYFPLQSYGF' of specific relevance towards the design and development of short peptide vaccine candidates for combat and care against coronavirus-2.


Sarcomas are a rare disease with high rates of recurrence and poor prognosis. Important discoveries about the biology of sarcomas have been done during the last decades, without a substantial improvement of systemic treatments. With the agnostic effectivity of immuno-oncological agents in different cancer indications, it is expected that sarcomas can also benefit from these treatments. This article gathers the available data on the specific immune tumor microenvironment of sarcoma and the immunotherapeutic strategies currently under investigation.Despite significant advances in multidisciplinary treatment strategies including surgery, radiotherapy, targeted therapy and chemotherapy there are yet no substantial improvements in the clinical benefit of patients with sarcomas. Current understanding of the underlying cellular and molecular pathways which govern the dynamic interactions between the tumor stroma, tumor cells and immune infiltrates in sarcoma tissues, led to the clinical development of new therapeutic options based on immunotherapies. Moreover, progress of the treatment of sarcomas also depends on the identification of biomarkers with prognostic and predictive values for selecting patients most likely to benefit from these new therapeutic treatments and also serving as potent therapeutic targets. Novel combinations with radiotherapy, chemotherapy, targeted therapy, vaccines, CAR-T cells and treatments targeting other immune components of the tumor microenvironment are underway aiming to bypass known resistance mechanisms. This review focuses on the role of tumor microenvironment in sarcoma, prognosis and response to novel immunotherapies. Sarcomas are very heterogenous tumors, with more than 100 histologic subtypes characterized by the evolving recognition of distinct morphological and genetic features . Based oThis review presents the rationale, design and clinical efficacy of newest therapeutic approaches, incorporating immunotherapeutic modalities for sarcomas. The immune system has the potential to identify and destroy nascent tumor cells in a process named cancer immunosurveillance . The proThe relatively non-immunogenic phenotype attributed to sarcomas could be due to the low-intermediate TMB displayed by some STS subtypes as compared to the other cancer types . On the TAA are cellular-membrane bound proteins characterized by restricted normal tissue expression profile and heterogeneous expression by cancer cells. Stimulation of a tumor-specific immune response after the recognition of TAA is the rationale of therapeutic vaccines. To date, several potential TAA have been identified in sarcomas. CT antigens are molecules exclusively expressed by germ cells and so the immune system cells recognize them as foreign antigens . There aTumor immune cell infiltrates, such as effector CD8+ T-cells, are essential in hindering cancer progression and may complement the classification systems of cancer, as in the case of breast cancer and melanoma ,27. ImmuAlthough the role of intratumoral CD8 + T-cell densities in sarcomas as prognosticators has not yet been firmly established, levels of PD-1 + CD8 +  and 2 patients achieving partial responses on pembrolizumab and nivolumab/ipilimumab with high TMB (12 and 15 mutations/mb) . A retroFurther proof-of concept studies are required to determine which specific biomarkers including TMB are clinically meaningful to predict the efficacy of anti-PD1 blockade in sarcomas.Several MAbs have been introduced in the therapeutic armamentarium of tumors aiming to manipulate critical signaling pathways that sustain their malignant phenotype and metastatic potential such as olaratumab and denosumab ,54. ThesDifferent MAbs have been tested for the treatment of sarcomas. The antiangiogenic agent olaratumab, a recombinant IgG1 MAb targeting the subunit alpha of platelet-derived growth factor receptor that has been tested in a phase III study in STS combined with doxorubicin vs. doxorubicin alone, did not improved overall survival over doxorubicin alone resulting in its removal from the market . Denosumab is a MAb targeting the receptor activator of nuclear factor kappa-B ligand. Patients with giant-cell tumors of the bone showed clinical responses to denosumab that has now become the standard therapy for this disease .Disialogangliosides (GD)-2 and -3 have become an attractive target in sarcomas, since their expression in tumors of neuroectodermal origin and in different histological subtypes of sarcoma was established . The effThe overexpression of a different targetable receptor, the insulin-growth factor-1 receptor (IGF-1R), has been described in tumors associated to the pubertal growth spurt . Its oveImmune checkpoint inhibitors (ICI) are robustly modulating cellular antitumor immunologic responses and therefore are considered as the mainstay of current and future therapeutic modalities for cancer. Nevertheless, the number of patients responding to ICI is lower than anticipated and among responders there is a substantial number of patients who develop immune resistance . ConsequA study testing the anti-CTLA-4 ICI ipilimumab, that in other cancers was shown to induce strong immune responses, has shown low or no response in sarcoma patients. This study was one of the first to indicate primary resistance to immunotherapy . SubsequA single arm, phase II clinical study (SARC028) with pembrolizumab in heavily pretreated patients with STS and bone sarcoma did not meet the primary endpoint of overall response . HoweverSeveral cell-intrinsic and -extrinsic factors may contribute to these forms of immune checkpoint blockade resistance. The percentage of sarcomas expressing PD-L1 is moderate with a wide range of variation among the tumor cells expressing PD-L1 which poses a serious obstacle for the use of PD-L1 as a prognostic biomarker . High TMGenerally, limited evidence exists on the activity of ICI in sarcoma with low clinical activity of anti-PD1 alone in most sarcoma subtypes suggesting that blocking the PD1 negative signaling alone is not sufficient to adequately reactivate the exhausted endogenous antitumor effector T cells in these patients . To thisThe greater understanding of the process of T-cell activation against sarcoma and sarcoma-associated antigens have paved the way for the development of newer strategies with strong potential for clinical application in the immediate future. It has been previously suggested the possibility to increase clinical efficacy for sarcoma patients by combining ICI with other treatment modalities, such as chemotherapy or radiation therapy, and targeted therapies . The ratRadiotherapy can sensitize tumor cells to ICIs by inducing immunogenic cell death, as a result of phagocytosis of tumor cells, processing of tumor antigens, and priming of CD8+ T cells . In addip = 0.03), regardless of age, gender and previous treatment [Chemotherapy has the potential to induce antigen release and immunogenic cell death . The optreatment .Another study evaluated the clinical impact of intra-tumoral infiltration of PD1-positive lymphocytes and PD-L1 expression in STS and found that both were associated with shorter event-free survival and poorer overall survival . OngoingSarcomas have diverse molecular characteristics and targeted therapies for these rare cancers are being investigated . Targete+ T-cells [The use of small-molecule therapies have been recently emerged as suitable candidates for combination therapies with ICI in overcoming some major ICI resistance constraints . Targete T-cells . These m T-cells . These mCombining anti-angiogenesis drugs that reduce the growth of blood vessels and immune checkpoint inhibitors that promote the activation of cancer-killing immune cells offers a promising new therapeutic regimen for patients with cancer . SeveralRecent phase II and phase III clinical trials provided evidence of the clinical effectiveness of combining immunotherapy with antiangiogenic therapy leading to the regulatory approvals of pembrolizumab along with axitinib or lenvatinib for patients with kidney and endometrial cancer respectively ,82. In tc259T-cells in HLA-A2+ patients with synovial sarcoma (NY-ESO-1+) indicated trafficking of these T-cells in the TME and maintained cytotoxicity in a subset of patients [Cellular immunotherapy is at this moment a “hot” field of research in the field of solid tumors, though at its infancy. Chimeric antigen receptor (CAR) modified T cells constitute an emerging, cellular adoptive immunotherapeutic modality with the advantages of bypassing restrictions of MHC presentation . The imppatients . Table 3In the hopes of extending this effort, other immune cell-based therapies are in current development. These include non-CAR/non-TCR gene modified cell therapies for cancer, such as the ex vivo activation of tumor infiltrating lymphocytes, and immunotherapies generated from less frequently studied cell types such as dendritic and natural killer (NK) cells . The latAn additional approach to expand and activate NK cells has been described with histone-deacetylase inhibitors (HDACi) . HDACi aAfter recruitment, TAMs are “polarized” by different signals in the TME to classic-activated (M1) or alternative-activated macrophages (M2), exerting pro-inflammatory and anti-inflammatory function, respectively . The besApatinib is a vascular endothelial growth factor receptor 2 inhibitor shown to have immunomodulatory effect in sarcoma, as it reduces PD-L1 expression in osteosarcoma cells . ApatiniA recent phase II clinical trial, investigated the combination of talimogene laherparepvec (T-VEC), that has been previously shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming, and pembrolizumab . The stuTherapeutic cancer vaccines aim at re-activating preexisting antitumor immunity which is mediated by memory T cells and/or generating novel antitumor responses among the pool of naïve T cells. To be successful vaccines should specifically target immunogenic peptides expressed by the tumor in the context of MHC class I and class II molecules. Sarcomas are considered to be generally poor immunogenic and therefore there is a limited application of vaccines which is usually combined with other therapeutic modalities. In a phase I trial, a vaccine consisting of autologous mature DCs pulsed with peptide mixtures with overlapping sequences from NY-ESO-1, MAGE-A1, and MAGE-A3 cancer testis antigens is designed for the treatment of relapsed high-risk neuroblastoma, Ewing’s sarcoma, osteogenic sarcoma, rhabdomyosarcoma or synovial sarcoma post chemotherapy with decitabine (NCT01241162). Safety and antitumor immune responses are the primary and secondary endpoint from this trial, respectively. In another phase I study, a vaccine consisting of mature DCs pulsed with whole tumor cell lysates is combined with gemcitabine for the therapeutic treatment of patients with STS and osteosarcoma (NCT01803152) with safety as primary endpoint and reduction in the numbers of MDSCs and generation of immunological responses, as secondary endpoints. Autologous DCs were intratumorally injected as a cellular vaccine in a phase I study in patients with STS combined with fractionated external beam radiation. This combined treatment resulted in the generation of significant antitumor responses but clinical efficacy has not been tested . In anotSarcomas represent a rare and heterogeneous group of cancers that even when treated radically and early they may progress to display intrinsic resistance to current systemic regimens. Single agent ICI studies have shown to be largely ineffective with only few patients responding and these are mainly with aggressive subtypes of sarcomas such as UPS. Several immunological studies have corroborated the poor immunogenicity and also the unfavorable immune-modulating nature of this malignancy. Novel strategies involving combination therapies with ICI and chemotherapy, radiotherapy or targeted therapies attempt to increase the immunogenic cell death. More advanced approaches manipulating the immune system such as polarization of macrophages, dendritic cells, NK cells and CAR T cells are also expected to shed more light on this heterogeneous disease. The presence of a large mutational heterogeneity has been recently reported among patients with the same type of sarcoma. This fact highlights the importance of identifying molecular biosignatures that can be used for the design of proper therapeutic approaches. This complexity of sarcoma molecular heterogeneity combined with the network of dynamic interactions of a variety of cell types within the TME underlines the requirement for precision therapies which likely will induce clinical benefits in sarcoma patients. For instance, cancer genomics could provide the platform for successful personalized oncology in sarcoma, introducing combinatorial treatments, targeting various components within the TME. In the near future, personalized therapy in oncology may be achieved through the customization of the right combinatorial treatment based on identified tumoral and personal features. Several translational and clinical studies are ongoing to identify and validate potential predictive biomarkers. In particular, in the case of sarcomas it is mandatory to explore therapeutic modalities which will integrate these biomarkers to overcome not only the obstacle of low immunogenicity of sarcomas but also therapeutic resistance.