Amyloid fibrils are mechanically robust and partly resistant to proteolytic degradation, making them potential candidates for scaffold materials in cell culture, tissue engineering, drug delivery and other applications. Such applications of amyloids would benefit from the possibility to functionalize the fibrils, for example by adding growth factors or cell attachment sites. The BRICHOS domain is found in a family of human proteins that harbor particularly amyloid-prone regions and can reduce aggregation as well as toxicity of several different amyloidogenic peptides. Recombinant human (rh) BRICHOS domains have been shown to bind to the surface of amyloid-β (Aβ) fibrils by immune electron microscopy. Here we produce fusion proteins between mCherry and rh Bri2 BRICHOS and show that they can bind to different amyloid fibrils with retained fluorescence of mCherry in vitro as well as in cultured cells. This suggests a “generic” ability of the BRICHOS domain to bind fibrillar surfaces that can be used to synthesize amyloid decorated with different protein functionalities. Amyloid formation is one particular type of protein folding associated with several severe human diseases4 but also with functional assemblies like bacterial surface fibers and spider silk6. Amyloid is formed by polypeptide chains in cross β-sheet conformation, i.e. the direction of the β-strands is perpendicular to the fibril axis7. The fibrils are thus highly regular but the atomic details of the architectures of determined high-resolution amyloid structures differ substantially9. Each amyloid is associated with fibrils made up of one specific protein and although properties such as size, native structure and location differ widely between amyloidogenic proteins, the ultrastructural appearance of amyloid fibrils is similar independent of constituent protein10.Proteins can misfold and end up in different types of aggregates, a phenomenon that is linked to many human diseases, but protein assemblies are also widely used in nature as the building blocks of functional materials with many attractive properties11. The β-sheet structural units that are the backbone of the fibrous structure provide the unique mechanical properties for nanofibers12. They can be built from a broad range of different proteins, including proteins from food production11. The β-structure of these nanofibers is energetically highly favored, which makes them particularly resistant and difficult to break down for the body’s own clearance systems. These properties open up for functionalizing protein nanofibers in medical applications. A great advantage is that the nanofibers can be equipped with further features by attaching functional sites, such as growth factors, specific biomacromolecules, fluorophores, etc., for novel applications e.g. as cell culture scaffolds or biosensors11.Amyloids can be utilized as proteinaceous artificial materials in form of protein nanofibers, which are a new class of nanomaterials with potential applications ranging from global health to materials science, including artificial tissues, scaffolds for cell cultures, biosensors as well as functional biocoatings13. The BRICHOS domain differs from other approaches previously tested to treat amyloid disease and was initially found to be expressed together with surfactant protein C (SP-C), one of the most amyloid-prone proteins known15. The fact that nature can produce α-helical SP-C led to the discovery that the BRICHOS domain in proSP-C acts as a molecular chaperone that prevents β-sheet aggregation during biosynthesis16. Mutations in the proSP-C BRICHOS domain result in amyloid formation of the SP-C part and lethal amyloid lung disease in early childhood16. All other known amyloid diseases occur late in life and these observations thus strongly support the hypothesis that the BRICHOS domain is capable of efficiently preventing amyloid formation under normal circumstances. The BRICHOS domain can prevent amyloid formation not only of its physiological clients but also of other amyloidogenic proteins, including Aβ and islet amyloid polypeptide (IAPP) associated with type 2 diabetes19. Importantly for potential synthetic applications, BRICHOS binds to the surface of Aβ amyloid fibrils, which results in that generation of neurotoxic Aβ species is markedly reduced23. Here, we investigate whether the ability of BRICHOS to bind to fibrillar surfaces can be harnessed for linking proteins fused to BRICHOS to amyloid fibrils, which could be a versatile way to generate functionalized nanomaterials. We tested this hypothesis by fusing BRICHOS to the red-fluorescent mCherry protein24 and confirm the co-localization on fibril surfaces by Thioflavin T (ThT) staining. Following this approach, we demonstrate here that mCherry-linked Bri2 BRICHOS can bind to the fibril surface of six different protein nanofibers, which include four typical human disease-related amyloid fibrils as well as a de novo-designed β-structure protein. These results hence provide the basis for decorating protein nanofibers with various functionalities, which can be implemented in designed protein-based materials.As amyloid fibrils are associated with human diseases, it is conceivable that endogenous defense mechanisms against fibril formation exist, and that a disease state develops when these defense mechanisms are malfunctioning. We have found an endogenous defense—the BRICHOS domain—against amyloid fibril formation and its toxic consequences, which holds potential to be translated to an efficient treatment of aggregation and neurotoxic effects of the amyloid β-peptide (Aβ) in Alzheimer´s disease24 downstream of human Bri2 BRICHOS, encompassing residues 113–231 in human Bri221 , and cleaved with thrombin to release recombinant human (rh) Bri2 BRICHOS-mCherry, which was isolated by a second IMAC step. The yield was approximately 100 mg per liter bacterial culture. The fluorescence properties of rh Bri2 BRICHOS-mCherry are very similar to those of mCherry alone  fluorescence . Green fluorescent protein (GFP)-tagged N-mutHtt with a 109 amino acid-long repeat (N-mutHtt109Q-GFP) was co-expressed with mCherry or mCherry-Bri2 BRICHOS in human osteosarcoma U2OS cells and human cervix carcinoma HeLa cells and the localization of the proteins were examined by fluorescence microscopy. While mCherry was typically excluded from the GFP-N-mutHtt inclusions or, alternatively localized at the rim of these structures, mCherry-Bri2 BRICHOS was enriched in the inclusions in the U2OS and HeLa cells , which is responsible for the neurodegenerative disorder Huntington’s disease42, IAPP and α-synuclein, which are implied in Alzheimer’s disease, type II diabetes and Parkinson’s disease, respectively, as well as the de novo-designed β17 protein, according to published protocols31. The fibrils were isolated by centrifugation and subsequently incubated with rh Bri2 BRICHOS-mCherry at a concentration that corresponds to 20% of the monomeric amyloid polypeptide concentration, at 37 °C for one hour. After washing, the rh Bri2 BRICHOS-mCherry incubated fibrils were analyzed by fluorescence microscopy. Green fluorescence (corresponding to ThT fluorescence) was observed for all tested amyloid fibrils incubated with rh Bri2 BRICHOS-mCherry. The results confirm amyloid-like properties for the different types of fibrils used. Red fluorescence was observed for rh Bri2 BRICHOS-mCherry suggesting that fusion protein bound to fibrils  and a part of the central core region R3 (V306-K317) of Tau (referred to as PHF6) implicated in Tauopathies (e.g. Alzheimer’s disease and Parkinson’s disease), which resulted in similar results . To this end, we incubated the rh Bri2 BRICHOS-mCherry decorated fibrils with a primary antibody against mCherry and a secondary antibody bound to 5 nm gold particles and studied them with negative stain EM. The immuno EM data Fig.  confirme42 fibrils on a carboxymethylated dextran surface and investigated binding by surface plasmon resonance (SPR). The SPR sensorgrams , while mCherry bound significanly weaker (KD = 7 μM). The dissociation constant for rh Br2 BRICHOS-mCherry is in the same range as previously determined for rh proSP-C BRICHOS binding to Aβ42 fibrils22.To quantify binding of rh Bri2 BRICHOS-mCherry and mCherry, we immobilized Aβ22. We show that a recombinant fusion protein between human Bri2 BRICHOS and mCherry can be efficiently produced using the solubility tag NT*FlSp25 derived from the N-terminal domain of spider silk proteins34. Rh Bri2 BRICHOS-mCherry shows the same fluorescence properties as mCherry alone, binds to the surface of Aβ42 (as well as to fibrils of the Aβ42E22G mutant), IAPP, α-synuclein, PHF6 and β17 amyloid fibrils and makes them fluorescent. The binding of rh Bri2 BRICHOS-mCherry to amyloid fibrils in vitro is accomplished simply by co-incubating the fusion protein and the fibrils in aqueous buffer at 37 °C for one hour under quiescent conditions, and specific binding of mCherry tagged Bri2 BRICHOS to amyloid-like inclusions of mutant huntingtin is observed in the cytoplasm of two different cell lines. These conditions will likely be tolerable by other Bri2 BRICHOS fusion proteins and other amyloid-like fibrils and nanomaterials. The approach presented here thus holds potential to be useful for decorating various fibrillar materials. Artificial spider silk-like fibers, with amyloid-like properties, are thought to have attractive properties for generation of novel biomaterials36 and can form fibers also when linked genetically to other proteins37. One drawback with fusing proteins to a fiber-forming protein is that the added protein needs to withstand conditions required for silk fiber formation, which can entail for example presence of organic co-solvents or non-physiological pH. Such obstacles might be circumvented by using Bri2 BRICHOS based fusion proteins to decorate premade fibers. Amyloid fibrils can self-propagate and potentially also induce other proteins to form amyloid structures38. Amyloid-like fibrils and/or oligomers that can be generated from them are potentially cytotoxic40 and the seeding and cross-seeding phenomena are thus potentially harmful if amyloid and β-sheet based materials are implanted in living organisms. It can be noted that Aβ42 or IAPP amyloid fibrils decorated with recombinant BRICHOS are markedly less prone to seed fibril formation and generate less cytotoxic oligomers compared to the corresponding naïve fibrils41. Moreover, addition of recombinant BRICHOS to mouse hippocampal slice preparations in vitro has been shown not to elicit detectable toxic effects and transgenic overexpression of BRICHOS in Drosophila fruit flies has no side effects on longevity or locomotor behavior43. It is thus conceivable that fibrils that have been decorated with Bri2 BRICHOS based fusion proteins have attenuated capacity to be cytotoxic and therefore can be well tolerated by cells and organisms.Amyloid-like fibrils are versatile materials that are used in nature for various purposes, and designed nanomaterials built up from regular β-sheets have many potential applications in materials science, biology and medicine. Here we show a potentially “generic” way to decorate amyloid-like fibrils with functional proteins. Our approach is based on the observations from immuno-EM that recombinant BRICHOS binds to the surface of Aβ and IAPP amyloid fibrilsEcoRI and HindIII restriction enzymes and the Bri2 BRICHOS-mCherry gene was isolated on a 2% agarose gel, extracted with QIAquick Gel Extraction Kit  and ligated into pT7-H6NT*FlSp plasmid that has previously been digested with the same restriction enzymes. To obtain mCherry, HiFi HotStart DNA polymerase  was used for PCR amplification with the Bri2 BRICHOS mCherry gene as template. The gene was amplified with forward primer 5′-CCG GAA TTC CCT GGT GCC ACG CGG TTC TGT GAG CAA G-3′ and reverse primer 5′-GGG AAG CTT ACT TGT ACA GCT CGT CCA TGC CGC CGG T-3′ at 65 °C as annealing temperature. The PCR product was cleaved and ligated into pT7H6NT*FlSp as described above. pET21a-alpha-synuclein (αSN) was a gift from Michael J Fox Foundation MJFF (Addgene plasmid #51486) and was used as template to obtain pT7H6NT*FlSp(TRS)αSN as described above with the forward primer 5′-GCG GGA ATT CAG AAA ACC TGT ATT TCC AAA TGG ATG TGT TTA TGA AAG G-3′ and reverse primer 5′-CCT GCC AAG CTT ATT ACG CTT CCG GTT CAT AGT CTT G-3′. The plasmid pGEX 2TK encoding for GST-IAPP (kind gift from professor Gunilla Westermark) was used as template to obtain pT7H6NT*FlSpmetIAPP as described above with the forward primer 5′-GCG GAA TTC AAT GAA ATGCAA CAC TGC CAC ATG T-3′ and reverse primer 5′-CCT GCC AAG CTT ACT AAT ATG TAT TGG ATC CCA CG-3′. All the plasmids were transformed into chemically competent E. coli Nova Blue cells by heat shock transformation followed by plasmid preparations and sequence verifications. For mammalian expression EGFP of the EGFP-N1 and EGFP-C1 vectors from Clontech were replaced by mCherry. mCherry-C1 was digested with XhoI, isolated on a 1% agarose gel and extracted with QIAquick Gel Extraction Kit . Bri2-Brichos was PCR amplified using 5′-AGT CCG GAC TCA GAT CTC GAG CTC AGA CAA TTG AAG AAA ATA TTA AAA TC-3′ as forward and 5′-GCA GAA TTC GAA GCT TGA GCT TAC AGT TTG TAA GTT TCC TTG-3′ as reverse primer, the Phusion DNA polymerase and 58 °C as annealing temperature. The PCR product was purified using the QIAquick PCR purification kit . The PCR product and the digested backbone were incubated together with the NEBuilder HiFi DNA Assembly Master Mix  and transformed into DH5α E. coli bacteria using heat chock transformation  followed by plasmid preparations and sequence verifications. An expression plasmid for GFP-tagged N-mutHtt was generated with a PCR fragment generated from pBacMam2-DiEx-LIC-C-FLAG-huntingtin-full-length-Q109 (Addgene #111730) with the Phusion polymerase using forward primer 5′-GTA TTT CCA AAT GGC GAC CCT GGA AAA GCT G-3′ and reverse primer 5′-GTG GAT CCC GTG GTC GGT GCA GCG GCT C-3′, and 71 °C as annealing temperature. The PCR product was used to generate a GFP fusion using NEBuilder HiFi DNA assembly kit . For generating Aβ42E22G, we used the forward primer 5′-GTT AGA ACC CAC GTC TCC AGC GAA GAA CAC CAG-3′, reverse primer 5′-GTT AGA ACC CAC GTC TCC AGC GAA GAA CAC CAG-3′ and pT7H6NT*FlSp Aβ42 as template with the QuikChange II XL site-directed mutagenesis kit  according to the manufacturers protocol. The pT7H6NT*FlSpAβ42E22G plasmid was transformed into chemically competent E. coli nova blue cells by heat shock transformation followed by plasmid preparations and sequence verifications.The synthetic gene coding for Bri2 BRICHOS with mCherry attached to the C-terminal and a hexa-glycine linker between was ordered from GenScript . The pU57 plasmid was digested with 21. Aβ42 and β17 were expressed and purified as described previously30. Expression of NT*FlSp -α-synuclein and NT*FlSpMetIAPP was performed as described previously34. Purification of αSN was performed essentially as described earlier for Aβ25. In brief, cell pellets from 1L LB culture were suspended in 8 M urea, 20 mM Tris-HCl pH 8, and sonicated until a clear solution was obtained. The solubilized lysate was passed over a 0.22 μm filter to remove insoluble debris and loaded on a HisPrep FF 16/10 column , washed with 15 mM Imidazole, 8 M urea, 20 mM Tris-HCl pH 8 and eluted with 200 mM Imidazole, 8 M urea, 20 mM Tris-HCl pH 8. The eluted NT*FlSp(TRS) αSN was dialyzed against 20 mM Tris and cleaved with TEV protease  in 1 mM DTT and 0.2 mM EDTA, 20 mM Tris-HCl pH 8 overnight at 4 °C. The cleaved protein was loaded on a Ni-NTA gravity column to remove NT* and the flow-through was applied on a 3 ml Resource RPC column  equilibrated with 0.1% NH3 and 2% Acetonitrile. The protein eluted as a single peak using a linear gradient from 2 to 80% Acetonitrile. The peak fractions were pooled, aliquoted to 2 mg per tube, lyophilized and kept at − 20 °C until used. Purification NT*FlSpMetIAPP was performed as NT*FlSp (TRS)αSN. NT*FlSpMetIAPP were cleaved with 100 mM CNBr at pH 1 by addition om 2 M HCL until final HCl concentration was 0.1 M and incubated at RT overnight. The pelleted IAPP was dissolved in 6 M Guanidine-HCl and applied to 3 ml Resource RPC column  equilibrated with 0.1% Trifluoroacetic acid (TFA) and 2% Acetonitrile. The IAPP peptide eluted as a single peak using a linear gradient from 2 to 80% Acetonitrile. The peak fractions were pooled, lyophilized re dissolved in hexafluoroisopropanol (HFIP) at 100 μM and kept at − 20 °C until used.Expression and purification of rh Bri2 BRICHOS-mCherry were done as previously described for rh Bri2 BRICHOS42E22G was expressed and purified as previously described for Aβ42 (Ref.25). Synthetic PHF6 (Tau R3 306VQIVYKPVDLSK317) was purchased as lyophilized peptide from GenScript  with N- and C-termini capped by acetylation and amidation, respectively.AβU2OS and HeLa cells were transiently co-transfected with N-mutHtt109Q-GFP and mCherry-Bri2 BRICHOS or mCherry with Lipofectamine 3000 (Life Technologies), following the manufacture’s instruction. Cells were fixed after 48 h in 4% PFA, stained with Hoechst and mounted with Mowiol.42, Aβ42E22G, α-synuclein and IAPP was incubated at 37 °C overnight in 20 mM sodium phosphate buffer pH 8 (for Aβ42) or 7.4 (for αSN and IAPP) with 10 μM Thioflavin T under quiescent condition in Eppendorf tubes. 100 μM β17 peptide was aggregated by addition of 2.5 mM CaCl2 in a volume of 200 μl in Tris-HCl pH 8 with 10 μM Thioflavin T and incubated at 37 °C overnight under quiescent condition. The lyophilized PHF6 peptide was dissolved in 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) to a concentration of 1 mM. 10 μl of HFIP dissolved PHF6 was lyophilized and dissolved in 200 μl, 20 mM ammonium acetate buffer at pH 7 with 200 μM heparin and 10 μM ThT to a final PHF6 concentration of 50 μM and incubated at 37 °C under quiescent condition in Eppendorf tubes overnight. The aggregated peptides were split into two tubes each and centrifuged for 5 min at 21,100× g. The supernatant was removed by carefully pipetting over a UV-table to avoid sucking up the pellet. The pellet was resuspended in 100 μl of either 14 μM rh Bri2 BRICHOS-mCherry or 14 μM mCherry (20 μM of each for β17 and 10 μM of each for PHF6), which correspond to 5:1 peptide:BRICHOS ratio and incubated for at least 1 h in 37 °C followed by centrifugation for 5 min at 21,100× g and supernatant was removed over UV-table. The pellet was washed twice in 20 mM sodium phosphate buffer, pH 8 (for Aβ42 and β17) or 7.4 (for α-synuclein and IAPP) by repeating the procedure above. The final pellet was resuspended in 10 μl sodium phosphate buffer (same as above).A volume of 200 μl of 70 μM of AβFluorescence emission and excitation scan were measured on a Tecan infinite M100 using Costar 96-wells black flat bottom plate. Both rh Bri2 BRICHOS-mCherry and mCherry were diluted to 5 μM in PBS. In the emission scan, the proteins were excited at 545 nm with 5 nm bandwidth and emission was recorded from 555 to 750 nm with 5 nm bandwidth in steps of 1 nm. In the excitation scan, the protein emission was fixed at 655 nm with 5 nm bandwidth and excitation was scanned over 400–645 nm with 5 nm bandwidth in steps of 1 nm.42 aggregation under quiescent conditions were recorded by ThT fluorescence as described previously23 using a POLARstar Omega plate reader . Measurements were carried out of 20 µl per well, containing Aβ42 monomer only, or Aβ42 monomer plus isolated monomers of rh Bri2 BRICHOS-mCherry or rh Bri2 BRICHOS, 10 μM ThT in 0.02% NaN3, 0.2 mM EDTA and 20 mM sodium phosphate, pH 8.0. Traces shown are averages of 4 replicates with standard deviations as error bars.Aβ44. The values of each line scan was normalized to its maximum value. For quantifying the percentage of cells that displayed co-localization of mCherry-Bri2 BRICHOS or mCherry at N-mutHttQ109-GFP inclusions, cells were scored manually from three independent experiments (n = 35–50). Four μl of resuspended pelleted amyloid fibrils of Aβ42, α-synuclein, IAPP, Aβ42E22G, PHF6, or β17 were transferred to a microscope slide  containing Vectashield mounting medium for fluorescence and cover slip was carefully mounted. Fluorescence images were collected with an EVOS FL Auto 2 imaging system  using GFP-channel  to visualize Thioflavin T fluorescence and TX Red-Channel  to visualize mCherry fluorescence. Fluorescence intensities were measured with ImageJ software44.Transfected cells were imaged using a Zeiss LSM 880 microscope equipped with a plan-Apo 63X/1.40 Oil objective. Line scan analysis was performed using ImageJThe amyloid fibril was incubated with mCherry or rh Bri2 BRICHOS-mCherry for 60 min at 37 °C in the same conditions as above. It was then centrifuged at 12,000 rpm for 10 min to pellet down the fibrils. The supernatant was discarded, and the pellet was washed 3 times with sodium phosphate buffer, and it was diluted to 10 µM for EM sample preparation. 5 µl diluted solution was applied on 200 mesh formvar coated nickel grid and excess solution was removed using blotting paper after 10 min of incubation. It was then washed twice with 10 µl MQ water. The nickel grid surface was blocked with 1% bovine serum albumin (prepared in PBS) for 30 min. It was then washed thrice with MQ water. 5 µl primary anti-RFP antibody  was applied to the grid and it was incubated for 60 min at room temperature. Then, the grids were washed thrice with MQ water. Further, the nickel grids were incubated with anti-mouse IgG-gold  (1:40 dilution in PBST) secondary antibody for 60 min. Extra solutions were removed and then it was washed thrice with MQ water and then stained with 1% uranyl formate for 5 min. Extra stained was blotted with blotting paper and it was air-dried. Transmission electron microscopy  was performed for analysis of fibril morphology using 2 k × 2 k Veleta CCD camera  0.15–20 images were recorded for each sample randomly. Images were obtained at a magnification of × 43,000 and × 87,000. For the manuscript × 87,000 magnified images were used.42 fibrils was measured with a BIACORE 3000 instrument. Sonicated Aβ42 fibrils were immobilized on a CM5 sensor chips (Cytiva) using standard amine-coupling chemistry. A fresh mixture of 0.05 M NHS and 0.2 M EDC was added to the sensor-chip surface for activation, followed by incubation with 5.6 μM Aβ42 diluted in 10 mM sodium acetate pH 4.5 and finally deactivation by ethanolamine. Blank channels for negative controls were prepared by omitting protein in the coupling step. Each experiment involved five different protein concentrations of either 313 nM–40 μM mCherry or 39 nM–5 μM Bri2 BRICHOS-mCherry and subsequent buffer flow to monitor dissociation. The experiments were performed with HBS-E  as running buffer and a flow rate of 25 μL/min. The chip surface was regenerated between each sample by injection of 30 mM NaOH for 30 s. Analysis of the SPR data was done as previously described33.Binding of Bri2 BRICHOS-mCherry and mCherry to AβSupplementary Figures.


Assess the sensitivity and specificity of computed tomography angiography (CTA) for carotid near-occlusion diagnosis interpreted in clinical practice against expert assessment.CTAs were graded by two expert interpreters for near-occlusion. Findings were compared with clinical reports in 383 consecutive cases with symptomatic ≥ 50% carotid stenosis. In addition, 14 selected CTA exams (8 near-occlusions and 6 controls) were analyzed in a national effort by 13 radiologists experienced with carotid CTA.In clinical practice, imaging reports were 20% (95% CI 12–28%) sensitive for near-occlusion, ranging 0–58% between different radiologists; specificity was 99%. Among the 13 radiologists reviewing the same 8 near-occlusions, the average sensitivity was 8%, ranging 0–75%; specificity was 100%.Carotid near-occlusion is systematically under-reported in clinical routine practice, caused by limited application of grading criteria when assessing CTA.Carotid near-occlusion is severe stenosis with distal artery collapse; this collapse is often subtle.• A fifth of near-occlusions were detected in routine practice. Many readers mistake near-occlusion for stenosis without distal artery collapse, either by not actively searching for subtle collapses or by not interpreting the collapse correctly when noticed.• On the other hand, the novice diagnostician should be cautioned to not over-diagnose near-occlusion; other causes of extracranial ICA asymmetry also exist such as distal disease and Circle of Willis anatomical variants.•  Carotid near-occlusion is a variant of severe carotid stenosis. In contrast to conventional ≥ 50% carotid stenosis, near-occlusion has a reduced artery size (“collapse”) distal to the stenosis –3. The cNear-occlusion can be shown by computed tomography angiography (CTA) , 2, 7, 8The purpose of this study was to assess the sensitivity and specificity of CTA for carotid near-occlusion diagnosis, interpreted in routine clinical practice compared with expert assessments.We evaluated a consecutive sample of CTAs from all patients aged ≥ 18 years performed at or transferred to the Radiology Department at the University Hospital of Northern Sweden, Umeå, Sweden, between 2010 and 2014 Fig. . CTAs weNear-occlusion was diagnosed when severe carotid stenosis was shown with a reduced distal internal carotid, compared with expected. Near-occlusions with full collapse showed a threadlike ICA distally, clearly smaller than the ECA . Near-occlusions without full collapse had a normal-appearing albeit relatively small distal ICA. In the main analysis, a case of near-occlusion was determined in accordance with the approach of previous studies , 8, 14, An important mimic that was accounted for was anatomical variance for distal ICA size associated with variations of the Circle of Willis , 14. TheOcclusion was diagnosed when no contrast was seen beyond the stenotic lesion, commonly with a rounded stump of contrast filling. Cases with collapsed contrast visible distal to the stenosis but had not yet reached skull base at time of image capture were diagnosed as near-occlusion with full collapse. This was on the basis that, to be filled at the moment of the early bolus slicing on CTA, contrast requires a distal outlet that may not be visible without delayed imaging. Among cases with conventional stenosis, degree of stenosis was measured using established NASCET criteria .As an additional explorative method, near-occlusion can be suggested when it fulfills 4 criteria proposed by Bartlett et al , i.e., sMeasurements were made by the observer (E.J.) utilizing NASCET methodology. Distal ICA was assessed and measured well beyond the bulb where the walls are parallel and ECA was assessed and measured just proximal to its terminal bifurcation behind the jaw, both usually in the same axial slice at C2 vertebral level  and 29 general radiologists. The symptomatic side was analyzed. In order to categorize the report findings, the reports were assessed by two stroke clinicians with near-occlusion expertise , blinded to the grade of stenosis but aware of symptomatic side. Any synonym or approximating descriptors of near-occlusion was considered but general expressions like “very severe stenosis” were not as they refer to the stenosis, not the distal ICA. Reports were categorized as near-occlusion when a near-occlusion term was used to clearly or possibly describe a severe stenosis associated with a small distal artery and near-occlusion was the only diagnosis. Reports with near-occlusion terms was clearly used to describing the severe stenosis, not weighing in a small distal artery, was not considered near-occlusion, even when small distal artery was also mentioned in passing. In addition, clinical records were assessed to understand what degree of stenosis the clinician used for the treatment decision.As an additional comparison, 14 selected cases were sent to other institutions: The radiology departments of all 52 Swedish hospitals managing acute stroke were contacted by telephone. From this, 13 radiologists from 11 hospitals agreed to participate in this study. Academic centers were overrepresented (43% participation) compared with non-academic centers (18% participation). All participating radiologists graded carotid stenoses with CTA as their routine; 9 estimated that they had evaluated > 100 CTA carotid exams in their lifetime. All five participants from academic centers were neuroradiologists; no neuroradiologist worked outside. None of the participants had any previous professional contact with this study group. Care was taken to not divulge the purpose of the study to the participants other than being told the study was assessing how carotid stenosis is graded in routine practice. Study results and purpose were presented to the participants after study completion with options to withdraw participation; however, none opted to do so and none claimed misinterpretation of their findings after feed-back.We selected 14 cases, sent to participating radiologists for interpretation. The selected cases were 4 conventional ≥ 50% stenosis, 5 near-occlusion without full collapse, 3 near-occlusions with full collapse, and 2 occlusions. All chosen exams had perfect inter- and intra-rater agreement and were judged as typical examples by both E.J. and A.F.. In six of the near-occlusions, the four features of near-occlusion were easily assessable and two were more difficult: One near-occlusion without full collapse had a very calcified stenosis and one near-occlusion with full collapse had a very small residual distal lumen, but visible from bulb to terminus. All eight near-occlusion cases fulfilled all four measured Bartlett criteria except the case with very calcified stenosis where stenosis diameter could not be determined.We sent axial source images and 3 mm MIP reformats in axial, coronal, and sagittal planes. Participants were instructed to treat these as routine cases  and provide an imaging report. The imaging reports were categorized as the original reports in clinical routine practice by two observers (E.J. and T.G.) blinded to case details and to each other. Disagreements between observers were resolved by consensus discussion.χ2 test, and one-way ANOVA with REGW-Q post hoc test. For reliability testing, kappa values were used. P < 0.05 was pre-specified as border for statistical significance. SPSS 24.0 was used in all calculations.Stenosis grading from the CTA expert review was the reference standard. CTA imaging reports  were compared with the reference standard using mean, standard deviation (SD), 95% confidence intervals (95% CI), 2-sided In the routine practice grading analysis, 384 CTA exams from 379 consecutive patients were included, but 1 image report was missing, resulting in 383 exams with data available (baseline data presented in Table Sensitivity of near-occlusion (including those with and without full collapsed) in the original report was 20% (95% CI 12–28%). Specificity of near-occlusion was 99% Fig. . Detecten = 19) and varied with diagnosis in the CTA report, but also by ultrasound findings .Of the 104 near-occlusion cases, 21 (20%) were detected, 33 (32%) included no near-occlusion term or mention of distal ICA, and 15 (14%) were mistaken for occlusion; remaining 35 (34%) had some mention or indication of near-occlusion in the report .In the national comparison, the 13 participants produced 182 imaging reports; 104 were near-occlusion cases. Near-occlusion sensitivity was 8% (95% CI 2–13%) and specificity was 100% Fig. . SensitiCombining the local and national CTA comparison, 16% (8/51) of radiologists that assessed at least one near-occlusion case detected any near-occlusion, higher for neuroradiologists (36%) compared with general radiologists (8%).Inter-rater reliability between the two expert reviewers for near-occlusion diagnosis on CTA was good: kappa 0.80; intra-rater reliability on 49 randomly selected cases was kappa 0.75 and 0.88 for each observer. The inter-rater reliability between the two readers of CTA reports was good .n = 11), contralateral occlusion (n = 12), or both (n = 2). All ten false negatives missed a single feature, all well explained by interpretive approach such as bilateral near-occlusion and stenosis proximal to bulb causing ECA collapse. Requiring ≥ 3 Bartlett criteria (including 3 of 3 in cases with a single missing feature) for near-occlusion diagnosis resulted in 100% sensitivity (102/102) and 87% specificity (244/279) compared with expert diagnosis.Exploring diagnosing near-occlusion was based on fulfilling 4 of 4 Bartlett criteria, 93% sensitive (87/94) and 97% specific (257/264) compared with expert diagnosis. Of the 94 cases with 4 of 4 Bartlett criteria, local imaging reports diagnosed conventional stenosis or thrombosis in 64 (68%), near-occlusion in 18 (19%), and occlusion in 12 (13%). However, 25 exams  were excluded from this assessment due to calcified stenosis  when the more easily reproducible 4 of 4 Bartlett criteria approach was used. As each case was assessed by different observers in the original report analysis, it was impossible to separate case and observer dependency. The additional national sample analysis revealed substantial difference in sensitivity (0–75%) despite reviewing the same cases, indicating that variation in sensitivity was caused by difference in observer proficiency. However, near-occlusions with full collapse and smaller distal diameter were more often detected in the original analysis, so the chance of a near-occlusion to be correctly diagnosed was also dependent on case details. Detection of at least one near-occlusion was more common for neuroradiologists than general radiologists, but some neuroradiologists missed all near-occlusions, even those with full collapse. Also, the national sample revealed that the low sensitivity of near-occlusion was not limited to a single center.Some reports included both a near-occlusion diagnosis and a percentage grade as if they were both correct at the same time. Near-occlusions should not be graded as percent stenosis as deriving percent stenosis is fallacious; these are mutually exclusive –3, 22. WUnfortunately, delayed imaging was not used by all, and conventional angiography was rarely used during the study period . If used, delayed images might prevent some near-occlusions to be mistaken for occlusions in the original reports. There were possibly cases of near-occlusion only separable from occlusion with delayed imaging, mistaken for occlusion by both the experts and clinical observers. However, interpretation education needs priority as many mistook no visible contrast in severe stenosis lumen and clearly patent arteries beyond the stenosis as occlusion with retrograde filling. With CTA aiming at arterial phase, it is near impossible for contrast to reach from skull base down to bulb region in this short time, rather a technical limitation as partial volume effect is more reasonable to presume. However, the vast majority (81%) of missed near-occlusions were called conventional stenosis—not occlusion.As the near-occlusion experts conducting the study did not participate in the clinical or diagnostic work during the time of the study, observation bias was reduced. Although we have sampled a consecutive cohort from a single hospital and added scattered observers from a single country, these findings may or may not be representative in other locales, depending on attention to detail of near-occlusions. However, we suspect these results are not specific to Swedish general radiologists or neuroradiologists. As the CTA findings varied with interpreter expertise, CTA findings are likely to vary greatly between centers.In conclusion, near-occlusion seems to be systematically overlooked in clinical routine practice, presumably due to lack of attention to detail and limited application of documented grading criteria when assessing CTA.


The ubiquitous presence of wrinkles in two-dimensional materials alters their properties significantly. It is observed that during the growth process of graphene, water molecules, sourced from ambient humidity or transferred method used, can get diffused in between graphene and the substrate. The water diffusion causes/assists wrinkle formation in graphene, which influences its properties. The diffused water eventually dries, altering the geometrical parameters and properties of wrinkled graphene nanoribbons. Our study reveals that the initially distributed wrinkles tend to coalesce to form a localized wrinkle whose configuration depends on the initial wrinkle geometry and the quantity of the diffused water. The movement of the localized wrinkle is categorized into three modes—bending, buckling, and sliding. The sliding mode is characterized in terms of velocity as a function of diffused water quantity. Direct bandgap increases linearly with the initial angle except the highest angle considered (21°), which can be attributed to the electron tunneling effect observed in the orbital analysis. The system becomes stable with an increase in the initial angle of wrinkle as observed from the potential energy plots extracted from MD trajectories and confirmed with the DOS plot. The maximum stress generated is less than the plastic limit of the graphene. Depending upon applications, wrinkling can be “either boon or a bane”. The ubiety of the surface textures significantly alters the mechanical, electronic, and optical properties, which can be exploited in various applications in the fields of electronic2, sensing and actuation3, energy storage5, chemical reactions8, and bioelectronics9. Given the literature, applications in flexible electronics stand out among the others. Initially, wrinkles in graphene were detected during the synthesis processes12 Usually, amid the synthesis process of graphene, water molecules sourced from the ambient air/transfer process can get diffused in the system. The diffused water can significantly influence the geometrical parameters of the wrinkles, and consequently, the properties of the graphene8 in addition to the substrate effect13. There are other ways for the water to get diffused in the system. For example, Raghav et al. had studied the wedging of a graphene bilayer by water molecules14. The first on-purpose fabrication of the wrinkles was demonstrated in 201115, followed by multiple studies on modeling the wrinkle theoretically/numerically11,17.Graphene like two dimensional (2D) materials are less resistant to buckling, and they tend to form surface corrugations such as wrinkles, ripples, crumples2O3 TFT displayed minimum current leakage of 10−13 A, attributed to the airgaps present underneath the wrinkles12. Additionally, the In–Ga–Zn–O (IGZO), pentacene, reduced graphene oxide(rGO), and rGO/ Polydimethylsiloxane(PDMS) based TFTs are observed to preserve the electrical performance and mechanical stability under the strain of 20% due to wrinkles’ existence21. Similar behavior was observed in silver, and rGO based interconnects18,22 It inspired the investigation of dependency of the device performance on individual geometrical parameters of the wrinkles. In an IGZO TFT and rGO interconnect, the wrinkles’ wavelength and amplitude were controlled in a way so the device can be bent up to 13 μm and strained up to 5% while preserving the electrical performance17,24. Nirmalraj et al.25 had demonstrated controlling the Pentacene growth on a MoS2 substrate by tuning the wrinkle height. Furthermore, the non-uniformity of the wrinkled 2D materials’ topology can be used for photon trapping in between trough and vertex. This lead to the design of a wearable laser device with a threshold below than ever reported26. TFTs are usually decorated with mechanical sensors. A crumpled rGO based piezoresistive pressure sensor developed high stretchability, strain insensitive resistance profile, and increased sensitivity27. In another study, a wrinkled rGO sheet improved the sensitivity and range at non-stretched state too28. In addition to pressure and position detection, strain measurements are equally important in flexible electronics. Wrinkle assisted crack structures can be used to design strain sensors29. While subjected to bending stresses, the resulting sensors showed high sensitivity, high stretching range, low limit of detection, high durability, wider strain range. A wrinkled 2D complex-based strain sensor is demonstrated to acquire a high gauge factor of 107130 and sustain the electrical signals over a wide range of deformation15. The intrinsic piezoresistive property and electron tunneling effect  are core components in flexible electronics. Major obstacles in developing flexible TFTs are fragility and the current leakage. Surface instabilities in thin-film TFTs are exploited to overcome these shortcomings. A Single-Walled Carbon Nanotube(SWCNT)/wrinkled Al33. The wrinkles’ patterns were observed to affect GMR and magnetic field sensitivity uniquely over a wide range of strain34. Often, there are sensitive parts of the device which should be shielded against the magnetic fields. A wrinkled polyurethane(PU)/MXene(Ti3C2Tx) complex was tested for its electromagnetic wave interference(EMI) capability in flexible electronics35. The power storage components are also needed to be able to withstand mechanical deformations. Wrinkled/crumpled supercapacitors based upon graphene/rGO are demonstrated to have high capacitance at strains up to 300% and 5,000 stretching cycles37. Further, wrinkled 2D material based bioelectrodes seem to show no degradation in charge capacity and impedance while used for in-vivo measurements38. Wrinkles in Polyimide/Silbione system has resulted in low elastic modulus and high tangent modulus to avoid fracture and mechanical constraint on the movement of a biological tissue over a substrate, respectively39. Crumples in a 2D material can undergo a geometrical transformation for various reasons. A majority of systems acquired the same type of wrinkle as one can observe while growing graphene on a SiO2 substrate. A related work by Lee et al.40 revealed that during the transfer of exfoliated graphene onto a SiO2 substrate, water molecules get diffuse between graphene and substrate  even at strains as high as 4.5%ate Fig. . The dif37, and change in electronic properties of different 2D materials caused by wrinkles40. Our study is composed of three phases. Phase I addresses the wrinkle evolution due to diffused water in between graphene with distributed wrinkles and a graphene substrate. We had varied the amount of diffused water and the initial angle of the distributed wrinkles in graphene and studied the formation of final localized wrinkle configuration and the stress generated in wrinkled graphene. Phase II deals with the evaporation of diffused water, and the effect of evaporation on the collapsing of the wrinkle, i.e. completion of wrinkle life-cycle. We observed that, upon complete evaporation, the resulting localized wrinkle undergoes motion that is composed of three fundamental modes—bending, buckling, and sliding as studied theoretically by Im et al.41 Lastly, Phase III uses Density Functional Theory (DFT) while considering the free-standing WGNR with four different initial angles and studies its influence on the electronic properties. Only quasi-periodic wrinkles are considered instead of a complex network of wrinkles that exist in the experimental setups. However, we believe that our studies will encourage researchers to study more complicated wrinkle structures.In this work, we provided a detailed explanation of water diffusion and its impact on a wrinkled bilayer graphene system characterized by geometrical parameters and electronic properties. Our results can explain the phenomenon of the water interaction with the graphene systemsthree phases based on which part of the wrinkle life-cycle is being studied, i.e., formation, evolution, collapse, and the approach used for analysis, i.e., MD or DFT. In the first phase, we investigated the wrinkle formation and evolution. In this phase, as shown in Fig. This study is divided into entclass1pt{minimaly Table .Figure 242. We performed an energy minimization of this system  potential was used for the C–C interactions2O)44, and Lennard–Jones type of pair potential for C and H2O interactions45. We used TIP4P potential before for water modeling37, and our computational results were in good agreement with the experimental findings3.The water molecules were added to the system Fig. b in two Phase I is summarized in the ‘Wrinkle Evolution’ part of the flow-chart shown in Fig. 46. We then performed MD simulation using NVE ensemble with the timestep of 1 femtosecond. Sufficient MD steps (at least one million steps) were performed to make sure the system reached its lowest energy configurations (discussed later in detail). After the end of the first phase simulations, we stored the system configuration and reused it as the initial structure for the next phase of the project, i.e., water drying case. We performed the stress analysis of the final stabilized graphene sheet with a localized wrinkle using the in-built stress computation algorithm of LAMMPS47. The detail of the stress calculation methods is provided in the supplementary information (section D).The methodology of Phase II, we studied how wrinkle collapses due to the drying of water. The ‘Evaporation’ part of Fig. In Phase III, the DFT based analyses were performed on one frame from each case considered from Phase I, as shown in Figs. 48. All calculations were done using the projector augmented wave (PAW) pseudopotentials and the Perdew–Burke–Ernzerhof (PBE) exchange–correlation functional49. The plane-wave basis sets were used with a plane-wave cutoff energy of 100 eV, and a kinetic energy cutoff for augmentation charges of 400 eV. The convergence threshold of Kohn–Sham equations was set to 1e−06. The gamma-centered k-point sampling grids obtained using the Monkhorst–Pack method, were 8 × 8 × 8 with a unit offset for the graphene unit cell. The valence electrons contain s and p orbitals for carbon. Before DFT calculations, all atoms in the cell, as well as the lattice dimensions and angles, were relaxed to the equilibrium configurations by using MD. For consistency, only one case corresponding to two water (H2O) layers was studied from each considered IAW, i.e., 6°, 11°, 16°, 21°  The formation and evolution of wrinkles for no-water, and water inside GDW and supported FG (without drying), (II) The collapse of the localized wrinkle during water drying, and (III) The electronic structure of free-standing WGNR for four different geometries.51 and/or transfer process52, graphene can inherent distributed wrinkles, and they tend to coalescence to form a localized structures, i.e., from ‘Graphene with Distributed Wrinkles (GDW)’ to ‘Graphene with Localized Wrinkle (GLW)’. For the simplicity of analysis, we considered here GDW only among other available morphologies  layered structures, such as graphene, same-sign (same-side) winkles attract each other to form a localized wrinkle. A similar observation was reported for the MoS2 bilayer system54. As shown in Fig. As discussed in “55 with a contact angle of (≈)125°, which matches with the literature56. In Fig. 57. However, since the droplet formation phenomenon is dominant here, the final wrinkle configuration is primarily determined by the water droplet.Figure ed video . The prez-component of the Cauchy stress. Considering the stress-field in Fig. z-component stress. Only the sidewalls, i.e., the base of the localized wrinkle, are stressed. Figure z-component of stress across the GLW . Here, stresses are averaged upon the whole line of atoms along the periodic y-axis. We note that for all three stress cases  to 18 GPa (tensile)  as the ratio of the height of the wrinkle to its base length, and (2) ‘Final Angle of Wrinkle, AR = 0, it means the sheet is flat (height = 0). The higher the value of AR, the lesser the radius of the curvature of wrinkle-vertex. Moreover, there is a directly proportional relationship between AR and AR and initial angle of water) for the differing number of the water layers. When there is no diffused water  in Fig. AR and 61. The final shape is the result of the interaction between two phenomena. For 2-, 4-, or 6-layer diffused water cases . (2) Bending Mode . However, the wrinkle bends sideways. (3) Sliding Mode  ode Fig. a: The twode Fig. b: The baode Fig. c: In thi63. We attempted to get insights into the domination of sliding mode during drying. As mentioned before, for a lower initial angle . However, once the water molecules are obliterated, then not only velocity jumps to a higher value of magnitude, but it also shows aA critical study is needed to analyze which mode  is more dominant at different stages of evaporation, given various initial angles, to tune the wrinkle according to the needs as targeted by other methodsx-axis. This ‘wrinkle-velocity’ pattern indicates that the water molecules diffused under the wrinkle behaves as ‘wrinkle-anchor’ (speed breaker). After the water molecules are completely evaporated, the sideways movement of the wrinkle dominates because the ‘wrinkle-anchor’ is removed. We hypothesize that the direction of the wrinkle movement after the complete evaporation is decided by the complex interplay of three different modes at the moment when the “last-set” of water-molecules is evaporated. However, further critical analysis is needed to validate this proposition.positive gradient with MD time steps, and the wrinkle prefers to move towards the positive 1 if we can control the evolution of the whole wrinkle-network by controlling the water content trapped under just one wrinkle, which may be or may not be the member of the original wrinkle-network.To understand further the ‘wrinkle-anchor’ phenomenon by the diffused water molecules inside the wrinkle, we considered another set-up, as shown in Fig. We performed the Molecular Orbital (MO) analysis of WGNR for all four angles  at the portion of the WGNR, where the the radius of curvature is high, i.e., the flat portion Fig. . For IAW65. The wrinkle is slender enough to confine the lowest unoccupied molecular orbital parallel to its direction. Further investigation is necessary to understand whether it’s just the effect of curvature or the presence of HOMO along the sidewalls of the wrinkle or both of these factors contributing to some extent to the formation of “electron-tunnel”-LUMO. This observation proves that not only the presence of wrinkles but its slenderness also plays a crucial role in the generation of “tunnel” LUMO orbital. It can be utilized to control the direction of electron flow in a (semi)conducting system, as observed by Sulpizio et al. in the form of electron flow following a macro hydrodynamic flow pattern, i.e., Poiseuille flow66.The Lowest Unoccupied Molecular Orbital (LUMO) is mostly observed in the portion of the GNR having high curvature Fig. . From IA67. For IAW of 16°, the asymmetricity is observed for LUMO. As shown in Fig. Similar to these ‘electron-tunnels’, ‘electron-channels’ are obtained in the IAW values of 6°, 11°, 16°. The ‘electron-channels’ is the overlapping of the atomic orbitals of the same energy level forming an “electron-flow-channel” along the direction of wrinkle, which can act as a tunnel to the flow of every extra electron added to the system. Consequently, by controlling the course of these channels, the direction of electron-flow can also be controlled, which has multiple applications in the semiconductor industries. The studies aimed for similar outcomes are available in the literatureIn Fig. The bandgap is observed to be decreasing linearly with IAW 6°, 11°, 16°), but then it increases suddenly for 21°. This can be attributed to the observation made in Fig. °, 16°, b68. With the increase in IAW, the angle of curvature of final localized wrinkle decreases. It means that the wrinkled portion of GNR is denser (number of atoms/volume) than the flat portion of GNR and hence more stretched bonds.In Fig. Consequently, the strained lattice increases the energy levels of the localized crystal orbitals, which results in changing the Fermi energy as a function of IAW. In Fig. As can be seen from potential energy plots Figure −S4, the The distributed wrinkles in graphene coalesce to form a ‘localized wrinkle’, whose configuration significantly depends on the initial pattern of distributed wrinkles and the amount of diffused water present in between the graphene and substrate. Upon evaporation of diffused water, the localized wrinkles, except with the highest initial angle, lose their angle of curvature values in the range from 7 to 63%.When water is distributed on a graphene sheet, due to hydrophobicity, distributed water molecules condense to form a water droplet. In the presence of diffused water, the final wrinkle configuration is the result of the competition between the water droplet formation process and the localized wrinkle formation process. For the lower initial angle of wrinkle, droplet formation is dominant, resulting in wrinkle with a high angle of curvature values, i.e., non-sharp vertex. For higher initial wrinkle angle, wrinkle formation dominates, and the resulted localized wrinkle tends to possess a lower angle of curvature, i.e., sharp vertex.The drying of water, diffused under the localized wrinkle, changes its configuration, e.g., curvature/vertex angle. The wrinkle is static until the complete evaporation, and after that, it acquires a motion. In case of diffused water under two localized wrinkles placed side-by-side, even after the complete evaporation under one wrinkle, it remains static until the water diffused under the neighboring wrinkle is completely dried.The wrinkle movement is the combination of three fundamental modes—bending, buckling, and sliding. The dominant mode depends on the configuration of the wrinkle., e.g., relatively narrowed or wrinkles with relatively a lower angle of curvature tends to bend. Further investigation is necessary to understand how three different modes influence wrinkle dynamics.The stress analysis reveals that the maximum stress is at the base of the wrinkle, and always below its plasticity limit. Therefore, wrinkle formation due to water diffusion does not cause any significant bond-breaking.“Electron-tunnel/channel” is observed along the vertex of the WGNR. By controlling the direction of WGNR, we can control the direction of the electron flow. This concept can be used in designing flexible electronics and optoelectronics.The direct bandgap is observed for all the wrinkle cases considered. The change in wrinkle geometry also generates lattice strains, which further changes the energy level of the crystal orbitals. The strain in lattice changes the Fermi energies which results in Fermi level modulations.In conclusion, we have performed MD and DFT simulations to geometrically and electronically characterize the wrinkle formation, evolution, and collapse in graphene when water is diffused in between graphene and substrate, and while it is evaporating/had evaporated. Our key findings are summarized below:Our study addresses the fundamental questions regarding the wrinkle formation, evolution, and collapse due to water diffusion/evaporation, and its importance in flexible electronics.Supplementary file1Movie_Figure4a.mp4Movie_Figure4b.mp4Movie_Figure4c.mp4Movie_Figure7a.mp4Movie_Figure10a.mp4Movie_FigureS8.mp4


Recent studies provide evidence that hepatocellular  adenomas  (HCAs) frequently take up gadoxetic acid (Gd-EOB) during the hepatobiliary phase (HBP). The purpose of our study was to investigate how to differentiate between Gd-EOB-enhancing HCAs and focal nodular hyperplasias (FNHs). We therefore retrospectively included 40 HCAs classified as HBP Gd-EOB-enhancing lesions from a sample of 100 histopathologically proven HCAs in 65 patients. These enhancing HCAs were matched retrospectively with 28 FNH lesions . Two readers  reviewed the images evaluating morphologic features and subjectively scoring Gd-EOB uptake  for each lesion. Quantitative lesion-to-liver enhancement was measured in arterial, portal venous (PV), transitional and HBP. Additionally, multivariate regression analyses were performed.p < 0.001) with significantly higher uptake scores assigned to FNHs (Cut-off: 75%-100%). Typical lobulation and presence of a central scar in FNH achieved an accuracy of 0.750 or higher in at least one reader (lobulation—AUC: 0.809 (R#1); 0.736 (R#2); central scar—AUC: 0.595 (R#1); 0.784 (R#2)). The multivariate regression emphasized the discriminatory power of the Gd-EOB scoring (p = 0.001/OR:22.15 (R#1) and p < 0.001/OR:99.12 (R#2). The lesion-to-liver ratio differed significantly between FNH and HCA in the PV phase and HBP (PV: 132.9 (FNH) and 110.2 (HCA), p = 0.048 and HBP: 110.3 (FNH) and 39.2 (HCA), p < 0.001)), while the difference was not significant in arterial and transitional contrast phases (p > 0.05).Subjective scoring of intralesional Gd-EOB uptake showed the highest discriminatory accuracies (AUC: 0.848 (R#1); 0.920 (R#2)—Even in HBP-enhancing HCA, characterization of Gd-EOB uptake was found to provide the strongest discriminatory power in differentiating HCA from FNH. Furthermore, a lobulated appearance and a central scar are more frequently seen in FNH than in HCA. High signal intensity during the HBP, hence significant Gd-EOB enhancement, is not uncommon for HCA and may be a pitfall in differentiating HCA from FNH.The scoring system we introduced was found to provide high discriminatory power in differentiating HCA from FNH and may overcome imaging pitfalls.Acceptable accuracies were also achieved with a lobulated appearance and presence of a central scar as both features are more common in FNH than in HCA.Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are among the most benign solid neoplasms of the liver and have the highest incidence in young adolescent to middle aged women . EspeciaMagnetic resonance imaging (MRI) is the imaging modality of choice for characterizing focal liver lesions (FLLs). Evidence is high for differentiation of benign liver lesions  using liver-specific contrast agents such as gadoxetic acid  or gadobenate dimeglumine  , 14–19.Published studies report significant hepatobiliary phase (HBP) Gd-EOB uptake for the majority of FNHs, resulting in iso- to hyperintensity to the surrounding liver, while most HCAs appear hypointense , 20, 21.The purpose of this study was to determine how significantly Gd-EOB-enhancing HCA and FNH can be differentiated .Our institutional review board  approved this retrospective study  and waived informed consent due to its retrospective nature. The study protocol conforms to the ethical guidelines of the 2002 Declaration of Helsinki. Imaging datasets were evaluated by two independent radiologists specialized in abdominal imaging (> 10 years of liver MRI experience). Primary endpoint was the discrimination between benign focal liver lesion, either FNH or HCA.In a first step, all patients with histopathologically confirmed HCA lesions (n = 100) who underwent Gd-EOB-enhanced liver MRI between January 2008 and March 2019 were retrospectively identified from our institutional databases. A “blinded” pathologist evaluated all macroscopic and microscopic features of the lesions, followed by reprocessing for immunohistochemical analysis. HCAs were classified into the four major molecular subgroups based on their genetic and phenotypic characteristics according to the Bordeaux classification from 2006 (HNF-1a-mutated adenoma (HHCA), inflammatory adenoma (IHCA—formerly telangiectatic FNH), β-catenin-activated adenoma (bHCA), and unclassified adenoma (UHCA)) , 27–29. In a second step, two readers rated intralesional Gd-EOB uptake in steps of 25%. Both readers in consensus identified 40 HCA lesions with an intralesional Gd-EOB uptake greater than 25%, which were defined as significantly enhancing lesions [Some of the HCA patients participated in previous studies , 19, 25.MRI was performed at either 1.5 T or 3.0 T MR scanner. Phased-array body coils were used in every patient. Standard imaging protocols included the following sequences: pre-contrast T2-weighted (T2-w) sequences with and without fat saturation (FS); T1-weighted (T1-w) sequences with and without FS (including in-/opposed-phase technique). After intravenous injection of Gd-EOB  and a 40 mL saline flush, multiphase T1-w 3D sequences with FS were acquired during breath-holds . The hepatobiliary phase was acquired with a 3D T1-w FS sequences 20 min after contrast administration [All images were read by two experienced radiologists specialized in abdominal / liver imaging (one with over 7 years and the other with over 10 years of MRI experience). Both readers were blinded to clinical data. The following qualitative parameters were recorded:Gd-EOB-specific characteristics:Intralesional HBP Gd-EOB uptake was rated subjectively as intralesional percentage of iso- to hyperintense areas on a five-point scale   Fig. .Fig. 2A/Arterial hyperenhancement (AHE): yes vs. no.Portal venous (PV) wash-out: yes vs. no.Contrast enhancement (CE) behavior:Largest axial diameter (mm).Lobulated: yes vs. no.Pseudocapsule (defined as a thin (mm) T2 hyperintense line around the lesions representing a compression of the surrounding liver tissue, vessels and a consecutive inflammatory reaction): yes vs. no.Central scar : yes vs. no.Intralesional fat deposition : yes vs. no.Atoll sign (defined as a hyperintense rim in T2/T2fs sequences probably representing dilated sinusoid spaces with the periphery of adenomas): yes vs. no.Qualitative MRI features:Some of these features are displayed in Fig. After analyzing the qualitative features of the lesions. Each of the two readers had to conclude a diagnosis of either FNH or HCA.For each liver lesion, a 2D region of interest (ROI) including the entire lesion at its maximum cross-sectional diameter was placed manually. First relative signal intensity (SI) for each lesion and the liver was measured:Therefore, ROIs were placed in the arterial phase sequence and cloned to the subsequent pre-contrast and post-contrast phase sequences . An additional circular ROI with a fixed 10-mm diameter was placed in healthy liver parenchyma sparing vessels and bile ducts. Furthermore, relative (Rel.) lesion-to-liver enhancement was calculated for the different contrast phases as follows:*As mentioned above some of the patients participated in previous studies with similar methods  .Statistical analysis was performed with XLSTAT . Proportional distributions were calculated using contingency tables. Descriptive parameters are given as mean and standard deviation. Based on histograms and quantile plots, normal distribution was not assumed for metric parameters, and therefore nonparametric tests were performed. Differences in contrast enhancement between FNHs and HCAs were analyzed using the Mann–Whitney U-test. Cross-tables and the Pearson chi-square test were used to investigate the association of categorical variables. To test inter-reader correlation, a pairwise two-sided Spearman rank correlation test was performed. Diagnostic accuracy in terms of sensitivity and specificity was tested throughout by performing ROC curve analysis. After identifying the parameters with significant univariate accuracy, a multivariate regression was performed for the three most accurate parameters. All tests were two-sided with a level of significance of 0.05 .*As mentDuring enrollment, we identified 36 patients with a total of 68 pathologically proven FNH or HCA lesions with at least 25% intralesional Gd-EOB uptake. There were 41% (28/68) FNHs and 59% (40/68) HCAs. By far the most common subtype according to the Bordeaux classification was inflammatory HCA, accounting for, 92.5% (37/40), while there were only 2.5% HHCAs, bHCAs, and UHCAs each (1/40) –29. Out 0.835 and p < 0.001 , while for FNH Reader 1 achieved an accuracy of 89% (25/28) and Reader 2 100% (28/28). For HCA, Reader 1 achieved 97.5% (39/40) and Reader 2 90% (36/40). There was excellent and significant interreader agreement with entirely enhancing (40/79) (score > 4) in HBP. Eighty-two percent of all FNH lesions (23/28) were classified as entirely enhancing lesions and 12.5% (5/40) as “only” significantly enhancing. In the HCA group, 87.5% of all lesions (35/40) were classified as significantly enhancing. The difference between the two groups of lesions was statistically significant. Interreader agreement of Gd-EOB scoring in HBP was excellent for both readers (p < 0.001–0.872).In the total study population, 60% (41/68) of all lesions were rated as significantly enhancing (scores of 2–3) and 40% were rated as p < 0.001). Interreader agreement of uptake pattern evaluation was good (0.604) and significant (p < 0.001) .During the arterial phase, the majority of all lesions, 89% (58/65) were classified as enhancing. There was no significant difference between FNHs  and HCAs . Wash-out during the portal venous phase was noted in only 9% (6/65) of all lesions—4% in the FNH subgroup (1/25) and 12.5% in the HCA subgroup (5/40)  of all lesions were classified as lobulated—89% (25/28) of all FNHs as opposed to only 27.5% 11/40) of all HCAs. A central scar was described in 35.5% (24/68) of all lesions. In the FNH subgroup, a central scar was observed in 46.5% (13/28) versus only 27.5% of all HCAs (11/40). The results for all qualitative MRI features are compiled in Table 1/40 of aAccuracies were evaluated for all binary parameters. Gd-EOB uptake behavior, assessed using the above introduced scoring system, showed the highest accuracies for both readers as compared to all other parameters . In line with the results of our contingency table analysis, lobulation and presence of a central scar achieved acceptable accuracies of 0.750 or higher for at least one reader  and p < 0.001 / OR: 99.12 (R#2). For both readers none of the other parameters turned out to be significant (p = 0.067–0-715 / ORs: 0.40–5.62) . For both readers only the HBP appearance was significant with p < 0.001) with a higher mean ratio for FNH than for HCA. Portal-venous wash-out was found to be stronger for HCA than for FNH (p = 0.048). Mean ratios in the arterial and venous phases did not differ significantly (p = 0.279 and p = 0.107). All results are displayed in Table ROI-based relative lesion-to-liver enhancement in the HBP was found to differ significantly between the two types of liver lesions p < 0.00 with a hOur results show that Gd-EOB MR imaging provides additional information for differentiating FNH and HCA based on enhancement during the hepatobiliary phase even in Gd-EOB enhancing HCAs. FNHs can be differentiated reliably based on their typical strong and homogeneous HBP uptake behavior, corresponding to more than 75% enhancement of the lesion area, hence defined as entirely enhancing lesion using the system presented here. Adding morphologic features such as a lobulated appearance and the presence of a central scar, can support the discrimination between FNH and Gd-EOB enhancing HCAs. Our scoring system of Gd-EOB uptake appears to be a promising tool for differentiate these two entities even in significantly Gd-EOB enhancing HCA providing a potential solution for not false diagnosing these lesions as FNH.Thomeer et al. (2014) reported areas of iso- to hyperintensity in 12 of 17 patients (71%) with histopathologically proven HCA of the inflammatory subtype (IHCA) after gadobendate dimenglumine injection [Agarwar et al. (2014) even conclude that IHCA can mimic the appearance of FNH on Gd-EOB MRI including HBP hyperintensity [Thomeer et al. and Agawar et. al., we found that the majority of significantly enhancing lesions were of the inflammatory subtype [Although both FNHs and HCAs are rare benign lesions of the liver, their non-invasive differentiation is important for determining further treatment algorithms. FNH does not have a risk for malignant transformation or bleeding and can be described as a so called “do not touch” lesions. In HCA, depending on their size and histological subtype, surgical resection is recommended due to the risk for malignant transformation or bleeding. It is therefore important to discriminate routinely between FNH and HCA with the help of MRI correctly , 28. Earnjection . They conjection . Agarwarntensity . These rntensity . In line subtype .Thomeer et al., however, we assume that HBP imaging still provides the highest discriminatory power to distinguish HCA and FNH in the noncirrhotic liver. In the present study, we only analyzed lesions with more than 25% intralesional Gd-EOB uptake. As reported before, we classified intralesional Gd-EOB uptake in steps of 25% [Unlike Grazioli et al. (2012) reported higher arterial signal intensity in FNH than in HCA, it must be borne in mind that IHCA shows greater arterial enhancement than the other subtypes, which is important for this study as the HCA subgroup mainly consisted out of IHCA [As expected, arterial hyperintensity provided high levels of sensitivity without any strong discriminatory power as both FNH and HCA are characterized by hyperintensity in the arterial phase , 19, 30. of IHCA , 31, 32. of IHCA , 31, 32.In our cohort, five HCAs were rated as entirely enhancing lesions. We believe that this small group of lesions is highly interesting as these are HCA lesions (4 × IHCA and 1 × bHCA) with the potential of mimicking FNH in Gd-EOB MRI. However, entirely enhancing HCAs are so rare that a statement on how to distinguish them from FNH is only possible in the context of further studies.Thomeer et al. we assume that, although HBP hyperintensities occur frequently in HCA, it is still possible to differentiate FNH and HBP and overcome MRI pitfalls by characterizing their uptake behavior more specifically, as done in our analysis. As also supported by our quantitative lesion-to-liver ratios, the Gd-EOB scoring system presented here may become a tool easy to use in daily clinical routine to discriminate between FNH and significantly enhancing HCA.The results of our quantitative analysis in terms of CE lesion-to-liver ratios corroborate our qualitative results regarding the discriminatory power of the intralesional Gd-EOB uptake scoring system as the higher mean value in FNH (110.3 ± 39.2), corresponds to a score of 4, and was significantly higher than in HCA (59.2 ± 38.0—p < 0.001). Referring to Our study has limitations. First, we performed a retrospective study and patients were not enrolled consecutively and although basic MRI sequences were consistent, acquisition parameters differed, as due to their rarity, the collective was enrolled during a time-period of eleven years. Also, the fixed delay after injection of contrast enhanced sequences could lead to missing the optimal timing for some lesions. Second, lesion-to-liver ratios were measured in ROIs and not volumetrically. Third, we only analyzed FNHs and HCAs. Hepatocellular carcinomas can also present as HBP enhancing lesions. To minimize this bias, only patients without cirrhosis were included. Fourth, although readers were blinded, they were aware of the study design, which may have introduced detection bias, especially as only FNH were included who were surgically resected.Even in Gd-EOB enhancing HCA during the HBP, characterization of Gd-EOB uptake was found to provide the strongest discriminatory power to differentiate HCA from FNH. The scoring system we introduce may facilitate interpretation of MRI and provide an easy-to-use diagnostic tool to overcome the imaging pitfall encountered in the differentiation of significantly enhancing HCA and FNH. Furthermore, a lobulated appearance and the presence of a central scar, which are more common in FNH than in HCA, can be used as additional features.


A large number of studies have examined expertise and gender-related differences in the mental representation of motor skills in different sports, like throwing technique in judo, front loop in sailing, and integration of routines in the mental movement representation in volleyball. Also, tactical behavioral studies were conducted in futsal and football. In addition, studies were also carried out to support the motor learning process through mental training in golf. The Structural Dimensional Analysis-Motoric (SDA-M) method was also used in the medical sector for the rehabilitation of stroke patients. So far, few studies have investigated differences in the mental representation of a specific motor skill by experienced athletes of other related sports. The goal of the present study is to examine group differences and similarities in the mental representation of the tennis serve between experienced tennis, badminton, and handball athletes as well as a control group without any sport experience. We want to assess the quality of mental representation of technical-related overhead motion task expertise. For this purpose, we used the SDA-M to measure the mental representation of the tennis serve of four different groups . As expected, badminton and handball athletes showed functionally well-structured representations, which were similar to the structure of the group of tennis athletes. Novices showed an unstructured mental representation. These outcomes confirm the relationship between mental representation and performance in the development of overhead motion. Furthermore, the results emphasize the importance of mental representations as an essential developmental aspect in learning motor skills, especially in learning technical-related motor skills. According to One of the most difficult fundamental motor skills is proximal-to-distal sequences (P-D sequence), like overhead motions, which can be observed in a large variety of motions of the upper limbs of humans in both everyday life and sport activities . Exampleoverhead motion  of d motion . A GMP cd motion . Accordid motion , p. 345,d motion , 2014. IThe optimal performance of these P-D sequences requires precise mechanics that involve coordinated kinetic chains to develop, transfer, and regulate the forces that are needed to withstand the inherent demands of the task and to allow optimal performance . The terIn this study the mental representations, including the representation units and the structural composition of these representations, are of main interest. Mental representations are the cognitive imagery of motor skills which are used to refer to memory-related structures and processes that allow an athlete to use their experience to improve their motor performances. Memory-related structures are all states and processes stored and accessible in the memory, and they are the basis of the movement execution .The cognitive architecture of complex actions refers to the interplay of higher levels of mental and lower levels of sensory motor, control, and representational systems, with mental representations playing a key role in motor control and learning . The resIn numerous studies, mental representations of complex sport movements have been found to be dependent on expertise e.g., . ExpertsThe transfer of motor learning was investigated by Comparing the P-D sequence of different motor skills, studies have found both similarities  and diffWith respect to mental representation, as an essential factor for motor control and learning, the question arises on whether mental representations may transfer across motor skills that involve a similar P-D sequence. So far, no studies have investigated differences in the mental representation of a specific motor skill by experienced athletes of other related sports.The aim of the present study is to examine the tennis serve’s mental representation of experienced tennis, badminton, and handball athletes and novices as well as to analyze the differences and the similarities between their mental representation structures. The results of this study could benefit the process of motor learning of technique-related motor skills and could give valuable insights in a possible transfer of mental representations across experts from sports that require the completion of similar, yet distinct, P-D sequence tasks.First, we assume that the tennis experts’ mental representation show the most functional links, whereas novices demonstrate an unstructured mental representation.Second, we expect that experience in a specific P-D sequence  should also be accompanied by a functional structured representation of the technically related tennis serve. Thus, we hypothesize that the mental representations of badminton and handball athletes show no significant differences but differ from those of tennis experts and novices.n = 10; Mage = 29.9 years, SDage = 8.5; four females, six males; training age = 24.1 years; training frequency = 9.4 h per week); badminton ; handball ; or novices  without prior sport experience in tennis, badminton, or handball.In this study, 40 participants were divided into four groups: tennis  were members of sports clubs and active athletes. Throughout the testing, all participants were healthy and in good condition. To take part in the experiment, the participants gave informed consent. The study involving human participants was approved by the Ethics Committee of Bielefeld University and adhered to the ethical standards of the latest revision of the Declaration of Helsinki.The storage of motor skills’ mental representation can be assessed using the Structural Dimensional Analysis-Motoric SDA-M;  method. The SDA-M method comprises four steps : In the The tennis serve can be subdivided into three distinct biomechanical and functional phases , 1999, nHD Writer AE 1.2 for each BAC from a video showing the tennis serve’s movement in slow motion. In order to ensure that the images correspond to the respective textual descriptions, the allocation was validated by two experienced tennis experts (coaches with B-/A-level) who did not take part in the study. In this way, it has been possible to provide clear and adequate stimulus material combining images and textual descriptions . The participants sat approximately 60 cm away from a 15-in. laptop screen. The stimuli had a size of 9.0–9.0 cm (250–250 pixels) and were shown on a laptop (Sony Vaio). Each BAC  was presented in an anchor position, which was displayed in the upper half of the laptop screen. The remaining BACs were successively displayed below them. For each pair of BACs , the participants were asked to decide whether or not the BACs presented on the screen belonged together during movement execution. Once all BACs had been assessed in relation to the anchor BAC, a different BAC took over the anchor position, and the procedure was repeated. The split procedure ended after all BACs had been in the anchor position . All parvia cluster analyses (dcrit = 3.51) for a significant alpha-level of p = 0.05. Statistically, all BACs, which form structures below the critical value, are clustered together. The lower the value of a link between two items, the shorter the distance is between the related BACs.Mental representation structures were analyzed by determining mean group dendrograms analyses . The redλ with λcrit = 0.68 makes statements about the manner, in which the cluster solutions have a significantly similar structure to a predefined reference . This means that there was a significant difference between clusters if λ < λcrit = 0.68. If λ is greater than λcrit, there is a significant similarity, which corresponds to an alpha level of 5% , whereas both groups significantly differ from the handball group (λ = 0.468 for both) as well as from the group of novices (λ = 0.565 for both). Additionally, statistical analysis of invariance demonstrate a significant difference between the handball group and the novice’s group (λ = 0.457).As can be seen in The ARI indicates that the mental representation structure of the tennis group is identical to the mental representation of the badminton group (ARI = 1.0) as well as less similar to the handball group (ARI = 0.7) and the group of novices (ARI = 0.266). The comparisons of the badminton group with the handball group as well as with the novices group demonstrate the same results . The handball group and the novices group vary widely (ARI = 0.285).The aim of this study was to examine the tennis serve’s mental representation of experienced tennis, badminton, and handball athletes and novices as well as to analyze the differences and the similarities between their mental representation structures. We assume that the tennis experts’ mental representation shows the most functional links, whereas novices demonstrate an unstructured mental representation. Furthermore, we expect that experience in a specific P-D sequence  should also be accompanied by a functional structured representation of the technically related P-D sequence (tennis serve). Thus, we hypothesize that the mental representations of badminton and handball athletes show no significant differences but differ from those of tennis experts and novices.The outcome of the hierarchical cluster analysis of tennis athletes is very much in line with the biomechanical functional structure of the tennis service motion according to Although the movement of the badminton clear corresponds to an overhead throwing movement in handball and has certain similarities to tennis serve , 1979, tThe group of novices, the group without any comparable sporting experience, shows an unstructured movement representation in their movement memory. With regard to the classification of the single BACs to the corresponding function phases, the participants of group novices obviously achieve fewer good results than the other three groups. Thus, no function phase is structured by any of the participants in an optimal manner. In contrast to the group of tennis, badminton, and handball athletes, the mental representations of novices were organized less hierarchically, were more variable among persons, and were less well-matched with functional and biomechanical demands, which are in line with the studies of hitting the ball (BAC 8), which is not necessary in handball. BACs 5 to 7  are well structured and correspond to the biomechanical structure of tennis serve.Reasons for the similarity in the cluster solution of tennis and badminton athletes can be found in the certain degree of similarity of the analyzed motor skill. The main function phase consists of the forearm and humerus actions, and the racket movement starts with the stroke and ends with the ball leaving the racket. The final swing can be characterized as the second auxiliary function phase, and it is used to slow down the forward movement and control the swing out of the main function phase which enormously accelerated the racket. In tennis and badminton, the racket position and aspects like the velocity, swing, and orientation of the racket have to be controlled. These racket performances, especially hitting the ball with the racket, are key aspects for movements characteristic and specific to racket sports, like tennis, badminton, squash, and table tennis. Tennis and badminton athletes have to hit the ball, whereas a handball player already holds the ball in the hand when throwing. In handball, the forearm and humerus actions, which form the forearm whip, are the most important component in the overarm throwing movement . The obvSo, reasons for the different cluster solutions of tennis, badminton, and handball group could be found in the use of specific sport equipment, i.e., racket in tennis and badminton, and the high requirement in coordination of all body segments to produce high accuracy at the distal end of a P-D sequence . That unWe conclude that the mental representations of the tennis serve in tennis, badminton, and handball athletes are quite similar but not identical. This may be due to specific adaptations based on technical and tactical requirements in different sport disciplines and, in particular, the different upper body movements and the use of a racket , p. 353.overhead motion, may be accompanied by benefits in mental representations of related motor skills  and therefore has positive effects in the quality of movement execution. This approach might also be conceivable in the opposite direction. Accordingly, developments in the mental representation of a specific motor skill  may benefit the mental representations to related motor skills , which could be linked with qualitative and quantitative development in performance.The overhead motion is an important part of the integrative (non-specific) concept of team ball games according to the game implicit learning model of This could provide important information to understand the transfer of mental representations and, therefore, the transfer of motor skills in the motor learning process and to supply an approach that is useful to practice and alter different overhead motions, like overhead throwing movement .Indeed it has been shown that a motor skill with relatively complex movement dynamics can be acquired implicitly, without the accrual of consciously accessible declarative knowledge and without a corresponding increase in verbal knowledge about the motor skill . This maTherefore, it remains to be shown how far the improvement of such basic skills is accompanied by an improvement of related motor performance. The results are also limited by the fact that the present study examined only the mental representation of the tennis serve and not the overhead throwing movement in handball or badminton clear. Further studies should perform motoric tests to investigate qualitative and quantitative data of different overhead motions. Another interesting avenue for further research that could benefit our understanding is how the P-D sequence evolves during learning in a new field of sport. To do so, studies should focus on learning a P-D sequence of different types of motion  and examine how this affects the outcome of the actual P-D sequence .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The ethics committee waived the requirement of written informed consent for participation.CM and MG contributed to the conception and design of the study and performed the statistical analysis. MG wrote the first draft of the manuscript. All authors wrote sections of the manuscript and contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BackgroundThe coronavirus disease 2019 (COVID-19) pandemic led to a need to introduce video telemedicine for outpatients as an emergency measure without widespread stakeholder consultation. The patient and clinician experience of video outpatient consultation during the peak of the pandemic was studied for acceptability and to gather recommendations to improve the service during continuing infection control measures.MethodsOutpatient video telemedicine was introduced over a 14-day period including the provision of equipment, systems integration and stakeholder communication. Patient and clinician experience were measured between 15 April 2020 and 5 May 2020.ResultsA total of 43 patients and 79 clinicians provided feedback. Of the patients, 86% were above the age of 30 years, with the largest patient group aged 51-70 years. Patient experience was positive. All (100%) patients found joining the video consultation easy; 93% of them recommended to use it for future consultations.Clinician satisfaction was >90% with sound and video quality. Patients were less satisfied than clinicians in that they had communicated everything they wanted to (86% versus 95%). All (100%) patients thought that the video telemedicine solution met their needs, but 25% of clinicians believed that the patient experience of a video consultation was worse than a face-to-face clinic appointment. The three significant factors identified for introducing video consultations were successful IT, improved patient experience and digital healthcare records.ConclusionsIn the COVID-19 crisis, video telemedicine played a central role in outpatient consultations with excellent levels of success. With some differences in satisfaction level, clinicians significantly underestimate the level of patient satisfaction with outpatient video consultation. The novel coronavirus disease 2019 (COVID-19) has changed the dynamics of delivery of healthcare across the globe when the World Health Organization (WHO) reminded healthcare authorities across the world to take specific measures to protect the community, patients and clinicians -5. In thTelemedicine does not preclude the need for physical examination but helps in promoting healthcare services at a distance using technology at a lower cost. Different modalities under telemedicine range from text messages and emails, remote patient monitoring and teleconferencing ,12. VideLike every change, a video telemedicine solution has positive as well as negative effects. Positive influences include the establishment of trust between a patient and clinician, safety and convenience. The addition of the video element, to a virtual clinic, has the ability to pick up non-verbal communication, making the video consultation effective as face-to-face (F2F) consultations. Negative influences include patient suitability and preference, and the lack of physical examination or ability to deliver a procedure or intervention -15.COVID-19 provided a unique opportunity to introduce a significant new way of working across the UK NHS. Through this study, we aim to understand the acceptability by both patients and clinicians and evaluate their experience to improve the service.SettingThis qualitative prospective study was carried out for outpatient video consultations during the peak of the UK COVID-19 pandemic. In response to national guidance issued regarding outpatient consultations during COVID-19 , the hosKotter’s model for leading changeWe used Dr. Kotter’s eight-step model of change methodology to lead and deliver the successful transition in our organisation .Following the issuance of national guidance on stopping all non-essential F2F outpatient consultations, an utmost need of the hour was to create a mechanism for continued virtual outpatient consultations to deliver safe and effective care for both patients and clinicians. This requirement “created a sense of urgency” for the change to start (Kotter’s step 1).A team of managers, clinicians and information and communication technology experts (ICT) was formed to “build the guiding coalition” (Kotter’s step 2). The team worked rapidly on the mechanism to deliver video conferencing. A high-level options appraisal was used to decide which video conferencing solution to implement for patient video consultations. A comparison made between different telemedicine options using score matrix and risks related to IG and associated with staff working from home analysed. After this brief comparison, “Attend Anywhere” was chosen as the video telemedicine solution because of broader applicability and quicker adaptation.For a successful “development of a vision and strategy” (Kotter’s step 3), it was necessary to engage at many levels of the organisation to implement the change. Strategies were divided into leadership roles, implementation oversights, clinical processes, technical and testing requirements, staff training, booking processes, patient engagement and communication, IG perspective, IT equipment availability, and finance and procurement decisions. Under each of the work-streams in strategic planning, activities were identified and tasks divided between the team members.At the start of the COVID-19 pandemic, a “volunteer army was ready to be enlisted” (Kotter’s step 4) as we took advantage of the national and international communication to inspire the wider team. Many routine hospital processes were suspended, leaving patients and staff available and motivated to help in response to the COVID-19 pandemic. We took advantage of the active situation, and a large, highly motivated group, including patients as volunteers, was engaged to focus on the need to deliver video outpatient consultations.Kotter’s step 5 is to “enable action by removing barriers”. Once again, the opportunity of the COVID-19 pandemic emergency measures for management decisions, finance and IG processes led to streamlining of process within organisations, which successfully removed many obstacles previously encountered. Rapid approval from IG, clinical leads and patient representatives identified patient cohorts to prioritise. The video telemedicine was tested using “mock patients” in a clinical setting after confirming that technical requirements could be met from both hospital and patient location. Staff members included in design and testing were those managers, booking officers, nurses and clinicians involved in the outpatient setting as in pre-COVID era. Engaging the stakeholders in design and testing generated the early short-term wins for the project (Kotter’s step 6) to deliver successful consultations from locations within the Hospital Trust, consultants working from home as they were clinically shielding and patient group leaders.Video outpatient consultation using telemedicine was successfully delivered and continues as part of the provision of routine services for patients to sustain acceleration (Kotter’s step 7). During the healthcare recovery phase of the COVID 19 emergency measures (three months after the institution of the emergency measures), consolidation and delivering more is the process of institutional change (Kotter’s step 8). The continued infection risks worldwide from COVID 19 supports the anchoring.DurationAll consecutive video telemedicine consultations carried out over the three-week period (15 April 2020 to 5 May 2020) were included. The further qualitative survey of clinicians to gather information on influencing factors to continue to improve the delivery of video consultations to our patient population was administered from 10-17 July 2020.ProcessThe patients were contacted to confirm if they were confident of receiving a video outpatient consultation. Patients who chose video telemedicine consultation were sent appointment letters along with a user guide. Information was added to the Hospital Trust website regarding video telemedicine, including links to joining a meeting, troubleshooting and user experience videos.Inclusion criteriaThe inclusion criteria included the following:1. Patients above the age of 18 years 2. Patients below the age of 18 years only if named guardian present during a consultation3. Have a valid email address and access to a computer/smartphone/tablet with a camera and sound capability with Google Chrome or Microsoft Edge.Exclusion criteriaThe exclusion criteria excluded the following:1. Patients under the age of 18 without a named guardian present.2. Patients with no access to computer/smartphone/tablet.3. Any appointment/consultation requiring “breaking bad news”.After the video consultation, the patients were sent out a feedback form by email. The clinician doing the video consultation received an automatically generated feedback form. The second clinician information-gathering questionnaire was administered three months after the initial introduction of the video telemedicine.The feedback questionnaire for both patients and clinicians are available in the supplementary data appendix.Patient feedbackA total of 43 patients provided feedback. Most  of the patients were above the age of 30 years Table .Mobile  and laptop n=14; 32.55%) were the two most commonly used devices. Google chrome and safari were the most commonly used browsers. Most of the patients were able to access the video consultation by themselves , whereas only a few required help . All the patients found joining the video call easy . Majority  of the patients of the patients opted to choose video consultation for future again, whereas none of them opted not to choose it again  and the video quality  . Patients were less satisfied than clinicians that they had communicated everything they wanted to (86% versus 95%). All (100%) patients thought that the video telemedicine solution met their needs, but 25% of clinicians thought that the patient experience of a video consultation was worse than a F2F clinic appointment.Clinician feedbackOverall, 45 times clinicians provided feedback for video consultation. Of the clinicians, 88.88% (40/45) found it easy to join the video call. Majority of them were happy with the sound quality  and the video quality 95.54%; 43/45)  is simple, dynamic, feasible and adaptable. There is a discrepancy between the patients’ positive enthusiasm for telemedicine and clinicians, believing that the patient experience is better with a F2F consultation. This suggests that some of the delays in delivering telemedicine for the UK NHS population arise from clinicians’ incorrect assessment of their patient wishes. This poses the greatest threat to continuing adoption after the crisis phase of COVID-19. Despite some inherent weaknesses and threats to its universal applicability, it has strengths and opportunity of future consultation equivalent to, and at times better than, F2F consultations.