Intracranial arterial calcification (IAC) may be present in the intimal or medial arterial layer. This study aimed to elucidate the link between the calcification and atherosclerotic disease in the intracranial vasculature.Consecutive patients with acute ischemic stroke were included. Bilateral intracranial segment of the internal carotid artery, M1 segment of the middle cerebral artery, intracranial segment of the vertebral artery, and the basilar artery were visualized by the multi-detector computed tomography (CT) and vessel-wall magnetic resonance imaging (vwMRI) within 14 days after stroke onset. IAC was into the intimal or medial pattern. Subsequently, on the vwMRI, we assessed the luminal stenosis, eccentricity, plaque burden, and intraplaque hemorrhage (IPH) as markers of atherosclerosis at each IAC site.p = 0.003) caused by atherosclerotic lesions. Compared with the medial IAC, intimal IAC was more often accompanied by eccentric plaques (p = 0.02), larger plaque burden (p = 0.001), and IPH (p = 0.001).Among 69 patients with stroke, IAC was identified in 35% of (161/483) artery segments, of which 61.5% were predominantly intimal calcification and 38.5% were predominantly medial calcification. About 79.8% of intimal calcifications and 64.5% of medial calcifications co-existed with atherosclerotic plaques. Intimal calcification was associated with luminal stenosis (Our multimodal imaging-based comparison study on intracranial arteriosclerosis demonstrated that intimal IAC, compared with medial IAC, was more often accompanied by the luminal stenosis, larger plaque burden, eccentricity, and IPH, providing strong evidence for clinical evaluation on the mechanism, risk, and prognosis of ischemic stroke. Intracranial arterial calcification (IAC) is commonly seen in major cerebral arteries –3 and esIntracranial arterial calcification mainly involves the intimal and medial layer, which could vary in both histopathological feature and imaging . HistopaThe study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. This was a retrospective study based on a prior prospective study. Patients who underwent computed tomography (CT), routine magnetic resonance imaging (MRI), magnetic resonance angiography (MRA), and vessel-wall magnetic resonance imaging (vwMRI) within 14 days after the onset of ischemic stroke or transient ischemic attack (TIA) from 2014 to 2018 at the Prince of Wales Hospital, Hong Kong were included in this study. The exclusion criteria were as follows: (1) other vasculopathies, such as dissection and vasculitis; and (2) incomplete image data or poor image quality.A routine non-enhanced brain CT was performed using a 64-slice multi-detector row CT system  with acquisition parameters as follows: slice thickness 5 mm; 120 kVp, 170 mAs, and 1 s per rotation. All axial images were reconstructed at 0.625-mm intervals. Each CT scan was obtained in axial mode, with tilting along the occipital-meatal line.The presence and patterns of IAC were assessed on reconstructed CT images by two readers (H.D. and L.Z.) who had more than 3 years of experience in brain CT imaging and were blinded to the clinical and MRI data. Each major intracranial artery segment was assessed, including the cavernous segment (C4) of the internal carotid artery (ICA), the supraclinoid and ophthalmic segment (C5-6) of the ICA, M1 segment of the middle cerebral artery (MCA), intracranial segment (V4) of the vertebral artery (VA), and the basilar artery (BA). IAC was defined as hyperdense foci over 130 Hounsfield units (HU) (refs). The patterns of IAC were categorized into intimal or medial according to a previously established scoring model , in whic3, acquired resolution 0.4 × 0.6 × 0.7 mm3, repetition time (TR)/echo time (TE) 23/3.5 ms. Parameters for T1w-VISTA: FOV 200 × 167 × 45 mm3, acquired resolution 0.6 × 0.6 × 1.0 mm3, reconstructed resolution 0.5 × 0.5 × 0.5 mm3, TR 1,500 ms, and TE 36 ms. vwMRI images were repeated after the injection of gadolinium-containing contrast agent  intravenously with a ratio of 0.2 ml/kg and an injection rate of 3.5 ml/s.Vessel-wall MRI was performed using a 3.0 T Achieva MR system  with an 8-channel head coil. The time-of-flight MR angiography (TOF-MRA) sequence and transverse 3D T1-weighted volumetric isotropic turbo spin-echo acquisition (T1w-VISTA) sequence were acquired. Parameters for TOF-MRA were as follows: field-of-view (FOV) 200 × 200 × 56 mmThe presence of atherosclerotic lesion was identified on reconstructed images using the OsiriX DICOM Viewer  by two readers (H.D. and L.Z.) who were blinded to the clinical data and CT findings. Images from TOF-MRA were used for identifying luminal stenosis . StenosiAfter the identification of IAC on CT and plaques on vwMRI, a side-by-side analysis was conducted to check whether the stenosis and plaques were at each corresponding site of IAC based on anatomy.T-tests were used for comparison of age and plaque burden between groups. Pearson's chi-square test or Fisher's exact test was used for comparisons of the distribution patterns of IAC, category of luminal stenosis, stenotic degree, eccentricity, and IPH between plaques with intimal calcification and that with medial calcification. Multivariable regression was used to evaluate the association between IAC and luminal stenosis, eccentricity, and IPH. A two-sided p < 0.05 was considered statistically significant.For statistical analysis, IBM SPSS  was used. Continuous variables were expressed by mean ± SD and categorical variables were presented as numbers and percentages. p = 0.001).In total, 79 patients were included after four patients were excluded due to incomplete images or poor image quality. It was found that 69 (87.3%) patients had calcification in one or more major intracranial arteries, and the other 10 patients showed no calcification. Patients with IAC were generally older than those without IAC  patients were men  and 25 were women . History of smoking was found in 22 out of 69 patients (31.9%). In addition, 54 out of 69 patients (78.3%) had hypertension while the prevalence of diabetes and hyperlipidemia was 39.1 and 50.7%, respectively. Among 10 patients without IAC, the number of male and female was four and six, respectively. Of the 10 patients, six patients had history of smoking, seven had hypertension, two had diabetes, and three had hyperlipidemia.Among the 69 patients with IAC, 65 (94.2%) had 1–4 calcifications and one patient had as many as 7 calcified arteries. A total of 460 segments were examined, out of which 161 segments (35.0%) with either intimal or medial calcification were identified in brain CT . Most IAOf the 161 IACs, 99 (61.5%) were categorized as predominantly intimal IACs and the other 62 (38.5%) as predominantly medial IACs . Figure p = 0.008) and less frequently involved the intracranial VA (p = 0.137).Intimal IAC was frequently detected in the cavernous (34.3%) and supraclinoid-to-ophthalmic (30.3%) segments of ICA and the V4 segment of the VA (24.2%), while medial IAC was more prevalently present in the cavernous (43.5%) and supraclinoid-to-ophthalmic (40.3%) segment of the ICA. Compared with intimal IACs, medial IACs were more predominantly distributed in the C4–C6 segment of the ICA  were identified as “non-calcified plaques”.p = 0.003) and non-calcified plaques  (p = 0.02) and non-calcified plaques . Plaques with intimal IAC had higher plaque burden compared with plaques with medial IAC  and non-calcified plaques . IPH was found in 35.4% of plaques with intimal IAC, much higher than that in plaques with medial IAC  and non-calcified plaques . No difference in the presence of luminal stenosis, plaque burden, eccentric pattern, or IPH was found between plaques with medial IAC and non-calcified plaques.Comparing atherosclerotic lesions assessed by vwMRI, 79.8% (79/99) co-existed with atherosclerotic plaque while 64.5% (40/62) had concurrent plaque. Luminal stenosis was more frequently found in plaques with intimal IAC than plaques with medial IAC  . There wOR 2.140; 95% CI, 1.001–4.578; p = 0.05), eccentric plaque , and IPH  were associated with intimal IAC, after adjusting for age, gender, hypertension, diabetes, hyperlipidemia, and smoking.The multivariable regression model revealed that the presence of luminal stenosis , whereas the MCA and the BA were rarely affected  . A possiHistopathological findings demonstrated that an intimal IAC was present prevalently in the advanced stage of atherosclerosis . Being gWith luminal stenosis exceeding over 70%, eccentric plaque in the extracranial carotid artery becomes associated with a significant increased incidence of ipsilateral stroke . Similarin situ thrombosis and distal embolism  also takes a dominant role in the ischemic events of anterior circulation. Under the coexistence of multiple atherosclerotic lesions in both ICA and MCA, it is difficult to identify the culprit lesion that accounts for infarction (or TIA), especially artery-to-artery embolism and hypoperfusion. On the other hand, as single subcortical infarction is prevalently led by the occluded perforating artery of MCA, its relevance to ICA calcification seems loose. Whether IAC has impact on territorial infarction requires further studies.The present multimodal imaging-based study revealed both similitude and difference between ICAD and IAC. Medial IAC is prevalently distributed along carotid siphon. Compared with plaques with medial IAC and non-calcified plaques, plaques with intimal IAC more frequently show luminal stenosis, eccentric plaque, larger plaque burden, and IPH, indicating a latent impact of intimal calcification on plaque vulnerability. Our findings may provide evidence for the clinical evaluation on mechanism, risk, and prognosis of ischemic stroke. The understanding of the mechanism in the presence of calcification will be deepened with further study upon the correlation of IAC and territorial infarctions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. The patients/participants provided their written informed consent to participate in this study.HD recruited patients, analyzed the imaging data, and drafted the manuscript. JL participated in the study design and recruited patients. WY acquired the vwMRI data and revised the manuscript. DB revised the manuscript. LZ analyzed the imaging data. LW and TL participated in the coordination of the study. XC conceived the study, participated in the study design and coordination, and revised the manuscript. All authors contributed to the article and approved the submitted version.This study has received funding by grants from the General Research Fund from Research Grants Council of Hong Kong  and the Health and Medical Research Fund of Hong Kong .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phosphorylated tau (p-tau) species in bodily fluids are among the most reliable molecular biomarkers for differential diagnosis and progression monitoring of Alzheimer’s disease (AD) . The ATNWe quantified endogenous p181tau in CSF samples of APPPS1 transgenic (tg) mice  that do not develop neurofibrillary tangles or extensive neuron loss . CSF p18We then used an immunoassay which allows the quantification of tau phosphorylated at threonine 217 with or without adjacent phospho-epitopes  , 5 see a. On aveThese observations imply that p-tau increases in CSF are not exclusively triggered by Aβ deposition but can also be induced by the deposition of Danish amyloid (ADan). Thus, it is tempting to speculate that CSF p-tau increases are a general phenomenon of secondary tauopathies, as opposed to primary tauopathies. Interestingly, AD and FDD but also Familial British Dementia, yet another cerebral amyloidosis with concomitant neurofibrillary degeneration ), all shSupplementary file1 (DOCX 101 kb)Below is the link to the electronic supplementary material.


Anti‐microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His‐5) was synthesized and the transport efficiency and anti‐fungal effect were measured to evaluate the promotion of His‐5 modified by TDNs.C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His‐5.Tetrahedral DNA nanostructures/His‐5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti‐fungal effect of the TDN/His‐5 complex was evaluated by determining the growth curve and colony‐forming units of C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti‐fungal effect against C. albicans.The results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into Our study showed that TDN/His‐5 was synthesized successfully. And by the modification of TDNs, His‐5 showed increased transport efficiency and improved anti‐fungal effect. TDN/His‐5 were assembled by TDNs and Histatin 5 by electronic attraction and internalized by Candida albicans, leading to the increased formation of intracellular reactive oxygen species, high potassium efflux, and enhanced antifungal effect against C. albicans. They can affect different tissues and systems, ranging from the mucosa to the haematological system.Nowadays, diseases caused by eukaryotic microorganisms have become global contributors to illness and death, and this threat is intensely debated.Positively charged short anti‐microbial peptides (AMPs) found in plants and animals are critical components of the innate immune system and defend against invading microorganisms.As for extracellular AMPs, the initial stage of their function depends on the passive combination between their positively charged domain and the negatively charged microbial membrane.What is more, distinct from the bacterial cell wall, the fungal cell wall is composed of mannoproteins, chitins and α‐,β‐glucans, which weakens the initial binding of fungal cell membrane with AMPs, leading to decreased AMPs delivery efficiency and anti‐fungal effect.Recently, tetrahedral DNA nanostructures (TDNs) have attracted increasing interest because of their editability and biocompatibility.C. albicans.In this study, Histatin 5 was selected as the representative of intracellular anti‐fungal AMPs and combined with TDNs to construct TDN/His‐5 complex, whose characteristics and anti‐fungal effects were tested. To our knowledge, this is the first study to design a combination of TDNs and intracellular AMP as anti‐fungal agents. Owing to electrostatic attraction, the cationic AMP His‐5 can bind easily to anionic TDNs, leading to more efficient uptake by 22.1Candida albicans SC5314 (wild type) obtained from our laboratory were commercially from the American Type Culture Collection.Candida albicans strains were propagated on liquid or solid YPD medium at 30°C. The yeast cells used in all experiments were in the exponential growth phase (OD600 = 0.6). For inducing hyphae, C. albicans cells were grown in RPMI 1640 medium containing 2.5% foetal calf serum, 2 mm L‐glutamine, 20 mm HEPES and 16 mm sodium hydrogen carbonate (pH 7.0) (RP medium) at 37°C.2.2m MgCl2.6H2O, 10 mm Tris‐HCl). The total mixture was placed into a PCR instrument, and heated to 95°C for 10 minutes then cooled rapidly to 4°C for 20 minutes.Tetrahedral DNA nanostructuress were synthesized by four single‐stranded DNAs (deoxyribonucleic acids) Table  purchase2.32O (ddH2O) and TDNs were mixed in different ratios  and incubated for 30 minutes at room temperature.Histatin 5  dissolved in doubled distilled H2.4:To verify stability of TDN/His‐5 complex in the serum medium, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) was utilized to assess the degradation profile of His‐5. His‐5 and TDN/His‐5 incubated in the 10% foetal calf serum medium for 6h were measured by MALDI‐TOF MS. And the proportions of peak areas (mass range from 2900 to 3000) to the total area in spectrum were calculated as the proportions of His‐5 in the medium. The mass percentage of His‐5 was calculated with following equationInitial His‐5 and TDN/His‐5 in the 10% foetal calf serum medium were measured as control groups. And the proportions were compared between the TDN/His‐5 groups and His‐5 groups.2.5g for 10 minutes, and the concentrations of unloaded His‐5 in the remaining solutions were measured and calculated by spectrophotometry at the wavelength of 214 nm. The loading efficiency of His‐5 onto TDNs was calculated with the following equation:The loading efficiency of TDN/His‐5 complex was determined using ultrafiltration and spectrophotometry. TDNs and His‐5 were incubated for 30 minutes at room temperature in different ratios . Then, the TDN/His‐5 solutions were ultrafiltered  at 9 600 C1 and C2 mean the initial and unloaded concentration of Histatin 5, respectively.2.6C. albicans was used to perform fungicidal activity assays. C. albicans cells were washed with PBS thrice and diluted to 106 CFU/mL. Then, the strains were seeded at an initial density of 5 × 105 CFU/mL, divided into three groups and treated with 400 nm TDNs, 20 µm Histatin 5, TDN/His‐5 complex (400 nm TDNs with 20 µm His‐5) for 1:50 mass ratio group; 200 nm TDNs, 20 µm Histatin 5, TDN/His‐5 complex (200 nm TDNs with 20 µm His‐5) for 1:100 mass ratio group, 100 nm TDNs, 20 µm Histatin 5, TDN/His‐5 complex (100 nm TDNs with 20 µm His‐5) for 1:200 mass ratio group, respectively, in a 96‐well plate in YPD liquid medium. In addition, YPD liquid medium seeded with C. albicans alone was used as a positive control, in contrast, non‐seeded YPD liquid medium with the TDN/His‐5 complex was used as a negative control. Fungal growth was measured with an automated spectrophotometer at 30°C, and the optical density at 600 nm was detected automatically over 24 hours. The OD600 value was detected once an hour, and the plate was shaken every 15 minutes to avoid precipitation.To evaluate the yeast killing ability of the TDN/His‐5 complex, exponential‐phase C. albicans were treated with 200 nm TDNs, 20 µm Histatin 5 or TDN/His‐5 complex of three mass ratio  in 96‐well plates as an density of 5 x 105 CFU/mL for 2 hours at 30°C in YPD liquid medium.Furthermore, 2.7C. albicans under the TDN/His‐5 complex conditions, the yeast cells were diluted to a concentration of 1 x 106 CFU/mL in a 12‐well plate in RP medium and treated with 200 nm TDNs, 20 µm Histatin 5 or 200 nm TDN/His‐5 complex (1:100 mass ratio) for 8 hours at 37°C.To determine the morphology of 2.8C. albicans were cultured in RP medium in 96‐well plates and diluted to a density of 106 CFU/mL. In addition, 200 nm Cy5‐TDNs, 20 µm FAM‐Histatin 5 and 200 nm Cy5‐TDN/FAM‐Histatin 5 (1:100 mass ratio) were added to different wells for 2 hours at 37°C. After the incubation, the cells were rinsed with PBS three times and stained with Hoechst for 30 minutes. Then, the suspension was rewashed and resuspended in PBS into a density of 107 CFU/mL. Five microliters of the samples was added to a slide and covered with a coverslip, which was observed with a CLSM .Cyanine‐5 (Cy4)‐labelled TDNs and 6‐carboxyfluorescein (FAM)‐labelled Histatin 5 were applied for the observation of cellular uptake of TDN/His‐5 by confocal laser scanning microscopy (CLSM). 2.96 CFU/mL) were treated with FAM‐Histatin 5 (20 µm) and TDN/FAM‐His‐5  3 or 6 hours at 30°C. Then, the samples were harvested and rinsed with PBS three times. The samples were resuspended in PBS at a concentration of 1 x 106 CFU/mL in flow tubes. And the untreated samples were used as the negative control group. All the samples were detected by flow cytometry  (Excitation wavelength: 492 nm and Emission wavelength: 518 nm for FAM).Exponential‐phase yeast cells , Histatin 5 (20 µm) and TDN/His‐5  for 3 hours. After that, the samples were resuspended with PBS three times , incubated with DCFH‐DA solution (1:1000) for 25 minutes and resuspended again. Then, the samples were stained with Hoechst for 30 minutes, rinsed with PBS three times and diluted into a density of 1 x 107 CFU/mL. Five microliters from each sample was added to a slide and covered with a coverslip, which was observed with a CLSM . The immunofluorescence images were analysed by Image J.Both yeast‐form and hyphae‐form 6 CFU/mL. 1 × 104 yeast cells were selected and analysed using a flow cytometer to deter the mean fluorescence intensity of DCF .Moreover, after incubation with DCFH‐DA solution as mentioned above, parts of yeast‐form samples were rinsed thrice with PBS  and diluted into a density of 1 x 102.116 CFU/mL in YPD liquid medium. Then 200 µL from each sample was incubated with TDNs (200 nm), Histatin 5 (20 µm) and TDN/His‐5  at 30°C for 4 hours. After that, the tubes were centrifuged, and the supernatants were collected and diluted 1:2 with ddH2O for testing. The supernatants were quantitated for their potassium concentration by atomic absorption spectroscopy .Exponential‐phase yeast cells were rinsed with PBS thrice  and diluted into a density of 1 × 102.12t test was used, and the significance level was set to α = 0.05.All the results presented are based on at least three individual experiments. Statistical analyses were executed in GraphPad Prism (GraphPad Inc). Student's 3In this study, we selected His‐5 as the representative of intracellular anti‐fungal AMPs and combined it with TDNs to construct the TDN/His‐5 complex. By the interaction between TDNs and the fungal cell membrane, cellular uptake and anti‐fungal efficacy of His‐5 were improved.3.1Conventionally, TDNs comprise four different single‐stranded DNA (ssDNA) and self‐assemble according to the specific sequence of ssDNA based on complementary pairing rules Figure .25 EigFurthermore, the zeta potentials and hydrodynamic sizes were measured to prevent excessive agglomeration of TDN/His‐5 complex owing to an imbalance of the mixture ratio, which may affect the transport efficiency. The average zeta potential of TDN/His‐5 complex at the peptide/TDN ratios of 50:1 and 100:1 was −8.13 and −2.37, respectively, whereas that at the ratio of 200:1 was reversed to positive charge  Figure  Table 2  was much smaller than that in Histatin 5 group (from 35.19% to 9.0.14%), that indicating more stable ability for His‐5 with the assistant of TDNs.  for 2 hours. The fluorescence signals of Cy5 in TDNs group and FAM in His‐5 group showed that both His‐5 and TDNs can be internalized by cells individually. Moreover, the fluorescence signals of both Cy5 and FAM in the TDN/His‐5 complex group were stronger than those in TDN and His‐5 groups, indicating a greater uptake of TDNs and His‐5. Statistical analysis showed the obvious increase in fluorescence intensity of Cy5 and FAM in the TDN/His‐5 group compared with that in the TDNs and His‐5 groups  was greater than that in the His‐5 group  after 3h of incubation Figure . After 6C, albicans and consequently promoting the pharmacological characteristics of His‐5. On the other hand, His‐5 can also enhance the transport efficiency of TDNs for C. albicans. Previous studies have proved that cationic penetrating peptide can assist DNA nanostructure like TDNs in cellular uptake.In this study, the surface of His‐5 was modified by TDNs, thus allowing the anti‐fungal intracellular AMP His‐5 to be internalized by The anti‐fungal efficacy of the TDN/His‐5 complex did not enhance gradually with the increasement in the proportion of TDNs in TDN/His‐5 complex. At the mass ratio of 50:1, the anti‐fungal effect of the complex decreased instead. Previous studies showed that TDNs can be internalized by cells and bacteria as the small‐weight‐drug vehicles.From our perspective, surface modification of AMP by TDN may have broad applications, especially for fungi that are insensitive to traditional AMPs.3.5C. albicans was monitored using the fluorescent probe DCFH‐DA.C. albicans cells. And the TDN group was not significantly different from the control group.To investigate the intracellular role of TDN/His‐5 complex, the early formation of ROS in both yeast and hyphal forms of Potassium efflux caused by His‐5, which resulting in volume dysregulation and cell death, was also measured in this experiment Figure .20, 33.6There are some limitations to our study. Only one pathogenic fungus was assessed, and detailed in vivo experiments were not conducted in this study, which we plan to perform in the future. In addition, the specific mechanism by which TDNs assist AMP in penetrating the fungal cell wall needs to be explored. We believe that the application of TDNs as the delivery vehicle of anti‐fungal AMP can be broadened by overcoming these limitations.There are no conflicts to declare.Bowen Zhang involved in conceptualization, investigation, methodology, and project administration and wrote the original draft. Xin Qin involved in data curation, formal analysis and investigation and wrote the review and editing. Mi Zhou involved in methodology, investigation and software. Taoran Tian involved in data curation, methodology and resources. Yue Sun involved in data curation and investigation and wrote the review and editing. Songhang Li involved in data curation, methodology and software. Dexuan Xiao involved in resources and methodology. Xiaoxiao Cai involved in funding acquisition, supervision and wrote the review and editing.Figure S1‐S2Click here for additional data file.


N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine  KRAS gene are almost consistently the initiator and present even in low-grade PanINs  for PanIN lesions, as well as PDAC, when crossed to additional genetic models , and PanIN formation  is the main SET domain-containing histone lysine methyltransferase (HMT) responsible for catalyzing H3K9 mono- and di-methylation (H3K9me1 and H3K9me2)  methylation is emerging as an important transcriptional regulatory and epigenetic pathway in pancreatic cancer . HistoneH3K9me2) . The facH3K9me2) . Indeed,H3K9me2) , in whicH3K9me2) . PharmacH3K9me2) . Howeversurvival . The curG12D induction experiments, cells were deprived of doxy at least 48 h for beginning experiments . Then, fresh media containing doxy 10 μg/ml was added, and cells maintained under culture during multiple time intervals . Human hTERT-E6/E7-HPNE  and hTERT-E6/E7-HPNE-KRASG12D  pancreatic cell lines were obtained from ATCC and maintained in appropriate media according to recommendations. All cells were cultured at 37°C in a humidified incubator with 5% CO2 and were used to a maximum of 30 passages. All cell lines tested negative for Mycoplasma with last testing performed in July 2020.Mouse iKras 4292 cell lines were obtained from Dr. Marina Pasca di Magliano [University of Michigan]. Cells wn = 3 experiments was completed using ImageJ and statistical significance determined by Student’s t-tests in GraphPad Prism 7.Mouse pancreas (∼100 mg) was cut in small pieces and homogenized in protein extraction buffer  with freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific). Homogenized tissue was moved to a 2 mL microcentrifuge tube containing 1 stainless steel bead (7 mm diameter) and further dissociated using TissueLyser LT (2 min at 50 Hz). The lysate was then sonicated twice (amplitude 5 for 10 s) and supernatant was cleared by centrifugation . HPNE and iKras proteins were collected from cells by lysis in protein extraction buffer supplemented with phosphatase and protease inhibitors. Equal amounts of protein from pancreas or cell lysates were electrophoresed on 12% SDS-PAGE gels and transferred to nitrocellulose membranes . Membranes were blocked in either 5% (w/v) milk or 3% (w/v) BSA for 1 h, and primary antibody incubations were performed overnight at 4°C with rocking. iKras 4292 cells were stimulated with doxy at the indicated intervals, fixed using 4% Formaldehyde and washed three times with 1× PBS. Cells were permeabilized using 0.2% Triton X-100 for 10 min, PBS washed and blocked with 1% (w/v) BSA in PBS-Tween 20 solution. Slides were then incubated with EHMT2, EHMT1 and WIZ primary antibodies at 4°C overnight and labeled using Alexa Fluor-conjugated secondary antibodies. For proximity ligation assays (PLA) assays, the Duolink PLA Starter Kit  was used according to manufacturer’s instructions. Coverslips were mounted using Prolong Gold antifade mounting media . Antibodies used are listed in RRID: AB_2552320) or control IgG antibody to Protein A/G Magnetic Beads (Thermo Fisher Scientific) through disuccinimidyl suberate crosslinking. iKras 4292 cells were grown and then stimulated with doxy for 24 h. Cells were lysed with IP Lysis/Wash Buffer (Thermo Fisher Scientific-Pierce), and lysates were incubated overnight with the antibody conjugates at 4°C. Immunoprecipitated complexes were washed, eluted, and run on a 4–15% CriterionTM Tris–HCl Protein Gel (Bio-Rad). The gel was subsequently visualized with BioSafeTM Coomassie Stain (Bio-Rad). Each gel lane was de-stained, dehydrated, dried, and subjected to trypsin digestion. Subsequently, liquid chromatography (LC)-ESI-MS/MS analysis was performed on a Thermo Scientific LTQ Orbitrap mass spectrometer at the Mayo Clinic Proteomics Core.Immunoprecipitation (IP) of EHMT2 was performed by conjugating the EHMT2 antibody  ethanol for histological analysis or freshly processed for cellular studies. B6.129S4-KrasTM 4Tyj/J  (B6.FVB-Tg(Pdx1-Cre)6Tuv/J  (Ptf1aTM 1(cre)Hnak/RschJ  (∗)5Amc/JB6.Cg-Tg (EHMT2 flox/flox (EHMT2fl/fl) animals were a generous gift received from Dr. Oltz  Cre drivers. Animals were weaned after 3 weeks and 0.4–0.8 cm length of tail was taken for genotyping. DNA was extracted from the tail pieces using Qiagen’s DNeasy® Blood and Tissue kit (Cat# 69506). PCR was performed as previously described  , B6.FVB-:014647) , Ptf1aTM:023329) , and B6.:004682)  were purDr. Oltz . All aniescribed  with proTo induce chronic pancreatitis, caerulein was administered via IP injection to 4-week-old mice once a day, five times a week, for a total of 4 weeks at a dose of 50 μg/kg. Animals were given 7 days with no treatment before sacrifice to allow recovery. Animal and pancreas weights were recorded at the time of sacrifice and tissue was taken for RNA, protein and histological analysis.Immunohistochemistry was performed on mouse pancreas tissue as described previously . PrimaryCAGGCre-ERTM and CAGGCre-ERTM;EHMT2fl/fl mice were euthanized and pancreas immediately injected with 2 mL of chilled collagenase P (1.33 mg/ml), dissected and cut into small pieces for digestion (20 min at 37°C with gentle shaking). Acinar cells were collected by centrifugation and resuspended in 3D culture base medium . 96-well plates were pre-coated with Matrigel  for 1 h at 37°C. Cell suspensions were mixed 1:1 with Matrigel and plated (100 μL/well). The cell/Matrigel mix was allowed to solidify for 1 h at 37°C before addition of 175 μL 3D culture media. For the first 48 h, DMSO or 4-OHT (10 mmol/L) was added, followed by EGF (20 ng/mL) or TGFα (50 ng/mL) for an additional 5–6 days  according to manufacturer’s protocols. Briefly, frozen samples were lysed with tissue Denaturation Solution and disrupted in the TissueLyser LT  with a single, 7 mm stainless steel bead. RNA isolation continued with Phenol:Chloroform:IAA and Acid-Phenol:Chloroform extractions to the aqueous phase and a final isopropanol precipitation. Precipitated RNA was cleaned up according to Qiagen’s protocol on the miRNeasy column , with an on-column DNA digestion and RNaseOUT  added to final elution to reduce degradation during storage at −80°C. Isolation of RNA from cells was completed according to Qiagen’s protocol for the RNeasy kit, including an on-column DNase digestion.p-values, called q-values, for assessing pathway significance, which queries different annotation resources to analyze enrichment for an input set of genes. The Molecular Signatures Database (MSigDB) hallmark gene set collection . To quantitatively determine the percentage of infiltrating immune cell types, we utilized a “digital cytometry” method called the quanTIseq deconvolution algorithm, which estimates the absolute proportions of relevant infiltrating immune cell types from bulk RNA-seq profiles , as well as the caerulein-treated animals dataset (4654 DEGs).RNA was quantified by Qubit (Invitrogen) and quality assessed with a Fragment Analyzer (Agilent), with the highest quality  utilized for library preparations. Pancreas RNA was prepared and sequenced in the Mayo Clinic Medical Genomics Core using the Illumina TruSeq RNA v2 library preparation kit and the Illumina High Seq-2000 with 101 bp paired end reads. Reads were aligned to the mouse reference transcriptome Gencode vM23 (GRCm38.p6) with at least 24 million mapped read pairs acquired per sample. Sequencing reads were processed through the GSPMC workflow including MapRseq3  and diffllection  was usedprofiles . The celprofiles  on DEGs 2 first strand kit (Qiagen) following manufacturer’s protocol. Real-time PCR was performed in a volume of 20 ul using SYBR Green Master Mix and CFX96 Real Time System (Bio-Rad). Primer sequences are shown in 2 Profiler PCR Array  was used to examine the expression patterns of 84 genes involved in cell cycle. The array was performed following manufacturer instructions. ddCT was obtained using Qiagen software and all groups were normalized to Pdx1-Cre mice, with an absolute FC ≥ | 1.5| for the generation of a heatmap using R-studio.Reverse transcriptase was performed using RTLSL-KrasG12D;EHMT2+/+ and LSL-KrasG12D;EHMT2fl/fl with both Cre/+Pdx1-Cre and P48 drivers were collected, washed in PBS (1×) and placed in a well of a 12-well dish containing 1 mL of fixative solution for 15 min. Pancreas were then rinsed twice with PBS (1×) and stained with β-galactosidase solution at 37°C overnight in a dry incubator. Next morning, pancreata were washed with PBS (1×), and images acquired.Senescence-associated β-galactosidase activity was assessed in fresh pancreas tissue using the Cell Signaling β-Galactosidase Staining kit, according to manufacturer’s protocol . Briefly, under terminal anesthesia, pancreas from Animal care and all protocols were reviewed and approved by the Institutional Animal Care and Use Committees of Mayo Clinic Rochester (IACUC protocols A00002240-16 and A24815) and the Medical College of Wisconsin (AUA00005963).RNA-seq datasets that support the findings of this study have been deposited to the public database repository Gene Expression Omnibus (GEO) under accession code GSE169525.P48Cre/+KrasG12D mice indicated that EHMT2 deficiency prolonged survival by reducing PanIN growth, which was accompanied by a decreased number of phosphorylated Erk-positive and Dclk1-positive cells, both populations that contribute to PDAC initiation , an inducible Cre to investigate EHMT2 inactivation ex vivo , as well as the model of pancreatitis-associated promotion   levels in Pdx-Cre;LSL-KrasG12D;EHMT2fl/fl animals . Similar findings were obtained with Cre/+;LSL-KrasP48G12D and P48Cre/+;LSL-KrasG12D;EHMT2fl/fl animals  . EHMT2 id by PCR . Notablyectively . Therefo animals . Similareas size , knockouc growth . Next, bormation , which iormation . In suppin vivo data, we next investigated whether EHMT2 is involved in the ADM process, using a genetically engineered ex vivo approach with acinar explants from a tamoxifen-inducible model of EHMT2 knockout. This model carries a CAGGCre-ERTM transgene, which has the chicken β-actin promoter/enhancer coupled with the cytomegalovirus immediate-early enhancer to drive high levels of Cre expression, allowing this enzyme to translocate into the nucleus  or presence (+) of 4-Hydroxytamoxifen (4-OHT) to inactivate EHMT2. Western blot showed that EHMT2 protein levels were reduced after 4-OHT treatment (ex vivo ADM formation. Phase microscopy of 3D cultures showed that EGF and TGF-α induced duct-like structures from acini isolated from CAGGCre-ERTM (−/+4-OHT) controls, demonstrating ADM formation  or after TGFα treatment  or TGFα  vs. 0 h; p < 0.01; G12D increased protein levels for both, H3K9 methyltransferases EHMT2 and EHMT1, peaking at 48 h , and hTERT-HPNE E6/E7 KRASG12D (HPNE-KRASG12D), we observed a similar increase in the EHMT2-EHMT1-WIZ complex along with di-methylation of its H3K9 substrate. Congruent with the data described above, HPNE-KRASG12D cells displayed higher protein levels of EHMT2, EHMT1, WIZ, and H3K9me2 compared to its HPNE counterpart  in wild-type and KrasG12D expressing mice to translate these effects to the in vivo situation. Pancreata were harvested from both, Pdx1-Cre;LSL-KrasG12D and Cre/+;LSL-KrasP48G12D mouse models. Upon quantification of positively stained nuclei compared to total nuclei, we found that Pdx1-Cre;LSL-KrasG12D animals had higher levels of H3K9me2 compared to control Pdx1-Cre mice  in fl/flPdx1-Cre;EHMT2, 790 DEGs  in Pdx1-Cre;LSL-KrasG12D and 878 DEGs  in Pdx1-Cre;LSL-KrasG12D;EHMT2fl/fl, normalized to Pdx1-Cre mice, with an absolute FC ≥ | 1.5| and FDR < 0.1  revealed four major expression patterns  were upregulated in EHMT2fl/fl and KrasG12D;EHMT2fl/fl mice, while 647 genes characterized cluster 2 (yellow) with genes downregulated upon EHMT2 inactivation, either alone or with KrasG12D. Cluster 3 (pink) displayed 65 genes upregulated in KrasG12D animals, and Cluster 4 (purple) was defined by 82 genes downregulated in KrasG12D animals, independent of EHMT2 status. Notably, ADM and PanIN-related mRNA markers, such as Gkn1, Gkn2, and Muc5a, were downregulated in Pdx1-Cre;LSL-KrasG12D;EHMT2fl/fl animals  and immunoregulatory/inflammation-related pathways , . Additional gene network enrichment and visualization analyses using semantic-based algorithms  , which often also result in senescence induction . These genes were downregulated by KrasG12D alone but markedly upregulated in both EHMT2fl/fl and KrasG12D;EHMT2fl/fl mice  , remaini orange, . Genes wnduction , as welllication . These g purple, . Clustere green, . Thus, cion fork  and pharion fork . Notablyice red, . EHMT2flEHMT2 inactivation upregulates the Cdkn1a/p21-Chek2 pathway, which when participating in prolonged cell cycle arrest, leads to senescence , we found that EHMT2 inactivation, which upregulated Cdkn1a/p21 mRNA, also increased the levels of its encoded cell cycle regulator protein  leukocytes or histiocytes compared to only 33.3% of KrasG12D;EHMT2fl/fl animals  presented with rare infiltration, while these cells were absent in the majority of KrasG12D;EHMT2fl/fl animals (66.6%) with few mice demonstrating rare (16.7%) or focal (16.7%) infiltration  or substantial (40%) infiltration. However, in P48Cre/+;KrasG12D;EHMT2fl/fl animals, infiltration of these cells was mostly absent (18.2%) or rare (72.7%) with a minor portion of focal (9.1%). Additionally, we examined the degree of inflammation at foci of acinar tubular complexes and atypical flat lesions, which was categorized as absent, mild, moderate or severe. All of the Pdx1-Cre;KrasG12D animals showed moderate (40%) to severe (60%) inflammation, compared to 66.6% of the KrasG12D;EHMT2fl/fl animals with only mild inflammation and few with moderate (16.7%) to severe (16.7%) inflammation  to severe (80%) inflammation, whereas those carrying inactivating EHMT2fl/fl alleles most commonly demonstrated mild inflammation (72.7%) with only 18.2% scored as moderate , while KrasG12D;EHMT2fl/fl animals had a lymphoplasmacytic predominant cell type (50% for Pdx1-Cre and 45% for P48Cre/+), rather than neutrophilic or mixed  when compared to fl/flPdx1-Cre;EHMT2  and Pdx1-Cre;LSL-KrasG12D;EHMT2fl/fl . Histopathology showed extensive and dysplastic PanIN lesions with caerulein treatment in KrasG12D animals, while pancreatic tissues from KrasG12D mice with EHMT2 inactivation displayed more limited dysplasia . We conclude that EHMT2 deletion antagonizes KrasG12D-mediated cell growth even after enhanced stimulation by pancreatitis.Prolonged or chronic inflammatory conditions promote the initiation and development of neoplastic and fibrotic events, serving as a major risk factor leading to PDAC . Pancreaysplasia . MeasureKrasG12D, and KrasG12D;EHMT2fl/fl animals and compared them with untreated KrasG12D mice as controls . The upregulated subset included not only 6 distinct AP-1 transcription factors, which participate in growth, but also 19 different collagen genes and 11 chemokine ligands . Of these, 2839 DEGs were shared with caerulein-treated KrasG12D mice, but 773 DEGs were unique to EHMT2 inactivation , depicted as a heatmap in KrasG12D mice regardless of EHMT2 status , which is congruent with caerulein signaling via the Gq pathway , H2-Ob (human gene HLA-DOB), H2-DMb2 (human gene HLA-DMB), Cd2, Cd96, Cd28, Cd8a, Ccl5, Lck, and Zap70 , congruent with lymphocyte infiltration of T-cell origin. Finally, cluster 3 contained genes downregulated in KrasG12D animals carrying EHMT2 inactivation , TNF-α signaling via NFκB , as well as genes upregulated in response to KRAS signaling  among others  , NK cells, myeloid dendritic cells, monocytes and M2 macrophages  that was not detected in their corresponding untreated cohort  . Notablynditions , were norophages . Surpristrophils . This reesponses . ImmunohEHMT2 deficiency impairs the progression of PanIN lesions and prolongs survival of P48Cre/+KrasG12D mice  can be found below: GEO with dataset number GSE169525.Animal care and all protocols were reviewed and approved by the Institutional Animal Care and Use Committees of Mayo Clinic Rochester (IACUC protocols A00002240-16 and A24815) and the Medical College of Wisconsin (AUA00005963).RU and GL conceived and designed the study. GU, TA, AM, and AS conducted the experiments, acquired and analyzed the data. GU, RK, AZ, TS, VA, and BP participated in formal analysis. TA assembled figures. GU, TA, AM, MBD, MZ, JI, RU, and GL contributed to data interpretation. TA, JI, RU, and GL wrote the original draft. GL supervised the study. All authors provided valuable intellectual input on experiments, as well as read, offered feedback and approved the manuscript.MBD is a co-founder and has ownership interests in Protein Foundry, LLC and Xlock Biosciences, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Head-up tilt test (TT) has been used for >50 years to study heart rate/blood pressure adaptation to positional changes, to model responses to haemorrhage, to assess orthostatic hypotension, and to evaluate haemodynamic and neuroendocrine responses in congestive heart failure, autonomic dysfunction, and hypertension. During these studies, some subjects experienced syncope due to vasovagal reflex. As a result, tilt testing was incorporated into clinical assessment of syncope when the origin was unknown. Subsequently, clinical experience supports the diagnostic value of TT. This is highlighted in evidence-based professional practice guidelines, which provide advice for TT methodology and interpretation, while concurrently identifying its limitations. Thus, TT remains a valuable clinical asset, one that has added importantly to the appreciation of pathophysiology of syncope/collapse and, thereby, has improved care of syncopal patients.  During these studies, some subjects experienced total or near-total transient loss of consciousness (TLOC) due to hypotension induced by TT (often accompanied by bradycardia/asystole)., A positive TT has diagnostic value in syncope/collapse when the history does not provide a conclusive explanation for symptoms.The methodology and interpretation of TT results have evolved since it was introduced into clinical practice.et al. Regarding TT for evaluating the syncope of unknown origin, Forleo Given the preponderance of evidence, and working independently (a few members of each group provided reviews of the other document), the European Society of Cardiology (ESC)et al. , Recent criticisms by Kulkarni Tilt-table testing was introduced into clinical evaluation of TLOC of unknown aetiology to assess susceptibility to vasovagal reflex. Such testing is unnecessary for diagnosis if medical history is classical and diagnostic of reflex syncope. However, that is often not the case, especially in older patients in whom the history may be inadequate due in part to retrograde amnesia in these older fainters.TLOC has four features that can be derived from history taking: (i) tendency to fall as expression of loss of motor control; (ii) amnesia for duration of TLOC; (iii) abnormal responses to speech/touch; and (iv) short duration (<5 min).TLOC differential diagnosis includes: (i) concussion, (ii) syncope; (iii) epileptic seizures; (iv) psychogenic spells resembling syncope [psychogenic pseudosyncope (PPS)] or seizures [psychogenic non-epileptic seizures (PNES)]; and (v) intoxication/metabolic disturbance (strictly not TLOC as duration is >5 min).Distinction between these diagnostic entities by careful medical history including eyewitness reports is often but not always possible., , In some patients with recurrent apparent syncope, in whom previous attempts have failed to establish a diagnosis, TT is the best next step and guidelines support this strategy.Figure 1). Thus, TT can play an important role in VVS diagnosis but much less in therapy selection., , , Multiple observations suggest that reported syncope/collapse associated with positive TT is comparable with spontaneous vasovagal syncope (VVS), although it should be accepted that tilt-induced syncope is not identical to the spontaneous attack. For example, the bradyarrhythmias seen on implantable loop recorders (ILR) are more prominent than during TT.et al.,et al. The Fainting Assessment Study (FAST),Finally, clinicians caring for syncope/collapse victims realize that in patients of all ages, recurrent unexplained syncope/collapse may provoke considerable anxiety in those affected and their families. In the case of faints due to VVS/OH/PPS, the patient's understanding that the physician or highly trained assistant, , Detailed discussion of TT protocols is provided in several practice guidelines and consensus reports., The 2018 ESC syncope guidelines, Increasingly, laboratories that undertake TT are encouraged to include active stand testing in assessing patients.Moving from supine to upright posture rarely induces syncope in normal healthy patients but may cause minor worrisome symptoms. For instance, a transient sensation of ‘greying out/dizziness/light-headedness/unsteadiness’ is common immediately after upright postural change . While usually harmless, this sensation may cause alarm/instability in some patients.Delayed OH is a far more important clinical problem, especially in older patients, debilitated patients, those with neurogenic OH, or in diseases that affect neurological responses, such as diabetes or alcohol abuse. In these cases, OH may be considerably delayed after change of posture that can result in fall injury. Active standing may not be tolerated for sufficient time to be diagnostic with additional fall injury risk during testing. TT avoids injury risk while providing the possibility of defining the diagnosis.Tilt testing has added greatly to our understanding of syncope mechanisms and different collapse patterns,et al.’s, , Since its clinical introduction, the utility of TT has not been without criticism, as discussed above in light of Kulkarni , , Mortality is not an issue in most patients who undergo TT. The typical TT candidate is in a low-to-intermediate risk category, in whom a diagnosis is needed but has not been revealed at initial assessment by clinical history, physical examination including orthostatic blood pressure measurement and 12-lead ECG.Tilt-test reproducibility and estimated specificity and sensitivity are summarised in the recent European guidelines., False-positive outcomes occur with TT just as with any medical test, with a rate, expressed in terms of specificity and sensitivity,, , Tilt testing to monitor the effects of therapy is not recommended by European, Although TT may not be necessary to secure a diagnosis, it can serve to teach patients about prodromes so they can learn to invoke preventive measures, notably physical counter-measures, to abort subsequent episodes., , , , In the case of treatment selection, pacing using the closed-loop system offers stimulation earlier in the vasovagal reflex than awaiting bradycardia and, thus, a potentially effective therapy requiring consideration.Table 1).To summarize, TT has many pros in terms of its diagnostic, educational, patient reassurance and choice of pacing therapy with few cons other than being a lengthy procedure; importantly, it has virtually no risks. In contrast, active standing is really valuable only in immediate/classical OH. In delayed OH, it cannot replace tilt on grounds of haemodynamics and tolerability , brain imaging, and EEG. European and North American guidelines are clear about this aspect of syncope investigation in recommending TT and advising against less effective, more expensive tests., Increasingly sophisticated, easy-to-place ILR/ICMs enhance diagnostic capacity but, even when chosen with care according to guideline recommendations,Tilt testing is a useful and necessary diagnostic tool. Practice guidelines endorse its value based on published and strongly vetted evidence. TT adds importantly to our ability to appreciate the pathophysiology of syncope/collapse and improves care of our patients. Neither active standing nor ILR/ICMs can replace TT; active standing is valuable in some forms of OH but to date has no demonstrated value for other syncope presentations, while ILR/ICMs complement syncope workup.Conflict of interest: R.S. is a consultant to Medtronic Inc., a member of speakers' bureau Abbott Laboratories Inc., and a stockholder in Edwards Lifesciences Corp. and Boston Scientific Inc. D.G.B. is supported in part by grant from the Dr Earl E Bakken Family in support of Heart-Brain research and reports consultant fees from Medtronic Inc. and Abbott Laboratories (SJM). A.F. reports personal fees from Biotronik Inc. and Medtronic Inc. H.A. is supported in part by research grants from Boston Scientific Inc., Medtronic Inc., and Abbott Laboratories. J.M.S. has funding from NHLBI Grant RO1HL134674. R.D.T. receives research support from Medtronic, consultancy fees from Theravance Biopharma, and lectures fees from Medtronic, Union Chimique Belge, and Novartis. B.O. reports personal fees from Amarin, Boehringer Ingelheim, Sanofi Aventis, Respicardia, and Lundbeck. J.G.v.D., M.B., F.d.L., P.B.L., R.A.K., A.M., S.D.R., and V.R. declare no conflicts of interest.


This study aimed to investigate the effect of a-tDCS on a RPE-clamp test, a 250-kJ time trial (TT) and motor evoked potentials (MEP). Twenty participants volunteered for three trials; control, sham stimulation and a-tDCS. Transcranial magnetic stimulation was used to determine the corticospinal excitability for 12 participants pre and post sham stimulation and a-tDCS. The a-tDCS protocol consisted of 13 minutes of stimulation (2 mA) with the anode placed above the Cz. The RPE-clamp test consisted of 5 minutes ergometer bicycling at an RPE of 13 on the Borg scale, and the TT consisted of a 250 kJ (∼10 km) long bicycle ergometer test. During each test, power output, heart rate and oxygen consumption was measured, while RPE was evaluated. MEPs increased significantly by 36% (±36%) post a-tDCS, with 8.8% (±31%) post sham stimulation (p = 0.037). No significant changes were found for any parameter at the RPE-clamp or TT. The lack of improvement may be due to RPE being more controlled by afferent feedback during TT tests than during TTF tests. Based on the results of the present study, it is concluded that a-tDCS applied over Cz, does not enhance self-paced cycling performance.Anodal transcranial direct current stimulation (a-tDCS) has been shown to improve bicycle time to fatigue (TTF) tasks at 70–80% of VO Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique, applied as a constant low-intensity current 1–2 mA) on the scalp through saline-saturated sponge electrodes [–2 mA on 2max [Recently, a-tDCS has been investigated as a potential ergogenic aid in sports performance. For instance, in isometric time-to-fatigue (TTF) tasks, a-tDCS has been shown to improve performance of healthy participants in both the upper –6 and lo2max –10 as we2max . In thes2max ,9,11,12.2max . It has 2max . Another2max ,11.Despite the performance improvements observed in TTF-tasks, it should be noted that this type of performance task may not adequately reflect the demands of real-world sports competition such as time trials (TT), where the primary aim is to complete a given distance in the shortest amount of time ,15. To tTo achieve the shortest time possible in competition, such as in a TT, athletes rely on pacing strategies . A pacinThe aim of the present study was therefore to determine the effects of a-tDCS applied over M1 on corticospinal excitability and self-paced cycling performance during a submaximal RPE-clamp test and a TT. Based on previous findings of a-tDCS induced downregulation of RPE during TTF-tasks ,9,11,12,The study was approved by the local Ethics Committee (VN 20170081) and carried out in accordance with the Declaration of Helsinki. Twenty-two healthy recreationally trained participants were recruited from February to April, 2018, and volunteered for this sham-controlled study. Two participants were excluded due to injuries obtained unrelated to the study. The remaining participants were all included in the final analysis and consisted of 16 males and four females that on average (± standard deviation) were: 26 (±4) years, 181.5 (±9.9) cm and 83.9 (±17.9) kg. Participants were excluded if they had any physical or neurological disorders that could affect their performance. All participants were given a detailed verbal and written explanation of the experimental risks before providing a written informed consent. Twelve of the participants also volunteered to receive transcranial magnetic stimulation (TMS) to assess changes in corticospinal excitability through recording of motor evoked potentials. These participants were screened prior to participation using the Transcranial Magnetic Stimulation Adult Safety Screen (TASS) .In the present study, participants completed one familiarization session, and three test sessions . The thrFor the 12 participants receiving TMS, the a-tDCS and sham test days started with surface electromyography (sEMG) electrodes being mounted on rectus femoris (RF) on the right leg. First, the skin was shaved, abraded, and cleaned with alcohol, before two surface electromyography electrodes  were mounted parallel to the muscle fiber direction according to the SENIAM guidelines. A reference electrode was mounted between the two recording electrodes. A Magstim stimulator  was used to determine the hotspot, resting motor threshold (RMT), and MEP amplitude of the RF. To determine the hotspot, three consecutive stimuli over Cz were delivered where after the coil was moved in ~1cm steps laterally, until the highest and most consistent MEPs were elicited. This position was marked on the scalp with a felt pen. Subsequently, RMT was identified, and defined as the minimal stimulus intensity at which 5 out of 10 consecutive stimuli evoked a MEP with an amplitude of at least 50 μV in the resting muscle Twenty MEPs were then elicited at 120% RMT stimulus intensity. MEP´s were recorded at two time points. Firstly, prior to the a-tDCS protocol and secondly five minutes post the stimulation protocol.A battery driven, constant current stimulator  delivered a-tDCS through two custom-made plate electrodes. Thin physiological saline saturated sponges were placed between the electrode and the skin. Impedance was kept below 5 kΩ. For the twelve participants who had received TMS, the anodal electrode (9 x 4 cm) was placed on top of the previously located hotspot of the RF muscle. For the remaining the participants, the anodal electrode was placed on top of Cz, as the electrode would then be certain to cover the hotspot of RF on both hemispheres, due to its size (9 x 4cm) . The catn being revolutions per minute and α being the resistance. Sampling frequency was 50 Hz. Before each cycling test, participants had a three-minute self-paced warm-up with incremental resistance, on the ergometry bike. Three blocks of one minute was performed with an α resistance of 0.025, 0.034 and 0.042. The warm-up led directly to a five-minute RPE-clamp cycling test at a self-selected α resistance. The RPE-clamp test was used to evaluate the influence of a-tDCS on RPE during self-paced cycling, by comparing the PO between conditions. During the RPE-clamp test, participants were instructed to maintain a constant RPE of 13. Participants were given visual feedback on time spent and brake resistance during the test. Following the RPE-clamp test, participants performed a 250-kJ TT, previously estimated to reflect a distance of ∼10 km [The Lode ergometry bike was set to linear mode . The power output (PO) was measured in Watt, and calculated through the formulaf ∼10 km . Particif ∼10 km . Partici2 and 5% CO2). The software used to run the Jaeger Vyntus CPX  automatically transformed the data into standard temperature (0°C) and pressure (760 Torr) [All of the physiological measurements were collected throughout the warm-up, RPE-clamp test, and TT. Heart rate (HR) was recorded by a HR monitor . Pulmonary gas exchange was measured breath-by-breath using a computerized metabolic measurement system  connected to a face-mask with a digital volume transducer. The system was calibrated prior to each test using standardized gas concentrations (15% O60 Torr) .Rating of perceived exertion was measured using a 15-grade Borg 6–20 scale [A custom made EMG amplifier  and a custom made LabVIEW©-based computer program  was used to collect EMG signals at sampling rate of 5 KHz, with a sensitivity of 500 mV/V and using a 5–1000 Hz band pass filter.2 (ml/min) signals were synchronized and averaged over a 1-minute epoch between the third and fourth minute of the RPE-clamp and for every 25-kJ completed of the TT, to coincide with RPE rating. Further, a ratio between PO/RPE was calculated for every 25-kJ epoch of the TT.The size of the MEP response in each participant was defined as the average peak-to-peak value of the 20 MEPs recorded in either the a-tDCS or Sham stimulation session. The relative difference between pre and post stimulation MEP amplitude was calculated in percent for each participant and each session. PO (W), HR (beats/min) and VO2, respectively. A one-way ANOVA was used to test for statistical differences between the a-tDCS, sham, and control conditions for time to complete the TT, as well as for average PO, average HR, average VO2, average RPE, and average PO/RPE ratio. A two-way repeated measures ANOVA (condition × time) was used to test for statistical differences in RPE, PO, and PO/RPEratio for each 25kJ interval of the TT. Post hoc testing was carried out using a Tukey’s post hoc test. With the exception of HR data from the RPE-clamp test, all data were found to be normal distributed using a Shapiro-Wilks test and visual inspection of histograms and QQ-plots. Due to the robustness of the ANOVA test, an ANOVA was used, even when data was not normally distributed. Sphericity of data was tested using the Mauchly’s test. If the assumption of sphericity was violated, the degrees of freedom was corrected using the Greenhouse-Geisser corrections (G-Gcor). Data are presented as mean ± standard deviation. Statistical significance was accepted at p≤0.05. All calculations were performed in SPSS Version 25.0 .To test for changes in MEP following either a-tDCS or sham stimulation a paired t-test was performed, comparing the magnitude of relative change of MEPs between conditions. For the RPE-clamp test, a one-way repeated measures ANOVA was used to test for statistical differences between the a-tDCS, sham, and control conditions for PO, HR, and VOThe relative increase in MEPs was significantly larger following a-tDCS  compared to sham stimulation  (p = 0.037) .2  = 1.09, p = 0.34  = 0.828, p = 0.44), HR  = 0.63, p = 0.54), or VOp = 0.34 .2  = 1.40, p = 0.25), RPE  = 0.441, p = 0.64), or Power/RPE-ratio  = 2.63, p = 0.08) of the TT  = 1.89, p = 0.16), PO  = 2.05, p = 0.14), HR  = 0.654, p = 0.52), VOf the TT .Similarly, the two-way repeated measures ANOVA found no significant differences between conditions for the 25kJ intervals with regards to RPE  = 0.60, p = 0.77) or PO/RPE ratio  = 1.75, p = 0.13). However, a main effect of time was found for PO  = 49.96, p = 0.01), with a post hoc showing a higher PO for the interval between 200–225 kJ (238 ± 65 watt) compared to each 25kJ interval between 0–175 kJ (p<0.01) and a higher PO for the 25 kJ interval between 225–250 kJ (292 ± 78 watt) compared to all of the previous intervals (p = 0.01) .2 during the RPE-clamp test or the TT.The aim of the present study was to determine the effects of a-tDCS on corticospinal excitability of M1 and self-paced cycling performance during a submaximal RPE-clamp test and a TT. The main finding was a significant increase in corticospinal excitability following a-tDCS. However, this did not lead to any significant differences in PO, HR, or VO2+ channels through subthreshold depolarization has been shown in a study performed on rats [2+. However, this remains hypothetical, as the present study did not examine these mechanisms directly.Motor evoked potentials increased significantly following a-tDCS, but not after sham stimulation. The 36% increase in MEPs is in line with findings from previous studies . The mec on rats . The incThe results of the present study showed that neither of the parameters during the RPE-clamp test and the TT were altered following a-tDCS, despite corticospinal excitability being significantly increased. As RPE has been proposed as a key regulator of pacing during self-paced exercise, a-tDCS was expected to improve performance, as it previously has been shown to downregulate RPE, allowing more work to be produced at the same RPE .In Borg’s model of perceived exertion, both an efferent and afferent component of RPE are acknowledged  as the RSidhu et al. showed using a pharmalogical approach that feedback from group III/IV muscle afferents decreases motor cortex excitability [The lack of improvements in the TT despite increased corticospinal excitability could be explained by the peripheral governor model by MacIntosh and Shahi . In thistability . Such evtability  that cenA significantly higher PO was found in the last two epochs of the TT. This is most likely due to the participants performing a final sprint in an attempt to maximize the result of the test . There w2 responses during the RPE-clamp test and TT did not differ between stimulation conditions. This result is in line with previous studies showing no alterations in cardiovascular or autonomic responses following a-tDCS stimulation of M1 [2 consumption following a-tDCS compared to sham stimulation and the control session, this may either indicate that increased excitability of M1 does not influence VO2 kinetics directly or that increased excitability of M1 does not influence RPE and power output, and thus no difference is observed in VO2. It should be noted however, that several phenomena described in the literature indicates that the central nervous system (CNS) may also serve as a limiting factor, as limitation by peripheral anaerobiosis can not explain that: 1) Less than 100% of muscle fibers are recruited at the end of a task [In the present study the HR and VOon of M1 ,12. Howeon of M1 . As the on of M1 ,9,12, fof a task , 2) intef a task , 3) RPE f a task , and 4) f a task . This cof a task . The stuThe coefficient of variation for the PO in the TT in the present study was 3.76%, ranging from 0.29–10.56%. However, performance increases has been shown to be less in TT-tasks, compared to TTF-tasks, as Amann et al.  found hyAt the RPE-clamp test, participants were instructed to ride at a constant RPE of 13 on the Borg scale. An RPE of 13 would approximately match a HR of 130 BPM for participants aged 30–50 years . ParticiThe data of the present study is based on 16 male and four female subjects, which results in a clear gender imbalance. This imbalance could potentially influence the results of the study. Post hoc analysis in to this matter, however, revealed no such influence. Furthermore, previous studies have not found any gender differences in male and female responses to a-tDCS .2, or RPE.In conclusion, a-tDCS administrated over Cz significantly increased corticospinal excitability in the present study. However, this did not lead to a significant increase in power output in the RPE-clamp test, nor did it cause an increase in power output and a decrease in time to completion of the TT. The increased corticospinal excitability observed in the a-tDCS session did not lead to any differences between tDCS, sham and control sessions in terms of PO, HR, VOThe use on non-invasive brain stimulation as an ergogenic aid in sports performance contexts are quickly becoming popular. While the literature has indeed shown positive effects of this type of stimulation, the performance tests most often utilized, such as time to fatigue tests, are not representative of the demands of real-world sports competitions such as time trials. Our results indicate that a-tDCS, can significantly increase corticospinal excitability as compared to a sham stimulation protocol. This increase in corticospinal excitability, however, have no effect on the performance during a self-paced 10 km cycling time trial or on cycling performance during a submaximal RPE-clamp test. Therefore, although a-tDCS is capable of inducing changes in corticospinal excitability, our results clearly indicate that this does not influence cycling performance in any meaningful way.


Contrast-enhanced magnetic resonance imaging (MRI) has the potential to replace angiographic evaluation of atherosclerosis. While studies have investigated contrast agent (CA) uptake in atherosclerotic plaques, exact CA spatial distribution on a microscale is elusive. The purpose of this study was to investigate the microdistribution of gadolinium (Gd)- and iron (Fe) oxide-based CA in atherosclerotic plaques of New Zealand White rabbits.post hoc analysis of archived tissue specimens obtained in a previous in vivo MRI study conducted to investigate signal changes induced by very small superparamagnetic iron oxide nanoparticles (VSOP) and Gd-BOPTA. For analytical discrimination from endogenous Fe, VSOP were doped with europium (Eu) resulting in Eu-VSOP. Formalin-fixed arterial specimens were cut into 5-μm serial sections and analyzed by immunohistochemistry  staining, anti-smooth muscle cell actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining) and elemental microscopy with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation μX-ray fluorescence (SR-μXRF) spectroscopy. Elemental distribution maps of Fe, Eu, Gd, sulfur (S), phosphorus (P), and calcium (Ca) were investigated.The study was performed as a IHC characterized atherosclerotic plaque pathomorphology. Elemental microscopy showed S distribution to match the anatomy of arterial vessel wall layers, while P distribution corresponded well with cellular areas. LA-ICP-MS revealed Gd and Fe with a limit of detection of ~ 0.1 nmol/g and ~ 100 nmol/g, respectively. Eu-positive signal identified VSOP presence in the vessel wall and allowed the comparison of Eu-VSOP and endogenous Fe distribution in tissue sections. Extracellular matrix material correlated with Eu signal intensity, Fe concentration, and maximum Gd concentration. Eu-VSOP were confined to endothelium in early lesions but accumulated in cellular areas in advanced plaques. Gd distribution was homogeneous in healthy arteries but inhomogeneous in early and advanced plaques. SR-μXRF scans at 0.5 μm resolution revealed Gd hotspots with increased P and Ca concentrations at the intimomedial interface, and a size distribution ranging from a few micrometers to submicrometers.Eu-VSOP and Gd have distinct spatial distributions in atherosclerotic plaques. While Eu-VSOP distribution is more cell-associated and might be used to monitor atherosclerotic plaque progression, Gd distribution indicates arterial calcification and might help in characterizing plaque vulnerability.The online version contains supplementary material available at 10.1007/s11307-020-01563-z. Atherosclerosis is the major promoter of cardiovascular disease with clinical manifestations including myocardial infarction, ischemic stroke, and sudden death . In clinImaging methods including MRI, CT, positron emission tomography (PET), catheter-based virtual histology intravascular ultrasound (VH-IVUS), optical coherence tomography (OCT), and near-infrared spectroscopy (NIRS) are currently being investigated regarding their value for characterizing morphological changes leading to vulnerable plaque –7. AmongIONP are extensively used in MR imaging of experimental atherosclerosis particularly for detection of angiogenesis, inflammation, and apoptosis , 10. IONvia the kidneys, studies have demonstrated Gd deposition predominantly around vascularized areas of the skin, brain, liver, and kidney [GBCA are known as nonspecific agents that result in increased MRI signal intensity through pronounced perfusion and vascular permeability. Although GBCA were developed to distribute within the vascular and extracellular space, and to be eliminated as intact molecules d kidney , 18. Sysd kidney –21. Althd kidney .Discrimination between stable and vulnerable atherosclerotic plaques by contrast-enhanced MRI entails better understanding of IONP or GBCA interactions with plaque ECM components. Thus, elucidating microdistribution of CA in atherosclerotic plaques at different stages of disease progression by an experimental workflow with improved tissue detection ability offers a sophisticated basis. In this regard, mass spectrometry and X-ray fluorescence (XRF) analysis have emerged as two cutting-edge elemental microscopy techniques offering the lowest limit of detection (LOD) for reliable quantification of elements in tissue specimens and imaging characterization at the highest spatial resolution, respectively , 23.In this study, we aimed to investigate the microdistribution of Eu-VSOP and Gd-BOPTA (gadobenate dimeglumine) in atherosclerotic plaques of New Zealand White (NZW) rabbits using immunohistochemistry (IHC) and elemental microscopy by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and synchrotron radiation μXRF (SR-μXRF) spectroscopy.The study was conducted in accordance with the requirements of directive 2010/63/EU and the German Animal Protection Act and approved by the local animal protection committee of the Landesamt für Gesundheit und Soziales . Experimental conditions were constant at all times  for IHC and LA-ICP-MS, or on ultralene foil for SR-μXRF analysis. Movat’s pentachrome, von Kossa, and Alcian blue pH 1.0) histologic staining, anti-smooth muscle cell (SMC) actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining procedures were carried out on adjacent sections as described elsewhere . Qualita.0 histol−1 and 200 ms acquisition time with daily-optimized output energies of 1.5 J cm−1. The method has been described elsewhere [40Ar16O with the most abundant isotope at mass 56 [LA-ICP-MS analysis was performed on the sections adjacent to IHC sections. For that, an ICP-MS  was coupled to a laser ablation system . Prior to analysis, sections were deparaffinized. Laser ablation was performed in continuous-line ablation mode with a circular laser spot size of 20 μm at a scanning speed of 100 μm slsewhere . Ablated mass 56 . Gd and  mass 56 .2  using a fixed-curvature Kirkpatrick-Baez mirror system. Flux was ~ 5 × 1010 photons/s (~ 180 mA SR current in multibunch mode). Acquisition time per pixel was 100 ms. Pixel size for collecting the XRF maps was set to 30 μm, 10 μm, 1–2 μm, or 0.5 μm depending on the size of the region of interest (ROI). Scans were acquired in continuous mode. Distribution maps of Fe, Gd, sulfur (S), P, and Ca were generated. XRF normalization, spectral deconvolution, and quantification were done using the PyMCA software  networks. Synthetic SMCs and stretches of yellow/green-stained collagen fibrils were observed covering the lipid pools. von Kossa staining revealed intimal calcifications, especially along the intimomedial interface of advanced plaques  with a LOD of ~ 0.1 nmol/g for Gd, and ~ 100 nmol/g for Fe. No Eu signal was detected in controls , Gd was detected Fig. . In healSR-μXRF analysis at 30-μm-step width detected Gd in 4 samples—one early lesion and 3 advanced plaques. Predefined ROIs within these specimens were further investigated for their Gd content at higher resolution down to 0.5 μm, which displayed increasing Gd concentrations Fig. . In compThe aim of this study was to investigate the microdistribution of Gd- and iron oxide-based CA in the atherosclerotic plaques of NZW rabbits after IV injection of Eu-VSOP and Gd-BOPTA. VSOP are increasingly being investigated in MR imaging of experimental atherosclerosis owing to their early uptake (< 2 h) into atherosclerotic plaques that correlates with accumulation of the ECM rather than phagocytosis , 16. ECMPublished data suggest that VSOP enter atherosclerotic plaques through the endothelium or macrophage phagocytosis . In endo. Remarkably though, compared to healthy arteries, maximum Gd concentrations were 4 and 21 times higher in early and advanced plaques, respectively. This indicates focal Gd hotspots. Advanced plaques were characterized by a large number of such hotspots in deeper regions of the intima corresponding to the intimomedial interface, which hosts large lipid pools and macrophages


Background: Carotid artery atherosclerosis is a major risk factor for ischemic stroke. This risk is related to plaque vulnerability and is characterized by plaque morphology, intraplaque neovascularization, and cerebral microembolization. Advanced neurosonology can identify vulnerable plaques and aid in preventing subsequent stroke. We aimed to assess the time course of cerebral microembolization and intraplaque neovascularization during 6 months of follow-up and to explore the utility of advanced neurosonology in patients with acute cerebral ischemia.Methods: Fifteen patients with acute cerebral ischemia and carotid artery plaques underwent comprehensive extra- and intracranial ultrasound examinations, including microemboli detection and contrast-enhanced ultrasound. The examinations were repeated after 3 and 6 months.Results: We examined 28 plaques in 15 patients. The ultrasonographic features of plaque vulnerability were frequent in symptomatic and asymptomatic plaques. There were no significant differences in stenosis degree, plaque composition, plaque surface, neovascularization, or cerebral microembolization between symptomatic and asymptomatic plaques, but symptomatic plaques had a higher number of vulnerable features. None of the patients had recurrent clinical stroke or transient ischemic attack during the follow-up period. We observed a decrease in cerebral microembolization at 6 months, but no significant change in intraplaque neovascularization.Conclusions: In patients with acute cerebral ischemia and carotid artery plaques, cerebral microembolization decreased during 6 months of follow-up, indicating plaque stabilization.Clinical Trial Registration:ClinicalTrial.gov, identifier NCT02759653. The non-contrast examinations were performed as described in the NOR-SYS protocol  approved the study, and written informed consent was obtained from all participants. We included 15 non-consecutive patients admitted between August 2016 and November 2017 with acute cerebral ischemia and carotid artery atherosclerosis detected on routine duplex ultrasound. Both symptomatic and asymptomatic carotid plaques were included. A carotid artery plaque was defined as symptomatic if it was related to a recent clinical event such as an acute cerebral infarction or a transient ischemic attack (TIA). All patients underwent cerebral computed tomography (CT) and computed tomography angiography (CTA) of the pre- and intracerebral vessels on admission and cerebral magnetic resonance imaging (MRI) on the same or next day. We used a standardized questionnaire from The Norwegian Stroke in the Young Study (NOR-SYS) adapted to our study that comprised medical history, vascular risk factors, and current medication  was used for statistical analyses. Continuous variables are presented as median (interquartile range) and categorical variables as numbers (percentages). Univariate logistic regression analyses were performed to evaluate whether ultrasonographic features of plaque vulnerability could predict symptomatic plaques at baseline and explore potential associations between the different plaque features. For these analyses, the vulnerable plaque features were dichotomized as previously described. Linear regression was applied to determine whether the number of vulnerable plaque features differed between symptomatic and asymptomatic plaques. We used clustering to adjust for dependency between different plaques in the same patient. To investigate changes in IPN and MES occurrence during follow-up, we fitted a multilevel mixed-effects logistic regression model and adjusted for dependency between repeated measurements in the same plaque and between different plaques in the same patient. In this model, the degree of IPN was dichotomized to none/limited or moderate/extensive in accordance with previous studies , 26. Ope2. The median number of vascular risk factors was 3 (interquartile range: 2–5). Eleven patients had MRI-verified acute cerebral infarctions with corresponding carotid artery plaques. Among the remaining four patients, two had clinical TIA from the anterior circulation with corresponding carotid plaques, one had a branch retinal artery occlusion with corresponding carotid plaque, and one had cerebral infarctions in the posterior circulation only without a fetal origin of the posterior cerebral arteries. None of the acute cerebral infarctions had a lacunar appearance on MRI. Atrial fibrillation or other competing sources of emboli were not detected in any of the patients. Thus, acute cerebral infarctions were considered to be related to the corresponding carotid plaques.Fifteen patients (age range: 44–70 years) were included. Of these, 13 had bilateral carotid plaques; therefore, 28 plaques were examined in total. Patient characteristics at baseline are presented in p = 0.04). We found no significant association between the degree of IPN and the occurrence of corresponding MES. The only total occlusion was an acute thrombotic occlusion at the site of a highly neovascularized atherosclerotic plaque with severe luminal narrowing. Repeated CEUS examinations revealed a minimal recanalization that remained non-visualized on CTA.Features associated with plaque vulnerability were frequently found in our study. Twenty-two (78.6%) plaques had iso-/hypoechoic or mixed composition, and 19 (67.9%) had an irregular or ulcerated surface. IPN was found in 19 (67.9%) plaques, and 10 (35.7%) of the plaques had MES in the corresponding vascular territory. The interrater reliability for MES evaluation was substantial, with a kappa score (AF and SJA) of 0.8. Degree of stenosis, plaque composition, plaque surface, degree of IPN, and presence of MES were not significantly different between symptomatic and asymptomatic plaques, but symptomatic plaques had a higher number of vulnerable features , but the degree of IPN was not significantly different. Surgically removed plaques were excluded from the follow-up analyses.None of the patients reported recurrent clinical stroke or TIA during the follow-up. Further, there were no other acute cardiovascular events in the same period. One patient died of an unknown cause before the 6-months follow-up visit, while the remaining 14 completed the follow-up period according to the study protocol. Six patients underwent unilateral carotid endarterectomy of a symptomatic carotid artery plaque, while the remaining patients received medical treatment. Changes in the degree of IPN and MES occurrence during follow-up are presented in Ultrasonographic features of plaque vulnerability were frequent in both symptomatic and asymptomatic carotid artery plaques. The frequencies of plaque echolucency, IPN, and MES occurrence were in the same range as reported in other studies , 27, 28.We found a significant association between an increasing number of vulnerable plaque features and symptomatic plaques. This finding is expected, as the individual features included are known to predict symptomatic disease , 6, 16. ®) as the contrast agent. We chose to use PFB microbubbles because PFB showed better contrast properties with higher signal intensity and more extended durability than sulfur hexafluoride, in our experience.We did not find an association between increased IPN and MES occurrence, likely because MES detection was done 3–5 days after admission and after the initiation of platelet inhibition. While changes to the degree of IPN are gradual, MES are most frequent in the early phase of acute stroke, and MES occurrence is, to a greater extent than IPN, affected by antithrombotic treatment , 30. OthWe observed a significant decrease in MES occurrence at 6 months. To our knowledge, no other study has examined the course of MES in the months after a stroke or TIA, but one study found a similar decrease in MES occurrence at 1 year compared to the baseline examination . This deHistopathological studies have found an inverse correlation between the elapsed time after an ischemic event and the degree of IPN, implying a decrease in IPN during plaque remodeling , 32. DurDue to the absence of recurrent ischemic events, it is not possible to determine the prognostic value of neurosonological surrogate markers of plaque vulnerability. However, our longitudinal data support the use of advanced neurosonology to observe plaque stabilization over time and to identify plaques that remain vulnerable and may need intensified medical treatment or surgical intervention. To our knowledge, this is the first study to assess the course of both cerebral microembolization and intraplaque neovascularization in the months following an ischemic event attributed to carotid artery disease. The main strengths of our study are the comprehensive cerebrovascular evaluation and the stringent follow-up with repeated standardized measurements. Our MES records were independently assessed by two experienced observers to increase data reproducibility, and the interrater reliability was substantial. However, the study also has some limitations. The sample size is small, reducing the possibility of subgroup analyses. Due to the small sample size and non-consecutive patient enrolment, the prevalence of vulnerable plaque features in our study may not be generalizable to all patients with carotid artery disease. Still, we believe that the temporal changes described in our study are generalizable to the target population. With respect to MES detection, it is possible that some cerebral microemboli may have originated from other sources such as atrial fibrillation, valvular heart disease, or aortic arch atherosclerosis. None of the patients had documented atrial fibrillation on Holter monitoring, but echocardiography and aortic imaging were not performed in all patients. Another limitation is the fact that MRI was not repeated during the follow-up period; hence, clinically silent recurrent infarctions may have been missed.This study adds to the current knowledge of the course of carotid plaque vulnerability in ischemic stroke. Our future aim is to include ultrasonographic features of plaque vulnerability in vascular risk evaluations to identify patients with high risk and a need for stringent follow-up.In patients with acute cerebral ischemia and carotid plaques, we observed a decrease in cerebral microembolization during 6 months of follow-up, indicating plaque stabilization over time. There were no significant changes in IPN during this period.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Regional Committee for Medical and Health Research Ethics of Western Norway (REC West). The patients/participants provided their written informed consent to participate in this study.AF and LT conceived and designed the study. AF, LT, UW-A, and HN contributed to the collection of data. SA and AF analyzed the data and drafted the manuscript. All authors contributed to the interpretation of the results, critically revised the manuscript, and approved the submitted version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients who undergo solid organ transplantation are at risk of opportunistic infection associated with immunosuppression. We report a case of confirmed donor derived visceral leishmaniasis (VL), in a patient following liver transplantation causing fever and pancytopenia. The diagnosis was confirmed by bone marrow biopsy, with confirmed positive donor serology, with no other route of transmission. To our knowledge, this is the first case report in the United Kingdom and Europe, of confirmed organ donor transmission of VL. This case report highlights an important consideration of donor derived infections, in the context of solid organ transplantation. Visceral leishmaniasis (VL) is parasitic infection common occurring in Asian, African and Central and South Americas. It often presents with fever, splenomegaly and pancytopenia. This clinical presentation is regularly observed in patients post liver transplantation. Common aetiologies for this include, graft rejection, immunosuppressive agents and opportunistic infections such as cytomegalovirus (CMV).This case describes donor derived leishmaniasis as a rare cause of fever and pancytopenia following liver transplantation in a patient from the UK. It highlights the importance to consider other less frequent causes of infection, including donor derived infections in those following solid organ transplantation.A 57-year-old man underwent orthoptic liver transplantation in March 2019 for alcoholic-related liver disease and portal hypertension, following 2 years of abstinence. The patient was born in Wales and his only travel history outside the UK was a transient visit to Calais, 15 year prior to his transplantation. He was a carpenter and did not work near the ports or places of travel in Wales.He required a 2-day intensive care admission following transplantation from a brainstem death donor. Immunosuppression was commenced with prednisolone, tacrolimus and mycophenolate (MMF) in conjunction with cotrimoxazole prophylaxis. Both the donor and recipient were CMV IgG negative, therefore, CMV prophylaxis was not required as per hospital policy.He developed an episode of biopsy-proven moderate acute cellular rejection requiring treatment with 3 days of pulsed steroids 1-month postliver transplant. His graft function gradually improved, with normalisation of alanine aminotransferase (ALT) coinciding with the development of pancytopenia 4 months post-transplantation. His MMF was reduced from 1 g two times a day to 500 mg two times a day in response to this.Despite this, the patient developed a worsening neutropenia and persistent pancytopenia, with the additional systemic symptoms of fever and rigours. He then was admitted to his local hospital with neutropenic sepsis and an acute kidney injury. He was transferred from his local hospital to our liver transplant centre for further investigation.He underwent a further series of investigations as summarised in Leishmania PCR test detected DNA from the Leishmania donovani complex. Furthermore, the patient’s Leishmania serology was strongly positive at a titre equal to or greater than 1 in 102 400 (a titre of 1 in 1600 or above is considered positive). His peripheral blood PCR for Leishmania was negative.A bone marrow biopsy was performed in view his pancytopenia and it showed amastigotes see . The LeiHe was given liposomal amphotericin (AmBisome) at a dosage of 4 mg/Kg for 21 days, with further doses at 24, 31 and 38 days to treat his leishmaniasis. Treatment of VL in immunocompromised patients is with Liposomal amphotericin B4 mg/kg daily on days 1 to 5, 10, 17, 21, 31 and 38, however, he received the longer regimen to maintain empirical antifungal cover. He underwent a repeat bone marrow biopsy following 6 days of treatment with AmBisome. This showed ongoing leishmanial infection, evidence of secondary haemophagocytosis and reduced myeloid activity.His fevers had settled and his pancytopenia slowly improved. He was later repatriated to his local hospital when his pancytopenia resolved. He was maintained on tacrolimus monotherapy immunosuppression for his liver transplant.He had not travelled abroad post operatively, therefore, reactivated or newly acquired leishmanial infection was extremely unlikely, however this was considered.The donor samples were investigated by National Health Service (NHS) Blood and Transplant to explore all routes of transmission. Leishmanial serology on serum from the liver donor was positive. Additionally, it transpired that the donor had travelled to India in the year preceding his death, and therefore the most likely transmission in this case is via the donor.Phlebotomus spp in the Old World and Lutzomyia spp in the New World).VL is caused by the Leishmania donovani complex: L. donovani in Asia and Africa; L. donovani infantum in the Mediterranean Basin, Africa, the Middle East, central Asia and China; and L. donovani chagasi (a synonym of L. donovani infantum) in Central and South America.VL is an obligate intracellular protozoal parasitic infection transmitted by sand flies  Donor infection via the graft which is extremely rare with no confirmed case reports in non-endemic countries, (2) Via blood transfusion which has been reported in endemic and non-endemic countries,The clinical presentation is characterised by persistent fluctuating fever, and splenomegaly. Other features include pancytopenia, hepatomegaly, weight loss, cough, diarrhoea and hypergammaglobulinaemia.9/L. Bone marrow aspiration is more commonly used and detects 85% of cases.VL is diagnosed through identifying amastigotes in tissue  or flagePCR should be performed in addition to microscopy on all samples. PCR on peripheral blood is useful for follow-up of VL in immunocompromised patients, but the yield from peripheral blood samples is too low for use in primary diagnosis in those with intact immunity.The drug of choice for VL is liposomal amphotericin B, with efficacy rates of >90%.Leishmania on retrospective evaluation and a lifetime travel history incompatible with de novo vector-transmitted or reactivated infection in the recipient. A recent case report from Brazil highlights a case of VL post liver transplantation via reinfection, from a patient in an area where VL is endemic.This case highlights donor-derived VL as a potential cause of pancytopenia after liver transplantation. We have performed over 5000 liver transplants in our centre and this is the first case of VL transmission via a donor. It is the first reported case in the literature of donor contracted Leishmaniasis in the setting of liver transplantation, as evidenced by positive donor serology for Due to the acute nature of organ donation, obtaining a full history, including travel can understandably prove difficult. Often this is carried out retrospectively, as and when required, from collateral sources. Storage of tissue and blood samples from organ donors is currently performed; they are stored for up to several years, but given the anonymity of donation, this is done by NHS Blood and Transplant with restricted access. In this case, we were able to obtain a donor travel history and serum samples which were tested. It is important that such provisions, for storage to occur.Leishmania from peripheral bloodTransfusion-related transmission has been described in earlier reports.In countries where Leishmaniasis is endemic, routine donor screening pre-liver transplantation is not recommended given the limited data on donor-derived infectionWhile our patient had classical features of VL, with persistent fever and pancytopenia, persistent splenomegaly is common following liver transplantation in a patient with portal hypertension and therefore can be overlooked in this setting. It highlights the need for a wider differential diagnosis in post transplantation patients.