Osmotic demyelination syndrome (ODS) is characterized by loss of myelin in various parts of the central nervous system. It is mainly caused by a rapid correction of hyponatremia, although other factors that may cause rapid rise in serum osmolality can also be associated with its development. Its prognosis is poor and the recovery rate is unknown. The authors report a rare case of a patient with multiple risk factors for ODS, without hyponatremia, who developed ODS and surprisingly recovered. This case report highlights the importance of recognizing risk factors for the development of ODS, even if the main one is not present. Osmotic demyelination syndrome (ODS) is a rare condition  characteClostridium difficile, for which aggressive fluid resuscitation was needed. He was given antibiotics for the pseudomembranous colitis—4 days of vancomycin to which he did not respond, and he was then switched to fidaxomicin. He also developed hypokalemia which was corrected with intravenous potassium chloride (60 meq a day for 5 days). Additionally, he had poor glycemic control for which insulin was instituted. Six days after this complication, he developed a slurred speech and a progressive decline in his level of consciousness throughout the course of the next two days. Neurologic examination revealed quadriplegia and, few days later, he developed a “locked-in syndrome.”A 55-year-old male with a history of insulin dependent type 2 diabetes mellitus was admitted to the nephrology department with anasarca. He had a nephrotic syndrome for at least 2 years caused by diabetic nephropathy and had history of nonadherence to medication. Despite the generalized edema, he was noticeably malnourished. The patient was treated with high doses of furosemide and was also given ceftriaxone for a urinary tract infection. After eight days of hospital admission, he developed hypovolemic shock caused by a pseudomembranous colitis due to 3, normal glucose and protein levels, negative Gram stain, Ziehl–Neelsen stain and bacterial culture, and negative PCR for Herpes simplex virus. Electroencephalogram revealed diffuse and symmetric slow wave activity. An MRI was then performed revealing heterogeneous T1-hypointense, T2-hyperintense, and FLAIR-hyperintense areas located in the pons, cerebellar peduncles (mainly in the middle cerebellar peduncles), which were compatible with ODS (There were no electrolyte disturbances in blood analysis by the time the patient developed these symptoms . The onlwith ODS . MRI alsAcinetobacter baumannii which was resolved with meropenem. Throughout the first month of recovery, he had a continuous and persistent program of physiotherapy and partially regained his legs and arms movements. When he was discharged, he was able to stand on orthostatic position and walk with bilateral support. He continued the rehabilitation program to regain complete autonomy.Supportive treatment was given to the patient, and, unexpectedly, he started recovering from coma over the following week. During this first week, he progressively recovered his level of consciousness with an intelligible speech. He also developed, during the recovery course, a bacteremia by Adams et al. were the first to describe pontine myelinolysis in 1959 . Today, The main risk factor for ODS is rapid correction of chronic hyponatremia, particularly when it is lower than 120 meq/L . CerebraFrequently, symptoms are delayed for two to six days after rapid correction of osmolality and it can present in various ways . AsymptoCT assessment of the skull base can be difficult due to beam hardening artifact. The preferable diagnostic method is MRI. Findings might be delayed up to four weeks after the initial symptoms . It reveThe most important therapy is prevention of rapid correction of hyponatremia, or in susceptible patients, prevention of rapid changes in plasma osmolality. Treatment is mainly supportive, but some case reports have shown that the use of intravenous immunoglobulin or plasmapheresis may be useful as they may remove myelotoxic substances from plasma .Overall, ODS has a poor prognosis with a high mortality rate. Patients who survive may not recover if in a coma and irreversible sequelae may persist . No clinWe reported an unusual case of CPM with no hyponatremia. After the unexpected diagnosis by MRI, we concluded that, even in the absence of the latter in our patient, the combination of multiple risk factors contributed to the development of ODS. First, he had a prolonged history of diabetes mellitus with nephrotic syndrome and poor glycemic control, which contributed to severe proteinuria and malnutrition. During the first days after admission, he was also given a high and prolonged course of diuretics to treat the anasarca. After the abrupt diarrhea that caused the hypovolemic shock and hypokalemia, he was given a large amount of fluids. From our point of view, this was the main trigger event that caused the rapid change in osmotic balance. After this event, the chronological presentation of neurologic manifestations is consistent with ODS.There are rare, but similar, cases reported of ODS without hyponatremia. Jacob et al. described a similar case in a patient with no risk factors besides aggressive fluid resuscitation after acute bleeding . BendersThis case report also emphasizes the need to always be mindfulness with the therapeutic measures we take, even when we think they are innocuous.


Welders demonstrate a significant prevalence of work‐related musculoskeletal disorders as indicated by high rates of illness‐related absenteeism. The aim of the study was to investigate the effects of a 24‐week exercise program on workload, physical performance, and overall health in welders.Seventy‐seven professional welders were assigned to either a control group (CG), an endurance training group (ETG), or a strength training group (STG). Both groups conducted a 24‐week, standardized and progressive endurance or resistance exercise training program. Before (TP1) and after training (TP2) all participants performed an experimental welding task (EWT) in order to test the hypothesis that training would reduce the relative load (%MVC) of eight skeletal muscles measured by surface electromyography. Secondary outcome measures included further EWT‐induced stress parameters and a series of health‐related outcome measures.P < .05 vs CG). Rate of perceived exertion and visual analogue scale were decreased, while increase of maximum EWT duration was found in participants of the ETG and STG after training (P < .05 vs CG). At T2, body fat (%) decreased and physical performance  increased in ETG and STG (P < .05).Results revealed a lower muscle load in participants of the ETG and STG for trapezius muscle at TP2 compared to T1 (Both regular endurance and strength training represent effective strategies for reducing workload and improving physical performance of welders. The results emphasize the importance of physical fitness for welders and might motivate health professionals in steel‐industry to offer access to exercise training programs. Since a randomization to the intervention groups was not accepted by some of the participants the investigators decided to adapt the design to a quasi‐experiment where the participants choose their groups themselves. Group 1 performed a 24‐week strength training (strength training group [STG]) which was specially designed for the demands during welding. Group 2 performed a 24‐week endurance training (endurance training group [ETG]) and Group 3 received no additional treatment (control group [CG]). All measurements were conducted before training (T1) and after training (T2). As main outcome measure, the relative load of trapezius muscle was chosen.2.2Seventy‐seven participants  completed the 24‐week intervention and were included into data analysis. Their age and anthropometrics are shown in Table 2.3The EWT was designed to simulate the occupational workload of welders by means of a standardized laboratory experiment and was performed without electricity and no welding light to avoid interferences with the surface electromyography (SEMG) and to increase work safety, respectively. The EWT was performed in two positions: First, the participants performed EWT in a standing overhead Position (StOP) (ASME: 4F) Figure . The pos2.4® Inc USA). Preparation and electrode  placement (middle of the muscle belly with 2 cm interelectrode distance) were consistent with the SENIAM guidelines for SEMG recordings.Surface electromyography (SEMG) was used to determine the relative muscle activity load throughout the EWT. SEMG signals were determined from shoulder‐neck , core (erector spinae m. at the height of lumbar vertebrae 1), and arm muscles  by a bipolar wireless eight channel system . Maximum heart rate (HRmax) was calculated at the end of all four passes. Maximum blood pressure (systolic [SBPmax]/diastolic [DBPmax]) was measured using an electronic blood pressure monitor  at the end of all four passes, too. Immediately after finishing each EWT position, the study participants rated their maximum perceived pain upward from the hips and maximum overall exertion by means of a 100‐mm visual analogue scale (VASmax) and Borg rating of perceived exertion scale (6‐20) (RPEmax), respectively.2.6Body fat (%) and muscle mass (%) were analyzed by bioelectric impedance analysis (BIA) .A bicycle exercise test  was performed until exhaustion . Therefore, we measured maximum bicycle performance (maximum workload in watt [W]) and relative bicycle performance in relation to body weight (BW) (W/kg/BW) as outcome measures to quantify endurance capacity. Maximum bicycle performance (W) was calculated according to the formula: workload last stage completed (W) + [time of the last uncompleted stage (sec)/stage duration (sec) × stage increments (W)].Maximum strength tests were performed to measure muscular capacity. Peak isometric torque (Newton meter [Nm]) during elbow flexion and extension, knee flexion and extension, as well as trunk flexion and back extension were measured by the m3‐Diagnos analysis station (Schnell). Two trials were completed for each position, with contractions lasting 5 seconds, separated by 30 seconds rest intervals. Participants were encouraged verbally to elicit their maximal effort . Peak torque values (Nm) were recorded, and the higher of the two repetitions was used for statistical analysis.In addition, health‐related quality of life was assessed by the SF‐36 questionnaire. Physical component summary (PCS) and mental component summary (MCS) were calculated.2.7The training program followed exercise guidelines for the general population from the American College of Sports Medicine.Participants of the STG performed a whole‐body strength training program and included a periodized variation of program variables. Precisely, during the first 12 weeks, participants performed the training in 3 sets of 20‐25 repetitions at 55%‐60% of their one repetition maximum (1RM). At the second 12 weeks, training was performed in 3 sets of with 10‐15 repetitions at an intensity of 70%‐75% of 1RM. There was a 60‐sec break between each set. The resistance training program considered those muscles which are stressed during welding most such as back, shoulder‐neck, and forearm and their antagonists, too. All participants received an introduction by a sports scientist and performed the strength training 2‐3 times a week self‐depended thereafter. The exercise intensity was adapted to the individual training process as shown by additional measurements of the 1RM at weeks 8 and 16. To increase the compliance the investigator contacted the study participants by telephone at week 4, 12, and 20  walking according to their own preferences thrice a week separated by a minimum of 24 hours rest. Endurance training program was designed in a standardized and progressively way and included a periodized variation of program variables, too. In weeks 1‐12, participants performed moderate intensity twice and vigorous intensity once. Duration of moderate intensity increased from 30 minutes each 4 weeks by 5 minutes up to 40 minutes, while vigorous intensity stayed consequently at 20 minutes. At the beginning of week 13 up to week 24, participants performed moderate intensity once and vigorous intensity twice. Duration of vigorous intensity increased from 30 minutes each 4 weeks by 5 minutes up to 40 minutes, while moderate intensity keeps consequently at 40 minutes. Therefore, training volume increased in both periods every 4 weeks by 10%. Moderate intensity was defined as 65%‐75% of individual maximum heart rate and a vigorous intensity was defined as 75%‐85% of individual maximum heart rate. The highest recorded heart rate at the end of the final stage of the bicycle exercise test was regarded as maximum heart rate. The participants controlled their training intensity themselves by wearing a heart rate monitor Polar® Heart Rate Monitor FT1 (Polar® Electro GmbH Büttelborn). In weeks 4, 8, 12, 6, and 20, the participants were contacted by telephone in addition to the training protocol . As all data were normally distributed (Kolmogorov‐Smirnov test), parametric tests could be applied for further analysis. Initial baseline measurements were analyzed by analysis of variance (ANOVA) to determine differences between the groups. To determine the interaction of group x time point and time effect a two‐way ANOVA with repeated measures (3 × 2) was calculated. Bonferroni‐Holm corrected tests were used for post hoc analysis to detect differences between groups and time points. Due to the explorative nature of the investigation of all secondary outcome parameters, no adjustment of the alpha error for multiple testing has been considered. If not indicated otherwise, results are given as arithmetic mean ± standard deviation (SD). The level of statistical significance was set at 3In total, attendance rate of ETG amounted to 77% ± 25% (55 ± 18 of 72 training sessions) and attendance rate of STG amounted to 66% ± 21% (40 ± 13 of 60 training sessions).3.1F1,73 = 5.93; P = .017) but no time × group  in standing overhead position (StOP). Post hoc analysis showed significant reduction of muscle load between TP1 and TP2 in STG   or ETG (P = .30). The two‐way repeated‐measure ANOVA revealed a significant effect for time  and time × group  in sitting bended position (SiBP). Post hoc analysis of time effect showed significant reduction of muscle load between TP1 and TP2 in ETG  and STG  , as well as STG and CG (P = .048). A significant time effect for infraspinatus muscle in StOP was observed, while post hoc analysis showed no significant difference (P = .078). In SiBP, analysis of the erector spinae muscle revealed a significant effect for time × group. Post hoc analysis revealed no significant differences between groups, too . A significant time and time × group effect were detected for pectoralis major muscle in SiBP. The post hoc analysis showed a significant difference within STG (P = .012) and between STG vs CG (P = .003)  Figure . There w3.2P = .018) and in SiBP (ETG: P = .030). The analysis of subjective exertion showed a significant time effect for RPEmax in StOP  and SiBP (ETG: P ≤ .001). Additionally, there was a time × group effect for RPEmax in StOP  and SiBP (ETG vs CG: P = .006). Furthermore, we detected a significant time effect for VASmax in StOP (STG: P = .012). Data analysis revealed further a significant time × group effect in StOP while there were no significant difference in post hoc analysis between groups. However, in SiBP there was a significant difference between ETG vs CG (P = .012) and STG vs CG (P = .015). Additionally, we found a significant time effect for EWT‐duration  and a time × group effect  in StOP  and time × group effect . Muscle mass (%) showed a time effect, too , but no interaction. Among the bicycle exercise test a significant time effect in maximum bicycle performance (W) , as well as relative bicycle performance (W/kg/BW) (ETG: P = .003) was found. Moreover, we detected significantly time × group effects for maximum bicycle exercise (W)  and relative bicycle exercise (W/kg/BW) (ETG vs CG P = .027). The results of maximum strength test revealed a significant time x group effect of trunk flexion . Additionally, there was a significant time effect  and time × group effect  for back extension (Table Body fat (%) revealed a significant time effect (ETG: Due to significant group differences for PCS at baseline no ANOVA was performed.4This study indicates that both a 24‐week strength as well as endurance training exert beneficial effects on work‐related stress and on different dimensions of health and performance in professional welders. Both exercise training interventions were effective in reducing the muscle workload, the degree of subjective exertion and the perceived pain intensity after EWT. In parallel, the maximum possible duration of the welding task increased when compared to control. Within ETG maximum heart rate decreased during welding task. Results slightly differed between StOP and SiBP relating to physical response. Strength and exercise capacity increased and body fat mass decreased following both interventions compared to control. Body muscle mass increased after strength and endurance exercise program. Overall health and performance parameter were improved equally by both interventions.Regarding the MVC measures relative load of the trapezius muscle was chosen as primary outcome measure. Former studies demonstrated that the upper trapezius muscle indicated one of the highest stress during welding especially in overhead positions.Surprisingly, an increase in relative muscle load of pectoralis major m. in SiBP was found. Albeit the pectoralis m. showed the lowest load of all measured muscles during EWT. This muscle is an important synergist of stabilizing muscle groups of the shoulder, such as rotator cuff, especially of the subscapular and deltoid muscles.Interestingly, the reduced muscular load of the trapezius muscle in SiBP was also detected in ETG. It is suggested that this is a result of ameliorated physical conditions as indicated by maximum bicycle and core strength test results and reflect improved welding ergonomics in general. We further assume that the alterations in relative muscle load may result in general health benefits and contribute to a reduced risk for shoulder‐neck disorders among welders in long‐term.Altered muscle activation went along within a reduction of RPEmax and VASmax. This is not surprising, since a lower level of muscular load induces less physical exertion and muscular pain. VAS seems to be a good indicator of musculoskeletal symptoms, and also RPE is commonly used to evaluate job related strains.The various effects of a regular resistance or endurance training on physical functions and health are well evaluated. Accordingly, the loss of body fat and increases in muscle mass are known reducing factors for cardiovascular diseases. Similarly, increased endurance capacity and muscular strength represents an important factor for overall health.Beside a positive effect on welders' health and wellbeing, companies are interested in a high productivity of their staff. In order to quantify working performance, EWT duration was analyzed, too. The present results show that a longer EWT duration could be achieved after both exercise programs in StOP. Consequently, we cannot exclude a bias on other outcome measures as a longer welding duration could result in a higher degree of fatigue.Overall, the attendance rate to the training program was only moderate to poor, especially in STG. Albeit, we did not evaluate the reason for missing training sessions. From the overall feedback, we conclude that training program itself represent no barriers for attendance. Nevertheless, even two to three sessions of endurance training or one to two sessions of strength training per week seems to reduce workload, improves physical performance, and promotes overall health in welders. However, given a low attendance rate in this study, the authors would expect that a higher exercise frequency would be more effective which is in line with other studies.Anyway, this study has some limitations. First, a randomization was not applied. Consequently, a selection bias may have occurred in terms of unbalanced training experience, prevalence of MSD, and work experience over the study groups. For instance, it cannot be excluded that participants chose to take part in a training group due to their own training experience. If this were the case, training adaptation effects would be underestimated since untrained individuals have higher adaptation potentials. Furthermore, the EWT was performed under standardized laboratory conditions increasing the internal validity of this study. Anyhow, the external validity was negatively affected since welders are generally exposed to additional stressors such as weight of protective clothing, fumes, and heat but we could not imitate these extreme conditions for technical reasons. Last, information about both, attendance rate and adherence to training were derived from the participant's self‐report, which could bias the results.In conclusion, this study demonstrated that a regular strength or endurance training induces various objective and subjective adaptations in welders. These data emphasize the benefit of a physical training in the prevention and health promotion of welders in terms of reducing workload, increasing physical performance, and improving overall health. Regarding our main outcome measures both exercise programs seem to have similar benefits, whereby strength training seems to be a bit more favorable. Secondary measures indicated differentiated effects for each exercise type without pointing out a superiority of one exercise type. While strength training has a greater impact on the muscular activation pattern endurance training exerts positive cardiovascular effects during welding. Both interventions lead to a decrease of subjective exertion. If these programs are also able to reduce WMSD, long‐term illnesses and painful conditions, and prevent work‐related absenteeism has to be evaluated in future studies.Approval of the research protocol: This study was approved by the local ethics committee of the Justus‐Liebig‐University Giessen, Germany. Informed consent: All participants gave written informed consent before enrolment. Registry and the registration no. of the study/trial: N/A. Animal studies: N/A. Conflict of interest: The authors declare that they have no competing of interests.All listed authors have significantly contributed to this work. CP, KK, and FCM conceived and designed research. CW, CP, TR, TF, and FCM conducted the experiment. CW analyzed the data and wrote the manuscript with CP and KK. All authors read and approved the final manuscript.


Despite the success of immune checkpoint inhibitors (ICI) for treating a variety of solid cancers, most gastric cancer patients are resistant to ICI monotherapies. Combinations of ICI with other therapies may be able to overcome this resistance. In order to develop combination immunotherapies, immunologically well-characterized preclinical gastric cancer models are required. To this end, in the present study, we characterized two murine gastric cancer cell lines, namely, YTN2 which spontaneously regresses, and YTN16 which grows progressively. Although anti-CTLA-4 monotherapy eradicated most YTN16 tumors, these were resistant to either anti-PD-1 or anti-PD-L1 treatment. Furthermore, we identified neoantigens in YTN2 and YTN16 tumors and conducted neoantigen-based immunotherapy for these tumors. In addition, the information on neoantigens facilitates the evaluation of tumor-specific immune responses induced by the combination therapies. These immunologically well-characterized gastric cancer models will contribute to the development of novel combination immunotherapies.+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8 Gastric cancer is the fifth most frequent cancer and the fourth most common cause of cancer death worldwide . Anti-PDFor developing targeted therapies for cancer, it is common for candidate drugs to be initially evaluated using cancer cell lines in culture and xenografts in immunodeficient mice. Given that ICI therapy targets the host’s immune system, immunocompetent animal models are needed to facilitate the development of new combination therapies. Because no immunologically well-characterized transplantable murine gastric cancer cell lines were available, from one tumor we established four subclones that could be transplanted into C57BL/6 mice [These earlier studies demonstrated that these gastric cancer cell lines might also be valuable for developing cancer immunotherapies. To this end, here we characterize two of these gastric cancer cell lines, YTN2 and YTN16, in terms of the expression of neoantigens derived from tumor-specific mutant proteins. Our findings may provide useful information for enhancing the development of models for novel effective immunotherapies for gastric cancers.b:Ig (H-2Db dimer), DimerXI:Recombinant Soluble Dimeric Mouse H-2Kb:Ig (H-2Kb dimer) were from BD Biosciences . FITC-conjugated anti-CD3ε, PerCP/Cyanine5.5-conjugated anti-CD4, APC/Cyanine7-conjugated anti-CD8, APC-conjugated anti-IFN-γ and Pacific Blue-conjugated anti-CD45 mAbs were from BioLegend .Six-week-old female C57BL/6N mice were purchased from Japan SLC  and kept in a specific pathogen-free environment. The animal use proposal and experimental protocols were reviewed and approved by The University of Tokyo Animal Care and Use Committee (ID:P15-125) and all animal procedures were conducted in accordance with institutional guidelines. The YTN2 and YTN16 cell lines [6 YTN2 or YTN16 subcutaneously into the right flank. Anti-PD-1 (200 μg), anti-PD-L1 (200 μg), anti-CTLA-4 (100 μg), and/or anti-CD8α (200 μg) were injected intraperitoneally. Tumor growth was monitored every 2 to 3 days with calipers, and tumor volume was calculated by the formula π/6 × L1L2H, where L1 is the long diameter, L2 is the short diameter, and H is the height of the tumor.Mice were inoculated with 5 × 10Genomic DNA was extracted from YTN2, YTN16, LLC1, and B16F10 cell lines using Allprep DNA/RNA mini kits  according to the manufacturer´s protocols. DNA was randomly fragmented by Covaris and adapters were ligated to both ends of the fragments. DNA was then amplified by ligation-mediated PCR, purified, and hybridized to the Roche NimbleGen SeqCap EZ Exome probe . The captured library was loaded onto the HiSeq 2000 and HiSeq 4000 platforms . After trimming of adapter sequences, reads were aligned to mm10 mouse reference sequences using the Burrows–Wheeler Aligner (BWA). Somatic variants were detected using SOAPsnp. Raw data were deposited in the Sequence Read Archive (SRA) database . WES data of MC38 was downloaded from the SRA database (SRR5684459).9, where Y is the number of fragments mapped to a gene; L is the length of the gene; N is the total number of mapped reads of a sample. Raw data were deposited in the Gene Expression Omnibus (GEO) database (GSE146027 and GSE184092).Total RNA was isolated using TRIzol RNA Isolation Reagent  and RNeasy mini kits (Qiagen). RNA-Seq libraries were prepared using TruSeq Stranded mRNA Library Prep (Illumina) according to the manufacturer’s protocols and sequenced on NovaSeq 6000 and HiSeq X systems (Illumina). The reads were aligned to mm10 reference sequences with STAR (version 2.7.0f). The mapped reads were counted with featureCounts (version 1.6.4). Fragments per kilobase of exon per million reads mapped (FPKM) was calculated by the formula Y/LN × 1050 and eluted ligand (EL) rank to H-2Db and H-2Kb. Presentation percentiles of MHCflurry (ver.2.0.1) [50 of NetMHCpan ≤ 250 nM, EL rank of NetMHCpan ≤ 0.5 and presentation percentile of MHCflurry ≤ 0.5 were selected. Peptides were synthesized by standard solid-phase synthesis using a Syro I  as described previously [First, we selected expressed mutations as follows: FPKM ≥ 30 and RNA variant allele frequency (VAF) ≥ 0.04. Then 8-, 9-, and 10-mer epitopes containing the mutated amino acid were inspected using NetMHCpan2.8 [eviously .5 RMAS cells incubated overnight at 26 °C were mixed with titrated peptide concentrations in 0.25% BSA-containing DMEM, incubated for 30 min at room temperature, and then exposed to 37 °C for 70 min. Cells were then stained with FITC-labeled anti-Kb mAb (B8.24.3) or FITC-labeled anti-Db mAb (B22.249) and analyzed by flow cytometry. Differences between experiments were normalized by including the reference peptides in every assay.Peptide binding was measured by a stabilization assay using TAP-deficient RMAS cells ,12. Brie9 YTN2 or YTN16 cells were lysed in buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF, and 1% octyl-β-D glucopyranoside . The peptide-MHC class I complexes were captured by affinity chromatography using purified monoclonal antibodies (clone Y-3 for Kb and 28-14-8S for Db) coupled to CNBr-activated Sepharose 4B . Peptides bound to MHC class I were eluted with a mild acid (0.2% TFA) and desalted using a Sep-Pak C18 cartridge  with 28% ACN in 0.1% TFA and ZipTip U-C18  with 50% ACN in 1% FA. Samples were dried using vacuum centrifugation and dissolved in 5% ACN in 0.1% TFA for LC-MS/MS analysis. Samples were loaded into a nano-flow LC  online-coupled to an Orbitrap mass spectrometer equipped with a nanospray ion source . The nonsynonymous mutations unique to YTN2 or YTN16 were translated in frame and the polypeptide sequences encompassing mutations with a maximum length of 61-mer amino acids were matched against a conventional protein reference database (Swiss-Prot). MS/MS data were searched against personalized custom reference databases using Sequest HT and Mascot  on the Proteome Discoverer platform (Thermo Fisher Scientific) with a tolerance of precursor and fragment ions of 10 ppm and 0.02 Da, respectively. A false discovery rate (FDR) of 0.01 was used in the Percolator node of the Proteome Discoverer version 2.2 software (Thermo Fisher Scientific) as the peptide detection threshold.Direct detection of neoantigens using mass spectrometry has been described previously . Briefly8 IFN-γ-treated  YTN16 cells were treated with citrate-phosphate buffers  for 1 min at room temperature. The eluted peptides were loaded onto a Sep-Pac C18 cartridge (Waters Corporation), washed with water, eluted with 60% acetonitrile, lyophilized using a Solvent SpeedVac (Thermo Fisher Scientific), and reconstituted in the citrate-phosphate buffer. Peptides of 3000 Da were then isolated by filtration through Centricon-3 ultrafiltration devices (Merck Millipore). The resulting flowthrough was then fractionated by reverse-phase high-performance liquid chromatography (HPLC). Briefly, bulk peptides were fractionated on a YMC-Pack ODS-A column  by the following gradient from solvent A (1% acetonitrile/0.1% HCl) to solvent B (80% acetonitrile/0.1% HCl): from 0 to 5 min, 0% B; from 5 to 10 min 0–12.5% B; from 10 to 70 min, 12.5–75% B. The flow rate was maintained at 0.5 mL/min, and fractions (0.5 mL each) were collected from 15 min to 70 min. The fractions were lyophilized. For T cell assays, the lyophilized samples were reconstituted in 50 μL HBSS and 10 μL were added to 1 × 105 cells from the mutated (m) Zfp106-reactive CD8+ T cell line in 200 μL medium. After overnight culture, the supernatants were harvested and IFN-γ was evaluated using Mouse IFN-γ ELISA Ready-SET-Go kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.MHC class I binding peptides were eluted from viable YTN16 cells as described previously . Firstly+ T cell lines, YTN16 tumor-bearing mice were treated with anti-PD-1 and/or anti-CTLA-4 mAbs. The spleens were harvested from mice that rejected the tumor, and 5 × 106 splenocytes were stimulated with IFN-γ (10 U/mL) and 1 × 105 irradiated (100 Gy) cells from the YTN16 cell line in the presence of 100 U/mL recombinant human IL-2 . The cells were re-stimulated with YTN16 on day 7 and harvested on day 12–14. They were screened for reactivity to YTN16 antigens by separately culturing them (1 × 105) overnight with 11 candidate short peptides at 1 μg/mL, harvesting the supernatants, and quantifying IFN-γ production by ELISA.To generate YTN16 cell line-specific CD8+ T cell line, splenocytes from mice that had rejected YTN2 were stimulated with IFN-γ-treated (10 U/mL) and 100 Gy-irradiated YTN2 cells four times weekly. For establishment of the mCdt1-reactive CD8+ T cell line, splenocytes of mice that had rejected YTN16 following treatment with anti-CTLA-4 mAb were stimulated with the 9-mer mCdt1 peptide at 1 μg/mL four times weekly. For establishment of the mScarb2-reactive CD8+ T cell line, splenocytes of mice that had rejected YTN16 after anti-CTLA-4 treatment were stimulated with IFN-γ-treated (10 U/mL) and 100 Gy-irradiated YTN16 cells. Seven days later, mScarb2-H-2Db dimer+ CD8+ cells were sorted on an SH800S  and stimulated with IFN-γ-treated (10 U/mL) and 100 Gy-irradiated YTN16 cells another three times weekly.For establishment of the mZfp106-reactive CD8b and H-2Kb dimers. For intracellular cytokine staining, 1 × 105 cells were stimulated with peptides, 1 × 105 YTN2 or 1 × 105 YTN16 cells in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich) for 4 h. After staining dead cells with the Fixable Viability Dye eFluor 450 (Thermo Fisher Scientific) and blocking Fc receptors with anti-CD16/32 mAb, the cells were stained with mAbs for cell surface antigens, followed by fixation, permeabilization, and staining with APC-conjugated anti-IFN-γ mAb (BioLegend) using Intracellular Staining Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X) (BioLegend) according to the manufacturer’s protocols.Tumors were cut into pieces and incubated in RPMI-1640  supplemented with 1% FBS, 10 mM HEPES, 0.2% collagenase  and 2 KU/mL DNase I (Sigma-Aldrich) for 40 min at 37 °C. All material was passed through a 70 μm cell strainer to obtain single cell suspensions. After staining dead cells using a Zombie Yellow Fixable Viability Kit (BioLegend) and blocking of Fc receptors with anti-CD16/32 mAb , the cells were stained with mAbs for cell surface antigens, H-2D+ T cells, 1 × 106 neoepitope peptide-pulsed DCs were subcutaneously injected into the flank of mice twice biweekly. Two weeks after the second vaccination, the splenocytes were stimulated with the corresponding peptides and IFN-γ production was determined by intracellular cytokine staining. For evaluation of anti-tumor effects of neoepitope peptide-pulsed DC vaccines, mice were inoculated with 5 × 106 YTN16 cells on day 0 into the right flank. Neoepitope peptide-pulsed DCs were injected into the left flank on day 5. Tumor growth was monitored every 2 to 3 days.DCs were prepared as described previously . Briefly6 YTN16 cells on day 0. On day 6, 1 × 107 neoantigen-reactive CD8+ T cells were infused intravenously. Tumor growth was monitored every 2 to 3 days.Mice were inoculated with 5 × 10Data are presented as mean ± SD. Statistical analyses were performed with Prism software . Comparison of results was carried out by one-way ANOVA testing with Dunnet’s multiple comparison test.+ T cell-mediated [+ T cells using anti-CD8α mAb prevented tumor rejection on anti-CTLA-4 antibody treatment (+ T cell responses.YTN2 and YTN16 are subclones derived from the same gastric cancer cell line established from an N-methyl-N-nitrosourea (MNU)-induced tumor [mediated . Thereforeatment c,d, indi+ T cell responses, we determined the specificity of tumor-reactive CD8+ T cells in YTN2 and YTN16 tumor-bearing mice. First, we established YTN2-reactive CD8+ T cell lines from the spleens of mice that had rejected YTN2 tumors, and YTN16-reactive CD8+ T cell lines from mice that had rejected their YTN16 tumors following treatment with anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 mAbs. After two rounds of weekly in vitro stimulation with irradiated tumor cells, we obtained YTN2- and YTN16-reactive CD8+ T cell lines  were lysed and peptide-MHC class I complexes were precipitated with anti-MHC class I antibodies. The bound peptides were analyzed by tandem mass spectrometry. From YTN2 tumor cells, 424 H-2Db-binding and 228 H-2Kb-binding peptides were identified , both G1966A and T1975C mutations were found on the same reads e. Theref 10−11 M f. Furthe peptide f. This s+ T cells and identified three neoepitopes ). In addition, the identification of specific neoepitopes eluted from tumor MHC molecules through immunopeptidomics, or LC-MS/MS-based immunopeptidomics, confirms that the neoepitopes are processed and presented at the cell surface [+ T cells was a 10-mer peptide rather than the 9-mer peptide predicted by in silico algorithms  and pep+ T cell responses. We successfully demonstrated the neoantigen-based immunotherapy-controlled tumor growth. Identification of neoantigens enables monitoring of the dynamics of anti-tumor immune responses and helps evaluate the effect of combination immunotherapies on tumor-specific immune responses. This information will contribute to the development of novel combination immunotherapies for gastric cancer.Here, we immunologically characterized two gastric cancer cell sublines derived from the same parental line but with very different behaviors. We provided information on the efficacies of ICI treatment and neoantigen-specific CD8


Origanum syriacum, Rhus coriaria, Eryngium creticum, and Cichorium intybus were among the most quoted species by informants. Sleeq (steamed leafy vegetables), Zaatar (breakfast/dinner food), and Louf (soup) were the most popular wild plant-based dishes.Wild food plants (WFPs) have been an important source of human nutrition since ancient times, and it particularly revives when conventional food is not available due to emergency situations, such as natural disasters and conflicts. The war in Syria has entered 10 years since it started in 2011, and it has caused the largest war-related crises since World War II. Nearly 60% of the Syrian population (12.4 million people) are food-insecure. WFPs are already culturally important in the region, and may be supplementing local diets during this conflict. Our study aimed to uncover the conflict’s effect on the use of WFPs and to know what species are consumed by local people during the current crisis. The fieldwork was carried out between March 2020 and March 2021 in the Tartus governorate located in the coastal region of Syria. Semi-structured interviews were conducted with 50 participants (26 women and 24 men) distributed in 26 villages along the study area. We recorded the vernacular names, uses, plant parts used, modes of preparation and consumption, change in WFP use before and during the conflict, and informants’ perceptions towards WFPs. We documented 75 wild food plant species used for food and drink. Almost two-thirds (64%) of informants reported an increase in their reliance on wild plants as a food source during the conflict. The species of  Food is located at the bottom of Maslow’s hierarchy of human needs, where any threat to these basic needs could impact human behavior and bring emergency reactions . DespiteSince ethnobotany has been described as the science of survival , its impIn the last two decades, the world experienced an increase in its number of conflicts. Only between 2000 and 2014, there was an average of 35 active conflicts every year around the globe . Since tIn Syria, as a part of the Mediterranean basin, cooked vegetables and salads made from wild greens have been particularly important as local traditional foods since ancient times . However2 and has a population of 1,114,000 inhabitants [The study was conducted in the Tartus governorate , one of abitants . The cliabitants . The regabitants . Tartus abitants . So far, the region has been relatively quiet and safe during the Syrian conflict. Tartus is entirely under the government control; however, it witnessed several security incidents . It is fThe study was conducted between March 2020 and March 2021. The participants were chosen using a combination of purposive and convenient sampling methods ,40. We aInformants were asked to list all the wild plants they used for food and beverage preparation, their vernacular names, the parts used, and mode of preparation and consumption. Respondents were asked to report any changes in their use of WFPs prior to and during the conflict. Perceptions of informants towards WFPs were documented using open-ended questions, such as: Why do you use WFPs? Why were you using WFPs before the conflict started?Verbal consent was always obtained before each interview, and the Code of Ethics of the International Society of Ethnobiology was followed . A reseaCollected data were compared with previously published ethnobotanical studies from the Eastern Mediterranean region and adjacent countries ,25,28,43Eryngium creticum and Malva sylvestris. On the other hand, species that grew in high mountains and remote areas, such as Gundelia tournefortii, witnessed a decrease in use during conflict years due to obstacles regarding movement and security concerns. According to our informants’ statements, traditional knowledge of WFPs was noticeably decreasing during the stable economic situation in pre-conflict years. However, it was revived after becoming necessary to secure food during the conflict. The majority of our study respondents (94%) used WFPs before the conflict, whereas 6% of them started to use those plants only after the conflict started a decade ago. Informants showed that WFPs have always been a part of their traditional diet. However, such meals served as complementary food before the conflict, while they became among one the main dishes in recent years. Almost two-thirds (64%) of informants reported an increase in their reliance on wild plants as a source of food during the conflict compared to the pre-conflict time. On the other hand, 34% of the informants reported a no-change status in their reliance, whereas 2% stated a decrease. The main reported reason behind the apparent increase of reliance on WFPs was the economic crisis that hit the whole country after the war, which affected the food prices and purchase power, while the reason for the minor percentage of decrease in reliance was the move from a rural to urban area. The conflict affected accessibility and WFP species selection; informants reported that during the conflict, they started to consume wild plant species that grew in anthropic sites and their surrounding orchards, such as Informants’ statements show a clear difference in perceptions and reasons for using WFPs during and before the conflict. The main motives behind the use of WFPs in the pre-conflict era were represented by the tendency to eat healthy and organic food, efforts to diversify the food and taste, and enjoyment of the activity of gathering plants from the wild. On the other hand, the difficulty to afford the market food products was the main reason for WFP use during the conflict. The majority of informants (58%) reported that the natural ecosystems helped to substitute market products through the gathered plants, and consequently, to save money. The mutual perception among the local people was that some of the young generation’s views on WFPs did not change during the conflict compared to the pre-conflict time. These young people, especially those who did not bear the responsibility of securing food for the household, perceived WFPs as non-prestigious food and felt ashamed to share information related to this food with their peers.Our study documented 75 wild plant species used for food and beverage preparation by the local people. One taxon was identified only down to the genus level. The total of 75 taxa belonged to 70 genera and 28 botanical families. The most-represented families were Compositae (15 species), Fabaceae (8 species), Lamiaceae (7 species), Apiaceae, and Rosaceae (5 species each). Compositae included plants that are mostly prepared as cooked vegetables. On the other hand, Lamiaceae mainly included the spices and herbs that are consumed, dried, and ground, whereas Rosaceae included the species that are consumed mostly as fresh fruits. Species mentioned only once or twice in the survey were excluded from further analysis.Origanum syriacum, Rhus coriaria, Eryngium creticum, Cichorium intybus, Micromeria myrtifolia, Allium ampeloprasum, Cirsium vulgare, Gundelia tournefortii, Scandix pecten-veneris, Malva sylvestris, Anchusa strigosa. On the other hand, the species Nasturtium officinale, Rumex acetosa, Thymus vulgaris, and Arum maculatum were used by 60–80% of informants. Sixty-four plant species were reported by at least 6% of informants . The folSleeq is the most popular wild plant-based dish in the study area. It is prepared from gathered wild leafy vegetables. The name Sleeq is probably derived from the word Saleeq, which in the Arabic language means boiled food; however, the dish is mainly prepared by steaming, rather than boiling. The same dish was also called Mhabbleh by some informants, which locally refers to steamed food. There is no limitation to what kind of or how many wild vegetables can be included in the preparation. The most common species used in the preparation of Sleeq are Scandix pecten-veneris, Cirsium vulgare, Allium ampeloprasum, Erodium acaule, Cichorium intybus, Anchusa strigosa, Gundelia tournefortii, Silybum marianum, Rumex acetosa, Anemone coronaria, Malva sylvestris, and Urtica dioica. Practically every wild leafy vegetable that is able to be gathered by the collectors can be included in the dish, since it is sometimes difficult to gather a sufficient amount from one species only. The young shoots are the main part used in the Sleeq. The dish is mostly prepared in a local traditional clay pan called Meqli (see the video abstract), where chopped onion is fried in olive oil, then cut wild leafy vegetables are added. Some informants add a small amount of chickpea and/or softly ground bulgur if available. Several informants reported that it is possible to use Sleeq as a topping on the dough to be prepared as pizza, with the same mentioned receipt excluding chickpea and bulgur. Sleeq is a seasonal food connected with the availability of wild leafy vegetables usually collected from January to April.Zaatar bread/pizza. The dried and ground leaves of Origanum syriacum, the main ingredient of Zaatar, are mixed with dried ground fruits of Rhus coriaria, and roasted seeds of Sesamum spp. in the following proportion: 1.0 of Origanum syriacum, 0.75 of Rhus coriaria, 1.0 of Sesamum spp. Dried fruits of Pistacia terebinthus/Pistacia atlantica and seeds of Foeniculum vulgare may be sprinkled as spices. Zaatar is usually stored dried in jars or plastic bags, and consumed over the whole year.Traditional food in many regions in Syria. It is a sour spicy dish consumed by mixing with olive oil, usually in breakfast and dinner meals, as well as in Louf is a traditional soup consumed only in some parts of the Syrian coastal mountains, especially by the Alawite and Ismaili cultural-religious groups. The soup has a sour-astringent taste with a thick texture. Arum maculatum is the main ingredient in Louf soup; its dark-green leaves are loosely cut and steamed for around half an hour. Afterwards, olive oil and bulgur are added, then the water of boiled Rhus coriaria is added after disposing of Rhus coriaria. Arum maculatum contains a high amount of calcium oxalate, which is a toxic compound [Arum maculatum is usually available from December to April. Some informants reported that they store it in glass jars in the refrigerator and consume it throughout the year.compound ; howeverCichorium intybus is boiled for around half an hour, then olive oil, crushed garlic, and a little bit of lemon juice are added . Wild vegetables are also sold in the local markets at a lower price than other cultivated vegetables; for instance, the price of 1 kg of wild leafy vegetables (Sleeq) varies between 0,28 and 0,42 USD. We observed that several informants attempted to cultivate some wild species  in their home gardens. Some people living in the cities and town centres (where access to wild plants is limited) depended on their relatives and friends in rural areas to gather WFPs for them.Changes in diet in the study area have mainly been represented by the decrease in buying market food products and increase in the use of WFPs, particularly leafy vegetables. Most commodities’ prices in Syria have increased tens of times since the beginning of the conflict. However, a few products are still affordable by the majority due to the government supporting these products. Bread, which is the main part of every meal, is one of these affordable commodities at 0.06 US Dollars per 1 kg (1 USD = 3500 Syrian Pounds according to the market exchange rate of late October 2021). Olive oil is produced by the majority of people in the study area where olive orchards dominate the landscape. Vegetable prices in the market vary between 0.3 to 1.4 USD for 1 kg, and prices for 1 kg of meat range between 2.9 and 5.7 USD. Those prices are considered relatively high and commonly unaffordable compared to the average monthly salary of 149,000 SP ≈ 43 USD . Hence, Gundelia tournefortii is documented in all compared studies; this is possibly due to its pleasant taste, as our study informants reported that it is one of the most preferred WFPs. It is usually steamed in olive oil with minced meat or chickpeas. Several plant species were found to be unique to the study area, such as Arum maculatum, which is a very popular and traditional soup (Louf) in some parts of the study area. All the species that comprise the most common wild plant-based dishes are culturally important and often frequently used. Despite that some of these species are less available nowadays in the wild, they still remain highly preferred, and this is possibly due to their pleasant taste or their importance in the local culture as a main part of the traditional diet. Based on the results and our observation, the sour-astringent taste was found to be preferred by many informants, especially from Alawites and Ismailis, which was demonstrated by the high use of some wild species, such as Arum maculatum and Rhus coriaria, which were described by some informants as “the sour of Phoenicians”.The total number of 75 documented WFPs in the study area represents relatively high diversity compared to the Eastern Mediterranean region. It demonstrates a richness in the wild-related food culture. Kawas et al.  documentScandix pecten-veneris, Gundelia tournefortii, and Nasturtium officinale, as well as other dark-green leafy wild vegetables, such as Arum maculatum) could play a significant role in preventing anaemia , olive oil (a significant source of unsaturated fatty acids), bulgur , and bread (a main source of carbohydrates) serves as a good source of essential nutrients for the human body. There is a deficiency of data on nutritional status in Syria, especially considering that the situation is getting worse every year with the continued conflict, economic sanctions, and conflict-related chaos and corruption. However, a report from WFP  showed a anaemia . In timey system . Some ofO. syriacum, G. tournefortii, and R. acetosa demonstrated a richness in nutrient content. More research will help determine the exact nutritional role that these WFPs play in supplementing local diets during the conflict. Understanding the perceptions of local people towards WFPs could help in planning successful promotion of some nutritive species. Furthermore, future studies should consider the sustainability of WFP use and how these plants could be protected during crises.The field study that we conducted among the local people in the coastal region of Syria showed a remarkable level of reliance on WFPs as a source of human nutrition. The study demonstrated the increased use of WFPs during the conflict compared to the pre-conflict time. Our results strengthened the findings of previous studies that ethnobotanical knowledge functions as a coping method for food shortage. We documented 75 wild plant species used in food and beverage preparation with a relatively high diversity comparing to other studies from the Eastern Mediterranean. The most common wild food-based dishes and their preparation mode were documented. Some species, such as


Radio frequency fingerprinting (RFF) methods are becoming more and more popular in the context of identifying genuine transmitters and distinguishing them from malicious or non-authorized transmitters, such as spoofers and jammers. RFF approaches have been studied to a moderate-to-great extent in the context of non-GNSS transmitters, such as WiFi, IoT, or cellular transmitters, but they have not yet been addressed much in the context of GNSS transmitters. In addition, the few RFF-related works in GNSS context are based on post-correlation or navigation data and no author has yet addressed the RFF problem in GNSS with pre-correlation data. Moreover, RFF methods in any of the three domains  are still hard to be found in the context of GNSS. The goal of this paper was two-fold: first, to provide a comprehensive survey of the RFF methods applicable in the GNSS context; and secondly, to propose a novel RFF methodology for spoofing detection, with a focus on GNSS pre-correlation data, but also applicable in a wider context. In order to support our proposed methodology, we qualitatively investigated the capability of different methods to be used in the context of pre-correlation sampled GNSS data, and we present a simulation-based example, under ideal noise conditions, of how the feature down selection can be done. We are also pointing out which of the transmitter features are likely to play the biggest roles in the RFF in GNSS, and which features are likely to fail in helping RFF-based spoofing detection. The purpose of any RFF technique is to identify genuine transmitters (or transceivers) and distinguish them from malicious ones. For example, the authors in [The radio frequency fingerprinting (RFF) concept refers to the process of identifying the hardware (HW) characteristic and HW-specific features or signatures embedded in the radio frequency (RF) waves transmitted over a wireless channel ,2,3,4. Ithors in  performeEspecially in the context of global navigation satellite systems (GNSS), intentional interference such as jamming and spoofing has been on the rise in recent years and can have significant adverse effects on the navigation performance of GNSS receivers, as discussed for example in ,13,14,15Future aviation applications, and in particular unmanned aerial vehicles (UAVs), will increasingly rely on GNSS-based navigation and positioning solutions ,15. SafeThere are many authentication and anti-spoofing methods in GNSS which are not based on RFF and such methods that have been widely studied in post-correlation, and especially at navigation levels ,17,18,19A schematic block diagram of the three domains  of a typical GNSS receiver is shown in A good survey of anti-spoofing methods based on the post-correlation and navigation data in GNSS can be found for example in . HoweverOffering a thorough survey of RFF methods applied with GNSS and non-GNSS wireless data in the literature, and discussing which of these RFF methods have potential in GNSS, and in particular in GNSS with pre-correlation data. Finding good anti-spoofing methods based on pre-correlation GNSS data could have tremendous benefits for the future GNSS receivers, by being able to detect and remove non-genuine signals even before processing them further in the acquisition and tracking loops. Our survey is unique in the current literature, as the RFF methods for GNSS have to date not been widely investigated and there is a current lack of unified surveys on this;Proposing a step-by-step problem definition of RFF in the context of GNSS signals, by delving in depth in the sources of possible transmitter hardware impairments, and also discussing the possible channel and receiver–hardware impairments; this problem decomposition into feature-by-feature investigation is also lacking from the current GNSS literature, to the best of our knowledge;Proposing a four-step generic RFF approach, consisting of: feature identification, feature extraction, data pre-processing, and data classification. Classical ML and transforms methods are used in this four-step methodology, but the four-step block diagram is rather novel;Presenting the mathematical models of different GNSS transmitter features, with a particular emphasis of five main identified features, namely: the power amplifier non-linearities, the digital-to-analog converters’ non-linearities, the phase noises of the local oscillators, the I/Q imbalances, and the band-pass filtering at the edge of the transmitter front-end; unified mathematical methods of the transmitter HW impairments are not found in the current literature to the best of the authors’ knowledge;Providing the equivalent transmitter block diagrams for GNSS and spoofers by incorporating the aforementioned five hardware effects into the models;Presenting an illustrative simulation-based analysis based under ideal conditions in order to emphasize the impact of each HW feature on the RFF performance. Three feature extractors to identify the transmitter HW impairments were used, namely the kurtosis, the Teager–Kaiser energy operator (TKEO), and the spectrogram. The classification accuracies given as examples are based on support vector machines (SVM). Such a simplified analysis allows us to identify the strongest features among the five considered ones and to point out the remaining challenges to overcome to achieve the feasibility of RFF methods under more realistic GNSS scenarios;Bringing in a qualitative discussion on the existing algorithms and providing a roadmap towards further research on RFF in GNSS for interference detection and classification.As seen in the discussions above, there is still a lack of surveys of RFF methods for GNSS transmitter authentication in the current literature, particularly on surveys of GNSS authentication relying on pre-correlation signals. In this paper, we are addressing this lack, via a comprehensive study of the literature in the past two decades, as well as via theoretical insights and the preliminary analysis of algorithms. Our contributions are as follows:The rest of this paper is organized as follows: Most of the GNSS signals use the code-division multiple access (CDMA) technique, with a received signal power around A spoofing scenario is illustrated in Simplistic spoofing attacks, such as those generated by a software defined radio (SDR) GNSS generator connected to an antenna. In this type of attack, the GNSS transmitter is not synchronized to the genuine GNSS satellites, which means that there are typically jumps in the carrier-to-noise ratios (CNR) and Doppler shifts measured at the receiver and such spoofing attacks can be identified in the pseudorange domain via various consistency checks algorithms, such as those described in [ribed in ,28,29;Intermediate spoofing attacks [ attacks ,31: thesSophisticated spoofing attacks [ attacks : these aSpoofing attacks are typically split into three classes, described in detail in :SimplistSpoofing attacks adversely affect the quality of positioning, navigation and timing (PNT) services of GNSS receivers, by introducing errors in the estimated PVT. For example, as shown in , an intePre-correlation link-level methods relying on signal samples before the acquisition stage, i.e., on I/Q data. This is the case addressed in this paper. Such pre-correlation anti-spoofing methods are still very rare in the literature;Post-correlation link-level methods relying on the despread signal, at the output of the tracking stage for a single satellite. Examples can be found in [found in ,34 and tNavigation or system-level methods relying on the pseudorange signals coming from all visible satellites. These are by far the most encountered anti-spoofing methods in the current literature and a few examples can be found in [found in ,28,29  and aiming to identify, based on RF fingerprinting approaches, whether the received signal comes from a genuine GNSS transmitter or from a spoofing transmitter.four-step methodology for the RFF-based pre-correlation spoofing detection and transmitter identification, as listed below. Each of these four steps is further detailed in We are proposing a Identification of relevant features—this step refers to first identifying the different RF ‘features’ created by the inherent hardware impairments in any transmitter. Several such features will be subsequently described in Feature-extraction transform—this steps refers to choosing a suitable feature-extraction transform to emphasize the selected features from the previous step. Several feature-extraction transforms are addressed in Data pre-processing stage—this step refers to choosing the most suitable format of saving the data at the output of the feature-extraction transform, namely as time-stamped vector data, in matrix form, as an image of certain size and number of pixels, etc. The data format selection will be influenced by the algorithms selected in the feature-classification step, as subsequently described in Feature classification—this step refers to applying a selected classification methods, such as based on analytically-derived thresholds or on machine learning algorithms when training data are available, and classifying the received signal into ‘genuine’ versus ‘non-genuine/spoofer’ classes. Several feature classification approaches are discussed in The workflow of an RFF algorithm based on the aforementioned four steps is illustrated in Phase noise (PN): PN is unavoidable in any wireless transmitter, as it is introduced by the transmitter clock instabilities; atomic clocks on-board genuine GNSS transmitters are intuitively expected to have lower phase noise than the clock of spoofers and other malicious transmitters [smitters ,37,38. PPower amplifier (PA) non-linearities: non-linearities close to the saturation region for PAs  can represent an important HW feature to distinguish between different transmitters. In addition to non-linearities, possible memory effects of the PA can also create differentiating features at the transmitter. PA models are discussed in I/Q imbalance: the I/Q imbalance in a transmitter is introduced in the translation of the baseband signals to passband signals due to the facts that the phase shift is not perfectly at Digital-to-analog converter (DAC) non-linearity: signal distortions are also possibly produced by the non-linear DAC operation at each transmitter. DAC models are discussed in Band-pass filter (BPF) passband and out-of-band ripples: the transmitter BPF filter also puts its ‘fingerprint’ on the transmitted signal and can act as a smoother of the other HW features. BPF models are discussed in A first step in building the equivalent block diagrams for a GNSS transmitter (genuine or spoofer) was to identify the possible sources of hardware (HW) impairments at the transmitter side for wideband-signal transmitters, based on works in ,41,42,43Each of these identified HW impairments is further detailed in the subsequent sub-sections.Typically, the phase noises are random noises, modelled via random time waveforms the form :(1)xSϕThe usually adopted model for GNSS signals is to ignore everything except the white-noise PN model at te noise . Withoutte noise  where a The on-board GNSS local oscillators are atomic clocks based on rubidium/cesium clocks . TypicalA typical measure of the PN PSD is through the so-called Allan variance given by (3)σA2, such as solid state power amplifiers (SSPA) or a travel-wave tube amplifier (TWTA) , while nL. When the wideband signals pass through the power amplifier, the bandwidth of signals is not negligible compared with the inherent bandwidth of the amplifier, and therefore a frequency-dependent behaviour occurs. This behaviour is called a memory effect. Regarding the non-linear model with linear memory, the two most encountered models are the Wiener model and the Hammerstein model, as described in [There are typically two classes of models for PA non-linearities : the memribed in . We illuWiener model:s is the sample index (assuming the q-th coefficient of a finite impulse response (FIR) filter, and Hammerstein model:Due to the difficulties of estimating the coefficients for FIR filters in both the Wiener and Hammerstein model, the memory polynomial  has becoIn order to maximize the power efficiency and the lifespan of the satellite payload, the GNSS signals are usually designed to exhibit a (quasi) constant complex envelope. For instance, this is achieved by including an inter-modulation product among the signal components. For this reason, it is reasonable to expect that the PA non-linearities will not significantly distort the genuine GNSS signals. This might not hold for many spoofing signals, which may simplify the signal generation by only emulating some of the signal components and/or omit the inter-modulation product. However, it shall be noted that a spoofer usually needs to generate low-power levels, hence it is easier to ensure linearity with LPA. The fact that the spoofer needs to transmit at a lower power than the GNSS transmitters is due to the fact that spoofers are usually within the range of a few tens of meters to a few km away from the GNSS receivers, while GNSS satellites are at more than 20,000 km away from the receivers.During the baseband-to-passband conversion, the I and Q components xIBased on , the DACThe clock additive effect based on , gtΔn haHere, we demonstrate two examples in K is the order of the filter.The band-pass filter (BPF) is equipped at transmitters to filter out undesired non-central frequencies signals. In this work, we model BPF using a finite impulse response (FIR) filter. A general form of an FIR filter output We use the window design method for the genuine GNSS transmitter BPF and the least squares method for spoofer transmitter BPF. An example of BPFs used for a genuine GNSS transmitter versus a spoofer transmitter is shown in The equivalent block diagrams of a GNSS  satellite transmitter and of a spoofer GNSS transmitter are depicted in ock unit a appeareock unit b does noThe GNSS power amplifier is typically an HPA, while the spoofer power amplifier is typically an LPA, as discussed in N genuine GNSS transmitters and M spoofers, l-th path of the i-th channel, and l-th path of the i-th channel. Above, i-th channel. Typically, A signal h, etc.) ,54 or toh, etc.) . Howeverh, etc.)  were donh, etc.)  were forFurthermore, the receiver from Equation ). As shoThe error vector magnitude is a time-domain transform that measures how far the estimated symbols at the receiver side may deviate from the true symbols. I/Q imbalance, thermal noise, in- and out-of-band leakage, and phase noise are all causes that can degrade the EVM metric, thus EVM has the potential to be a good feature-extractor transform to capture hardware impairments from the received signals.In general, EVM is applied in the context of demodulated signals, as follows: let us assume that a symbol When the input to the EVM transform is the I/Q sampled data, one can apply the EVM as follows: Kurtosis is a measure of the Gaussian behaviour of a random variable and it is defined asAn example of a histogram for the kurtosis results of genuine GNSS transmitter and spoofer is shown in The Teager–Kaiser energy operator (TKEO) is a transform which can estimate the instantaneous energy of a signal, and thus may uncover features that are distinguishable in power or energy. The TKEO transform fined as (19)TTKETKEO has been previously used in the context of RFF in GNSS in  with prom is the time sample index, the f is the frequency, and The short-time Fourier transform (STFT) Clearly, A wavelet transform decomposes an incoming signal into some ‘coarse’ and ‘fine’ coefficients, based on shifted and scaled versions of a so-called ‘mother wavelet’ function. Unlike the Fourier transform that cannot offer compact support in both the time and frequency domains, a wavelet transform can offer a compact/bounded support in both tome- and wavelet-domains. Wavelet transforms have been extensively used in watermarking and image-processing applications, and have been reported to be able to identify ‘hidden’ features; thus, they look like relevant feature extractors for RF fingerprints. Wavelet transforms, in the context of RF fingerprinting, have been previously used, for example in ,60,61. TFeature classification methods can be typically split into two main classes: (i) methods based on thresholding or the direct sorting of the outputs of the feature extraction stage; and (ii) methods based on machine learning (ML) classifiers. The second category was by far the category most encountered in RF fingerprinting, as shown previously in The threshold-based classification is also known as a traditional hypothesis testing and can be implemented through well-known algorithms such as likelihood ratio testing (LRT) or Gaussian likelihood ratio testing (GLRT) ,84. The If the feature-extraction transform outputs scalar or vector values  or by using an initial training base with genuine and spoofing signals and derive a threshold based on the training database. Another challenge in this threshold-based approach is that most of the transmitter features are ‘hidden’ and not distinguishable through classical hypothesis testing, as the probability distribution functions of Equation  would ovIn our work, we used the optimal false alarm rate from ML-based classification to calculate the detection rate. By comparing this detection rate with the optimal one from ML-based classification, we evaluate the performance of the threshold-based method.Machine learning (ML) methods have been widely used in the literature as methods of classification in RF fingerprinting approaches or for transmitter identification and authentication : As the problem we address here is a classification problem with two classes: spoofer absent .Typically, a Gaussian kernel takes best into account the irregular boundary in the I/Q GNSS datasets. As the dimensions of raw I/Q data are typically huge, some forms of dimensionality reduction can be typically employed. One such form is known under the name of PCA is a common method to pre-process data for the purpose of reducing the dimension of the target dataset before the classifications. The first few principal components implies the most dominant features existing in the dataset, whilst using PCA is an effective way to improve the classification performance.For example, The convolutional layer applies a convolution operation between the input signal matrix and a filter (or kernel) . For example, The pooling layer will reduce the number of parameters; it is essentially a sampling method. The common pooling methods are max pooling, average pooling, and sum pooling. Here, we provide an example of max pooling in The fully connected layer is the actual neural network, by using the activation function, such as the sigmoid (or logistic function), we are able to label the outputs. A common fully connected layer is made of three parts, the input layer, the hidden layer(s) , and the output layer. Convolutional neural networks (CNN), the most frequently encountered category of deep learning classifiers, have been widely applied in image identification and pattern recognition. Recently, CNN classifier has also started to be considered as a promising method for the radio identification and RFF ,87. A geOther approaches of ML-based classification less encountered in the context of RFF are: linear discriminant analysis (LDA) ,82, logiLDA is usually used to separate two or more classes or to achieve dimensionality reduction. The basic idea behind LDA is to find a projection of the input data such that the separation of classes could be maximized. This method is limited, however, by the condition that both input classes follow normal distributions. LR usually works with classes characterized by linear features and it is not well suited to non-linear features as those created by power amplifiers and digital-to-analog converters. The studies in , appliedAn in-house-based simulator was built based on Matlab 2020b version and Python 3.7.5. The Matlab modules were used to generate I/Q samples based on a GNSS and a spoofer model, each having five types of transmitter features: PA non-linearities, DAC non-linearities, I/Q imbalance, phase noises, and BPF. The parameters of genuine GNSS transmitters are typically not available in open access, as they are protected via IPR. In the absence of such GNSS exact parameters for these HW features, we adopted various models from the literature. For example, the PA non-linearities were modelled according to , and theA two-millisecond observation window of Galileo E1 band signals was used in the examples shown in this section. In order to deal better with smaller The results of the classification are presented via the confusion-matrix metric. In our simulator, each feature can be active or inactive, making the simulator flexible to be able to down select or identify the ‘strongest’ features, as well as their overall impact when they act jointly . Moreover, as seen in Unlike the typical narrowband terrestrial signals typically studied to date with RFF techniques see , the GNSBased on our literature research and the preliminary theoretical analysis, The most promising combinations, based on our preliminary analysis, are the kurtosis and thresholding combination, and the spectrogram and SVM combination. Potential good results may also be expected, based on a current literature search and theoretical analysis, from kurtosis and SVM combination, as shown in This paper presented a survey of RFF methods for spoofing mitigation in GNSS receivers. While the survey of methods and the methodology presented in here can be generally applied also in a non-GNSS context, the focus in our paper has been on GNSS pre-correlation data, as the pre-correlation anti-spoofing methods are still rare in the current literature.Addressing the impact of the signal mixtures from signals from various satellites and various frequency bands: typically, the received signal is a mixture of all satellites visible in the sky at the considered moment, and possibly, of one or several spoofing signals. One approach to look at a single signal at a time would be to first despread each signal from each identified pseudo-random code, and then apply successive or parallel interference cancellation methods to identify each signal, one by one. The errors in the estimation of the signals from various satellites would, of course, affect the quality of the re-constructed signal, and possibly, the accuracy of the RFF-based classification. Another approach would be to create huge training databases with all possible mixtures of satellites in the sky and to use those databases in the classification process;Evaluating and mitigating the impact of channel multipath and fading effects: each wireless channel (from satellite or spoofer) has its own random signature, determined by the multipath delays, Doppler spreads, and fading effects. As these effects are random in nature, they will, most likely, not provide additional ‘features’, but will have a negative impact on the strength of the transmitter features. The effect of the wireless channels upon the RFF algorithms can be further investigated via simulation- or measurement-based approaches and it remains a topic of future investigation;Understanding the impact of the receiver HW features upon the RFF methods: while the same receiver is capturing either genuine GNSS signals or a mixture of genuine signals and spoofer(s), and thus the same receiver effects are present in both situations (spoofer present or spoofer absent), the receiver also has local oscillators, ADC and filter blocks, etc., and each of them can introduce additional phase noises, non-linearities and I/Q imbalances. Intuitively, such effects will have a negative impact upon the classification accuracy compared to an ideal receiver (without any HW imperfections), but such effects need to be further analysed based on measurements or simulated data.Dealing with the negative impact of high noise levels on RFF performance, especially when dealing with low-power signals such as those in the pre-correlation domain: GNSS signals in urban scenarios, such as GNSS receivers on-board of drones flying through tall buildings, can be received at relatively low CNRs, and these low CNRs are likely to act as smoothers of the transmitter features, to the point of fading them out. It remains an open research question what the CNR threshold is above which the RFF methods with pre-correlation GNSS samples are likely to work;Validating through real-field measurements the promising RFF performance for authenticating GNSS signals.A four-step methodological approach has been proposed in One of the main contributions of our paper was presenting a step-by-step methodological approach proposed to be adopted for a designer wishing to build an RFF algorithm in a GNSS receiver. The identified transmitter HW features are likely to be reflected not only in the pre-correlation data (illustrated in our examples through the paper), but also in the post-correlation and navigation domains, thus our four-step methodology also paves the road towards more advanced RFF GNSS processing in all three domains , with a future aim to offer robust and hybrid anti-spoofing solutions. An additional contribution of this paper has been to present an ample survey of existing RFF methods in the literature used with both GNSS and non-GNSS signals and already showing promising results. As described in this last section, several challenges are still to be overcome towards the success of RFF methods, especially when relying on the low-power GNSS I/Q raw data. It is our belief that this survey bridges the missing gap between the RFF studies in the non-GNSS context and the anti-spoofing methods studied to date only at the post-correlation and navigation levels in the GNSS context. It is our intent that this paper sheds new light on how to approach an RF fingerprinting process to identify hidden transmitter features, by first decomposing the problem into the relevant transmitter features and then by selecting the most suitable pair of feature-extraction transform and classifier algorithm in order to classify the transmitters according to their features or HW impairments. While many challenges still remain in the RFF GNSS research field, it is also the authors’ belief, based on our understanding of the research problem, that by combining various authentication methods, at different levels , one is more likely to obtain good results than by using a single authentication method. The simulation-based results presented here are only for some selected illustrative parameters and are useful in the context of down selecting the most important HW features of a GNSS transmitter. We saw that, even under ideal conditions such as 100 dBHz carrier-to-noise ratio, the phase noise and the DAC nonlinearities are not differentiating features, while P non-linearities, I/Q imbalances, and band-pass filters carry the potential of being good RF ‘fingerprints’. For the sake of a reduced complexity of simulations, the observation window used in our simulations was of 2 ms. Further investigative studies at a lower


Colon capsule endoscopy as an alternative to colonoscopy for the diagnosis of colonic disease may serve as a less invasive and more tolerable investigation for patients. Our aim was to examine patient-reported outcomes for colon capsule endoscopy compared to conventional optical colonoscopy including preference of investigation modality, tolerability and adverse events. A systematic literature search was conducted in Web of Science, PubMed and Embase. Search results were thoroughly screened for in- and exclusion criteria. Included studies underwent assessment of transparency and completeness, after which, data for meta-analysis were extracted. Pooled estimates of patient preference were calculated and heterogeneity was examined including univariate meta-regressions. Patient-reported tolerability and adverse events were reviewed. Out of fourteen included studies, twelve had investigated patient-reported outcomes in patients who had undergone both investigations, whereas in two the patients were randomized between investigations. Pooled patient preferences were estimated to be 52% (CI 95%: 41–63%) for colon capsule endoscopy and 45% (CI 95%: 33–57%) for conventional colonoscopy: not indicating a significant difference. Procedural adverse events were rarely reported by patients for either investigation. The tolerability was high for both colon capsule endoscopy and conventional colonoscopy. Patient preferences for conventional colonoscopy and colon capsule endoscopy were not significantly different. Procedural adverse events were rare and the tolerability for colon capsule endoscopy was consistently reported higher or equal to that of conventional colonoscopy. Early-stage detection and removal of colorectal pre-cancerous polyps is an effective measure for the prevention of colorectal cancer (CRC) . To dateThe existing knowledge on patient discomfort and minor AE with CCE is scarce as patients only have renewed contact with the hospital if a subsequent COC is mandatory and, even so, patient-reported outcomes (PRO) are rarely addressed. Patient acceptance and preference towards CCE are of outermost importance for a broader clinical implementation. Hence, we find it relevant to conduct a systematic review and meta-analysis to collect and present existing data on PRO and the preference for CCE compared to COC.We conducted a systematic literature search in PubMed, Embase and Web of Science to identify all relevant citations for studies in which acceptability, tolerance or preference was expressed by the patients , after COC and CCE were performed. The primary outcome of our analyses were the preference proportions, whereas acceptability and tolerance were secondary outcomes. According to the PICO search strategy, search terms were included in three areas: investigation, comparator and outcome. The terms within each area were combined using the Boolean “OR”; the three areas were combined in strings using the Boolean “AND”. Free text search terms with truncation were included. The literature search was concluded on 12 January 2021. The complete search strings are available in full text articles;articles reporting PRO after undergoing both COC and CCE or randomized controlled trials (RCT);articles in English/Danish/Italian/French/Spanish language.Inclusion criteria were:non-RCT articles in which patients underwent either COC or CCE alone;the following article types: reviews, conference papers, case reports.Exclusion criteria were:After identification and exclusion of duplicates, references were independently screened by three authors . Each author screened two thirds of the references in title and abstract, excluding those not meeting the inclusion criteria. In case of discrepancy, the reference was included for full text evaluation. After this first step, the process was repeated on included references by evaluating full texts with the same modality.® Crohn), cleanliness and completion rates for both COC and CCE. COC procedures were considered complete when the caecum was reached; CE procedures were considered complete when the CE was excreted during the recording time or when the anal verge or the hemorrhoidal plexus were visualized. PRO and AE were also collected. Regarding PRO, the preference for COC and CCE was expressed as proportions (%). AE were limited to events related to the procedure itself, excluding those related to bowel preparation (BP).All data were extracted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . We collA study assessment of the included studies was performed by three independent reviewers  through the STROBE assessment tool and the Cochrane Collaboration’s tool (according to the study design) ,14. STROn = 30). For Cochrane, within each bias category , the bias level was rated as low, high or unclear.For STROBE, we arbitrarily designated studies as of low-, medium- and high transparency and completeness according to the tools’ outcome scores: for STROBE, the cut-offs were respectively 60% and 80% of the total maximum points  but not indicating preference were included in the denominator. Non-responders were excluded. The significance level was set at 5%, and 95% confidence intervals (CI) were calculated. All pooled estimates were calculated in random effects models using the Freeman–Tukey double arcsine transformation. In sensitivity analyses, we repeated the preference proportions calculations, first, after exclusion of studies from which only a subset of the sample could be included, and second, after exclusion of studies with samples under 50 individuals. IHandbook . PotentiHandbook  were perHandbook .n = 2) studies they were randomized for either. Included studies are described in n = 10) studies reported patient preference for CCE and eight (n = 8) reported patient preference for COC. Four studies did not report patient preference but described patient-reported AE and/or tolerability.The initial literature search resulted in 1632 references. After the removal of duplicates this was reduced to 1326 and, additionally, 1274 were excluded after title and abstract screening. Full text screening was performed for 52 references and 14 were eventually included in the study  11,18,1,29,30. In = 9) studies were evaluated as having high transparency and completeness and five (n = 5) studies as medium, see Nine   for CCE 2 = 88.32% and 85.81), univariate meta-regressions were performed to identify possible sources. None of the tested variables resulted in statistically significant effects, although age mean yielded the lowest observed p-values for both CCE and COC preference analyses  moderate/severe AE  was reported in CCE from patients. More retentions could be expected as several of the included studies report retrieving capsules in the following colonoscopy if not excreted. Mild patient-reported AE from CCE included difficulty with camera ingestion and abdominal discomfort. In comparison, two (n = 2) moderate/severe AE in COC were reported from patients . Mild patient-reported AE from COC included local phlebitis due to intravenous access, abdominal pain, rectal bleeding and elevated blood pressure. In conclusion, moderate to severe procedural AE were rarely reported by patients for both CCE and COC.Very low proportions of AE (often none) were reported in the included studies. Only one (n = 13) ulcerative colitis patients rated overall tolerability for CCE and COC on a ten-point scale to be 7.9 average for both procedures, even though they rated abdominal pain 4.9 for COC and 1.0 for CCE on the same scale [Several studies reported results that were collected using visual analog scales (VAS) and/or numerical scales. Median overall rating reported from 1 (very bad) to 10 (very good) was 8 for CCE and 7 for COC in 148 individuals . On a nume scale . Similarme scale . In 31 ime scale .Our systematic review with meta-analysis shows no significant difference in patients’ preference between COC and CCE. About half of the included patients, having tried both modalities, declare an equal preference for the two colonic imaging modalities. One would have expected that patient preference would sway towards CCE as it can provide a virtually painless exploration of the colon, with similar diagnostic yield and accuracy to COC in many clinical situations. Nevertheless, all that glitters is not gold: as highlighted in the results of our study, the watershed of PRO for CCE versus COC remains blurred; the stigma surrounding colonoscopy, often raising well-known barriers in the CRC screening populations, failed to show any apparent inclination towards CCE as one may have expected, concerning PRO. On the other hand, a substantial proportion of patients still preferred CCE even though COC is considered the golden standard. The reality may be that each investigation should target the patient in question and not be considered a one-test-fits-all strategy.In a recent interim analysis , we askep-value. As speculation, a slight tilt in preference towards COC in older patients may be explained by the higher pre-test probability of colorectal pathology, eventually leading to therapeutic COC after CCE anyway. Taking into consideration possible publication bias, the funnel plot for COC preference seems a bit skewed to the left , whereas for CCE it is more homogenous.In the current study, no statistically significant possible sources of heterogeneity were highlighted regarding patients’ preference. The age of the patients in both CCE and COC groups was the variable with the lowest Taking into account these data, when coming to tolerability parameters, surprisingly, the tolerability was consistently reported higher for CCE. It is unclear why this does not translate to higher patient preference for the modality. One explanation may be that the patient-reported advantages for COC outweigh the drawbacks and these are therefore accepted. Moreover, patients with known disease who are familiar with COC, may be more accepting of the procedure and any level of relevant discomfort, or since the tolerability is very high for both procedures, the relative higher tolerance for CCE has no actual behavioral relevance. Moreover, if the tolerance is very high for both, the risk of two examinations following a positive CCE could be considered too much of a risk.Both examinations also require two stints of BP as the CCE report is not available immediately after excretion, enabling COC under same preparation. As BP is a documented barrier to COC and the increased cleansing regimen for CCE is a reason for preferring COC ,32, the 2 statistics indicated heterogeneous subsets of patients, and different collection methods were used in the assessments of PRO , although no statistically significant sources were identified. Finally, for some of the included studies, only a subset of patients could be included in the meta-analysis in this review as only those subsets underwent both investigations. This introduces a risk of overrepresentation of patients with positive CCE as those excluded in the present study were discharged after negative CCE. It is expected that COC is preferred in patients with pathology as they would prefer going straight to COC since it is not possible to perform a biopsy or polypectomy with CCE.This study comes with some limitations. The presence of small study effects for pooled preference proportion for COC (as backed up by the Egger’s tests) introduces the risk of publication bias. The IThe preference proportions for CCE and COC were not statistically different in this meta-analysis, with an estimated 52% preferring CCE and 45% preferring COC. Procedural AE are rare in both CCE and COC. The tolerability for CCE is generally reported higher or equal to that of COC. As a reminder for future studies, it should be emphasized to include both patients with and without positive CCE/COC to be able to estimate preference proportions for the general population.