Antiplatelet therapy may increase the risk of symptomatic intracranial hemorrhage after endovascular treatment for ischemic stroke but may also have a beneficial effect on functional outcome. The aim of this study is to compare safety and efficacy outcomes after endovascular treatment in patients with and without prior antiplatelet therapy.We analyzed patients registered in the MR CLEAN Registry between March 2014 and November 2017, for whom data on antiplatelet therapy were available. We used propensity score nearest-neighbor matching with replacement to balance the probability of receiving prior antiplatelet therapy between the prior antiplatelet therapy and no prior antiplatelet therapy group and adjusted for baseline prognostic factors to compare these groups. Primary outcome was symptomatic intracranial hemorrhage. Secondary outcomes were 90-day functional outcome , successful reperfusion  and 90-day mortality.n = 937) of the 3154 patients were on prior antiplatelet therapy, who were matched to 477 patients not on prior antiplatelet therapy. Symptomatic intracranial hemorrhage occurred in 74/937 (7.9%) patients on prior antiplatelet therapy and in 27/477 (5.6%) patients without prior antiplatelet therapy adjusted odds ratio 1.47, 95% confidence interval 0.86–2.49. No associations were found between prior antiplatelet therapy and functional outcome , successful reperfusion , or 90-day mortality .Thirty percent (We found no evidence of an association of prior antiplatelet therapy with the risk of symptomatic intracranial hemorrhage after endovascular treatment, nor on functional outcome, reperfusion, or mortality. A substantial beneficial or detrimental effect of antiplatelet therapy on clinical outcome cannot be excluded. A randomized clinical trial comparing antiplatelet therapy versus no antiplatelet therapy is needed. Although pre-stroke disability and large baseline infarct core are known causes of these poor outcomes, incomplete microvascular reperfusion—a potentially reversible process—might contribute to these poor outcomes as well. One of the causes of incomplete microvascular reperfusion is the formation of microthrombi occluding the distal capillary bed. These microthrombi are abundantly present after focal cerebral ischemia in the distal vascular territory. The formation of microthrombi might be promoted by vessel wall damage caused by EVT.3 Use of antiplatelet drugs could potentially reduce periprocedural formation of microthrombi by inhibiting platelet aggregation and inflammation of the vessel wall, which could ultimately improve microvascular reperfusion. On the other hand, one randomized trial showed that antiplatelet therapy increases the risk of symptomatic intracranial hemorrhage (sICH) when administered early—within 90 min—after intravenous treatment with alteplase. However, this trial did not focus on the subpopulation of patients with ischemic stroke caused by a large vessel occlusion undergoing EVT. In these patients, the beneficial effect of platelet inhibition could counterbalance the detrimental effects of increase risk of sICH. As interventionists are familiar with periprocedural use of antiplatelet agents during non-stroke neurovascular procedures (i.e. stenting), this treatment might be an easily applicable therapy of adjunctive value in EVT for stroke. However, as antiplatelet agents are not administered systematically during EVT for acute ischemic stroke and current evidence is limited to small observational studies investigating the association of prior antiplatelet therapy with sICH risk and functional outcomes, there are conflicting results. The evaluation of risks and benefits of prior antiplatelet therapy in a large cohort of patients treated with EVT could provide useful information for clinical practice. The aim of this study is to compare safety and efficacy outcomes, after EVT of patients with and without prior antiplatelet therapy.Approximately 50% of patients with ischemic stroke do not recover to functional independence after endovascular treatment (EVT). For the present study, we selected patients who were registered between March 2014 and November 2017 and adhered to the following criteria: age 18 years or older; treatment in a center that participated in the MR CLEAN trial; presence of a proximal intracranial occlusion in the anterior circulation confirmed on computed tomography angiography (CTA) , intracranial carotid artery terminus (ICA-T), middle cerebral artery (M1/M2), or anterior cerebral artery (A1/A2)); and groin puncture within 6.5 h after symptom onset and known data on prior antiplatelet therapy. The current observational study was guided by the STROBE statement.We used data from the MR CLEAN Registry that is a prospective national multicenter study including all consecutive patients treated with EVT for ischemic stroke in the Netherlands. The complete methods and definition of variables of the MR CLEAN Registry have been described elsewhere.http://www.mrclean-trial.org/docs/latestprotocol.pdf. Data cannot be made available, as no patient approval has been obtained for sharing coded data. However, syntax files and output of statistical analyses (R 3.5.0) will be made available upon request.The central medical ethics committee of the Erasmus MC, University Medical Center Rotterdam, the Netherlands, evaluated the study protocol and granted permission to carry out the study as a registry (MEC-2014-235). This approval extends to all participating centers in the Netherlands. Coded data were obtained and stored at Erasmus MC, and scientific analyses were approved and supervised by a central writing committee. The MR CLEAN Registry study protocol is available on Prior antiplatelet therapy was defined as the use of any antiplatelet agent at baseline, reported by the local investigators. We compared outcomes of patients that were on antiplatelet therapy prior to the EVT procedure to patients not on antiplatelet therapy. Data on which specific antiplatelet agent was used before EVT were not prospectively collected in the MR CLEAN Registry. For insight into dual antiplatelet usage, we retrospectively evaluated all available patient discharge letters on this specific issue. Acute administration of any antiplatelet agents during EVT is not part of common practice in the Netherlands. At the discretion of the treating physician, acute administration of antiplatelets was possible in those patients requiring immediate carotid artery stenting; this concerns only a few patients and was not recorded in this registry. Patients with non-cardioembolic ischemic stroke received antithrombotic medication according to local guidelines . In patients who also received intravenous thrombolytics, antiplatelet therapy was delayed until >24 h after stroke thrombolysis.The primary outcome was the occurrence of sICH, before final follow-up assessment at 90 days, defined as neurological deterioration ) and a compatible cerebral hemorrhage seen on imaging assessed by an independent imaging core laboratory. successful reperfusion of the distal macrovascular territory  at the end of the EVT assessed by an independent imaging core laboratory, NIHSS score 24–48 h after intervention, and within 90 days occurrence of mortality, progression of ischemic stroke, new ischemic stroke, extracranial hemorrhage, and cardiac ischemia.Secondary outcomes were functional outcomes at 90 days (range ± 14 days) on the modified Rankin Scale (mRS), which is a seven-point ordinal scale ranging from 0 “no symptoms” to 6 “dead” ,χ test for categorical variables, independent samples t-test for normally distributed continuous variables, and Kruskal–Wallis for non-parametric testing. Any mRS score (except for occurrence of death) assessed within 30 days of symptom onset was considered invalid and treated as missing. We assume “missing” in any (both safety and efficacy) outcome assessment to be distributed at random. For the purpose of unbiased estimation of associations of outcome with baseline characteristics, we used multiple imputation by chained equations and pooled data over five imputed datasets.10 All baseline data and outcomes that are reported are crude and not imputed. A description of the exact imputation settings used is provided in Suppl. File 1. To reduce possible confounding by indication, we performed propensity-score matching, using a within approach, performing propensity-score matching within each imputed dataset averaging the effect estimates. The propensity score for each individual was defined as the probability of being on the treatment (prior antiplatelet therapy) given the patient’s baseline characteristics and comorbidities. Variables used to retrieve the propensity score were required to be factors potentially related to the choice of treatment assignment remaining inclusive. In case of uncertainty whether a variable was related to treatment assignment advantage was given to this variable. Subsequently we performed nearest-neighbor matching on the derived propensity score with replacement setting a caliper of 0.25 SD of the logit for propensity score.13 To assess whether the propensity-score model has been adequately specified, we evaluated baseline characteristics distributions before and after matching and evaluated propensity-score densities graphically. Within the propensity-score-matched cohort, we performed binary and ordinal logistic and linear regression analyses as appropriate and additionally adjusted for important prognostic covariates to optimally reduce residual imbalances in observed covariates between patients with and without prior antiplatelet therapy.16 The selection of covariates was based on prior knowledge and included NIHSS at admission, treatment with intravenous alteplase, location of the intracranial occlusion, Alberta Stroke Program Early Computed Tomography Score (ASPECTS) at baseline, CTA collateral grade at baseline, and time from onset to reperfusion. Sensitivity analyses using binary and ordinal and linear logistic regression for the full cohort (without propensity-score matching) were performed. Additional subgroup analyses on associations of antiplatelet therapy were performed for history of myocardial infarction, history of prior stroke, treatment with both intravenous alteplase and EVT, treatment with EVT only, successful reperfusion, and for prior oral anticoagulant use. Associations are presented as (adjusted common) odds ratio (a(c)OR) with 95% confidence interval (CI). Because there is consensus on the ordering of the outcome scale in this case (each score on the mRS is more favorable than a one point lower score), the common OR can be presented and interpreted as a summary estimate of the treatment effect, even if the underlying proportional odds assumption would be violated. Therefore, we decided not to formally test this assumption. All statistical analyses were performed with R version 3.5.0 .Differences in baseline characteristics were assessed for both categorical and dichotomous variables using A total of 3154 patients were analyzed, of which 937 patients (30%) were on prior antiplatelet therapy . Based oOn average, 477 patients (number of matches over five imputations ranged between 474 and 478) not on prior antiplatelets were matched to 937 patients who were on prior antiplatelet therapy. In the propensity-score-matched cohort, baseline characteristics were more similar between groups compared to the full cohort suggesting that reasonable balance was obtained . Also, vIn the propensity-score-matched cohort, no significant difference in sICH risk was observed between patients who were on prior antiplatelet therapy and those not on prior antiplatelet therapy .19 Our study negates the observation in the MR CLEAN trial of a substantially increased risk of sICH in patients on prior antiplatelet therapy.Our findings are consistent with the results of two smaller observational studies evaluating prior antiplatelet therapy in EVT-treated patients, both reporting no significant associations on risk of sICH and functional outcome. The authors suggested an association with early re-occlusion prevention. These findings formed the rationale for a trial of acetylsalicylic acid directly after intravenous alteplase treatment. This trial was halted prematurely because of futility and increased sICH risk. These results are not generalizable to patients undergoing EVT, because these patients will be more susceptible to re-occlusion and induction of microthrombi by vessel wall damage, and are therefore at higher risk of ischemic complications.The safety of antiplatelet agents in patients treated with intravenous alteplase has been investigated in several studies. In the National Institute of Neurological Disorders and Stroke trial on the effect of intravenous alteplase, clinical deterioration was less common in patients who were on prior antiplatelet therapy.Given the liberal inclusion criteria of this registry and the broad area of common support in propensity scores after matching, we consider the results of this study are generalizable to the larger EVT-eligible population of ischemic stroke patients with an intracranial occlusion of the anterior circulation who are treated within 6.5 h from symptom onset.Our study has some limitations. First, despite (I) propensity-score matching to obtain properly matched groups of patients with and without prior antiplatelet therapy and (II) additional covariate adjustment to increase robustness of the outcomes, it is still possible that our results are hampered by confounding indications. Factors relating to the patients vascular may not be captured completely in the model. Additionally, occurrence of sICH is impacted by several other factors such as the post (peri)procedural blood pressure management or follow-up infarct volume. These unmeasured characteristics may confound differences between patients with and without prior antiplatelet therapy. Second, re-occlusions were not scored systematically on follow-up imaging in this registry after EVT. Instead, we reported occurrence of stroke progression and new ischemic stroke, which showed a non-significant trend toward lower occurrence in patients who were on prior antiplatelet therapy. Third, in this study, we were not able to address the question whether dual antiplatelet therapy is associated with different safety and efficacy results as compared to single antiplatelet therapy due to low numbers, which warrants further study. Finally, the compliance with prior antiplatelet therapy in our cohort is unknown. If the compliance was poor, which is conceivable as antiplatelet therapy is paradoxically used in the prevention of stroke, this may have influenced our results.Multicenter randomized clinical trial of endovascular treatment for acute ischemic stroke; the effect of periprocedural medication: acetylsalicylic acid, unfractionated heparin, both or neither; ISRCTN76741621), patients are being randomized to intravenous acetylsalicylic acid and/or unfractionated heparin to investigate whether this will improve functional outcome after EVT. In this trial, intravenous acetylsalicylic acid is administered during EVT, overcoming the issue of non-compliance. This trial will provide new randomized data to answer the question whether platelet inhibition is safe and beneficial for this group of severely affected ischemic stroke patients.The results of our observational study do not exclude the possibility of a sizeable beneficial effect of antiplatelet therapy in ischemic stroke patients undergoing EVT. In the ongoing MR CLEAN-MED trial (In this observational study, we did not find evidence that prior antiplatelet therapy is associated with sICH, functional outcome, reperfusion, or mortality after EVT for ischemic stroke. Our results do not exclude a beneficial or detrimental effect of antiplatelet therapy on outcome after EVT for ischemic stroke. A randomized trial is therefore justified to evaluate the safety and efficacy of antiplatelet agents administered during EVT.Click here for additional data file.Supplemental material, sj-pdf-1-wso-10.1177_1747493020946975 for Prior antiplatelet therapy in patients undergoing endovascular treatment for acute ischemic stroke: Results from the MR CLEAN Registry by Rob A van de Graaf, Sanne M Zinkstok, Vicky Chalos, Robert-Jan B Goldhoorn, Charles BLM Majoie, Robert J van Oostenbrugge, Aad van der Lugt, Diederik WJ Dippel, Yvo BWEM Roos, Hester F Lingsma, Adriaan CGM van Es, Bob Roozenbeek and on behalf of the MR CLEAN Registry investigators in International Journal of Stroke


Long acting injectable (LAI) antipsychotics are commonly used in the treatment of schizophrenia to improve adherence and clinical outcomes. Concerns have been reported in relation to their non-systemic or injection site adverse effect profile. As such, this study aims to review and evaluate all evidence reporting injection site adverse effects with LAI antipsychotics.An electronic search was systematically conducted through four databases  in order to identify studies investigating injection-site reactions associated with LAI antipsychotics. Unpublished studies such as conference proceedings and clinical trial registries were also searched. The search was limited to literature published in English without year limits.Of a total of 189 citations that were identified from the electronic database search, 12 were selected for inclusion in this review. Various injection site reactions were reported in these studies, including pain, bleeding, and swelling. Overall, the studies reported a low incidence of these injection site reactions. Only a minority of the included articles compared injection site reactions between different LAI antipsychotics.Injection site pain was the most commonly reported injection site adverse effect across all articles reviewed. The low incidence of injection site adverse effects associated with LAI antipsychotics indicates that these formulations appear to be well tolerated by patients. More head-to-head trials comparing second generation LAI antipsychotics are needed. Long acting injectable (LAI) antipsychotics  are concentrated formulations which, following intramuscular injection, release the antipsychotic drug slowly over time. This allows an effective maintenance dose of the antipsychotic to be delivered with injections at intervals that range from 2 weeks to up to several months.10First-generation LAI antipsychotics consist of the antipsychotic esterified to a decanoate, which is dissolved in an oily vehicle.15Considering the relatively limited attention that has been paid to non-systemic side effects associated with LAI antipsychotics, and which are rarely reported in clinical trials, this study aims to systematically review all the available evidence reporting injection-site adverse effects associated with the administration of LAI antipsychotics. This review of local adverse events of single LAI antipsychotics will provide evidence-based recommendations for their safe use in practice and may help clinicians in tailoring their choice of treatment.Appendix 1), which outlined the different steps of this systematic review of the literature, an electronic database search was conducted from database inception using PubMed, SCOPUS, Embase and Cochrane databases in order to identify studies investigating injection-site reactions associated with LAI antipsychotics. Search terms included “LAIAPs”, “long acting depot”, “antipsychotics”, “neuroleptics”, “local reactions” and “injection site adverse effects”. To avoid publication bias, unpublished studies such as conference proceedings and clinical trial registries were also searched. In addition, a quick update of the literature was undertaken to identify articles published between the time of the initial search and the time of publication.Based on a pre-determined protocol . Studies were selected for full review based on the following inclusion criteria: articles in English that investigated injection-site reactions associated with LAI antipsychotics or studies reporting any form of adverse effects relating to injection-site administration of antipsychotics. By injection site adverse effects, only local reactions of the skin and underlying tissue were considered. As such, non-localized adverse effects resulting from the intramuscular administration of the antipsychotic (such olanzapine-induced post-injection delirium/sedation syndrome) were excluded. Articles were also excluded if injection-site related adverse effects were not included in the study outcomes. The full text of all potentially relevant articles were retrieved and distributed among three reviewers  to confirm eligibility. The quality of the articles was assessed using the Critical Appraisal Skills Programme (CASP) Systematic Review checklist.Of the 189 citations identified in the initial electronic database search, 25 full-text articles were selected to undergo a more comprehensive review based on the inclusion/exclusion criteria. However, following the full-text review, 13 articles were excluded because these articles did not provide information on injection-site adverse effects. Therefore, a total of 12 articles were selected for full review and data extraction using a standardized data collection tool.ore*1Hay 9517CrossThe quality assessment scores reported in In this study, a systematic review was conducted on all the available evidence reporting injection-site adverse effects associated with the administration of LAI antipsychotics aiming to provide evidence-based recommendations for their safe use in practice. The most commonly reported injection site reaction across the 12 articles included in this study was pain at the injection site. In an indirect comparative study of injection-site pain associated with first-generation LAI antipsychotics versus paliperidone palmitate (PP), researchers suggested that PP may be associated with lower mean pain severity than first-generation products.Most studies investigated in the current systematic review were of relatively high quality . All theIn conclusion, the majority of the articles discussed in this review reported that patients tolerate LAI antipsychotics formulations relatively well. The most commonly reported injection site reaction across all the studies was pain at the injection site, especially with oil-based formulations of LAI antipsychotics. Several strategies have been recommended to minimize injection site pain, such as selecting appropriate injection administration techniques, using suitable needle specifications, increasing the interval between injections and administering low volume of highly concentrated formulations of LAI antipsychotics. Noticeably, such strategies should be further investigated in order to assess their influence on improving injection-site reactions. Similarly, the selection of the muscle site for injection administration is an important consideration that is worth exploring further. Although the gluteal region is the preferred site for administering depot antipsychotics, the deltoid area is less intrusive for patients; hence, this may influence patient acceptability and compliance.32–34Although this review generated important findings, some limitations in the literature review process need to be highlighted. First, the article by Kern Sliwa et al


Rather, we propose that the observed activations reflect a local transformation from a feature-based visual representation to a representation of “elemental affect,” computed through information-processing mechanisms that detect deviations from an observer’s expectations. Furthermore, we found modulation by aesthetic appeal in subcortical reward structures but not in regions of the default-mode network (DMN) nor orbitofrontal cortex, and only weak evidence for associated changes in functional connectivity. In contrast to other visual aesthetic domains, aesthetically appealing interactions with natural landscapes may rely more heavily on comparisons between ongoing stimulation and well-formed representations of the natural world, and less on top-down processes for resolving ambiguities or assessing self-relevance.During aesthetically appealing visual experiences, visual content provides a basis for computation of affectively tinged representations of aesthetic value. How this happens in the brain is largely unexplored. Using engaging video clips of natural landscapes, we tested whether cortical regions that respond to perceptual aspects of an environment  were directly modulated by rated aesthetic appeal. Twenty-four participants watched a series of videos of natural landscapes while being scanned using functional magnetic resonance imaging (fMRI) and reported both continuous ratings of enjoyment (during the videos) and overall aesthetic judgments (after each video). Although landscape videos engaged a greater expanse of high-level visual cortex compared to that observed for images of landscapes, independently localized category-selective visual regions  were not significantly modulated by aesthetic appeal. Rather, a whole-brain analysis revealed modulations by aesthetic appeal in ventral  and lateral  clusters that were adjacent to scene and motion selective regions. These findings suggest that aesthetic appeal  Interactions with the natural environment can be highly impactful and aesthetically rewarding. Natural landscapes are rich sources of beauty, pleasure, awe and fascination , aesthetWhereas a wealth of detail is known about how the human brain represents what is in our visual environment  is in the parahippocampal gyrus (PHG) or adjacent collateral sulcus (CoS); when identified by a contrast of activation for scenes vs. images of isolated objects, this region is referred to as the “parahippocampal place area” PPA; . It is tSeveral studies have reported increased activity in or near the PPA in response to preferred scenes, but the evidence is far from conclusive. faces is represented in regions of the face-selective network have reported mixed results, with some studies showing effects in fusiform face area . Natural landscapes have a strong capacity to invoke aesthetic engagement . ImportaThe use of videos of natural scenes adds another feature, motion, whose representation in visual cortex is also well characterized. Similar to the scene-selective areas, it is unclear whether area hMT+ , a core region for the computation of visual motion , is modumPFC), orbitofrontal cortex (OFC) or nodes of the default-mode network (DMN). Many studies have reported that aesthetically appealing stimuli are associated with greater fMRI activation in portions of prefrontal cortex that support valuation and emotion (mOFC) for visual artwork  when compared to neutral videos  or orbitofrontal cortex.However, the question of whether portions of the prefrontal cortex or DMN are engaged by aesthetically appealing natural scenes is an unsettled issue. Using a whole-brain activation analysis, one study reported effects in prefrontal regions for aesthetic appeal of diverse scenes  while anest ROI; , whereasl videos . Using mndscapes  or aboutndscapes ; howeverndscapes . In contnsistent . We therOur third aim was to assess whether feature-selective visual regions show increased functional connectivity with reward or DMN regions for aesthetically appealing experiences with natural landscapes. One possible mechanism by which visual information is transformed into a representation of aesthetic appeal is through an increase in coordinated activity between visual regions and subcortical reward, prefrontal or DMN regions. An increase in functional connectivity between content-related visual regions and ventromedial prefrontal cortex (vMPFC) has been reported during valuation judgments of t-shirt designs , and alsIn addition to increasing engagement  and alloTo test these hypotheses, twenty-four observers watched 30 s video clips of natural landscapes while being scanned using fMRI. During the clips, observers were asked to continuously rate their enjoyment using a hand-held dial. After the clip ended, observers then used the same dial to rate the overall intensity of their aesthetic experience. We found that ROIs sensitive to specific perceptual features such as scene layout and motion were strongly activated by the landscape movies, but were not clearly modulated by overall aesthetic appeal. Rather, a whole-brain analysis revealed several regions sensitive to aesthetic appeal of dynamic natural landscapes that were adjacent to, or only partially overlapping with, these ROIs representing specific perceptual features. Beyond the visual system, we observed that subcortical regions of the basal ganglia were modulated by aesthetic appeal, but that no modulation by aesthetic appeal could be detected in prefrontal and DMN regions using reasonable thresholds. Finally, functional connectivity between visual ROIs and reward or DMN ROIs was only weakly modulated by aesthetic appeal, failing to survive a stringent corrections threshold.per se are not represented in core visual regions that are selective for specific visual features such as scene layout and motion, but rather in cortex adjacent to these regions. Additionally, we present evidence that, at least for movies of natural landscapes, the neural basis for aesthetic appeal does not depend on either large-scale activation in prefrontal cortex nor on functional connectivity between content-selective visual regions and reward or DMN regions.These results suggest that features that drive aesthetic appeal Twenty-six participants were recruited for this study and paid for their participation. Two participants were excluded, one due to excessive motion (mean framewise displacement > 0.5 mm) and one due to missing behavioral data, leaving a final group of 24 participants . Informed consent was obtained in accordance with a protocol approved by the Medical Ethics Committee of Goethe University Frankfurt and was signed by all participants before the experiments.Stimuli were 30 s video clips of landscapes collected from video streaming websites  or from non-narrative cinematic films. The movies consisted of aerial drone shots, point-of-view shots or time-lapse photography depicting different types of natural landscapes . They had clear artistic intent conveyed through the selection of depicted content, light conditions, use of camera techniques, and presence of changes throughout the duration of the clip . To ensure that aesthetic engagement was mainly driven by the landscape content, videos did not include human beings, animals or other objects. Forty movies were clipped to 30 s length using Adobe Premier Pro and saved with the same aspect ratio (16:9), resolution (1280 × 720 px) and video compression method (H.264). Movie stimuli were presented using PsychoPy2 (v1.85.2) and MovieStim3  at the cParticipants completed a 6-min resting state scan in which they were asked to fixate a central fixation cross on a gray background. In the following four runs (8 movies each), participants viewed 32 landscape movies in an order counterbalanced across participants. Each movie trial began with a 10 s fixation period, followed by a 30 s movie presentation with no fixation requirement, followed by a 4 s response period . A fifthHow much are you enjoying the clip at each moment?”). After the video clip finished playing, participants were given 4 s and asked to make a summary aesthetic judgment . Participants were instructed to give ratings of their subjective aesthetic experience of the clips and that they should base their judgments on their personal experience. They were told that they might have a more intense aesthetic experience for many different reasons, such as a clip being experienced as beautiful, profound or emotionally moving. Responses were collected using a custom-made MRI compatible rotary dial  positioned at their side. The dial had stops at the 9 and 3 o’clock positions and haptic feedback on the 12 o’clock position indicating extreme negative, extreme positive and neutral enjoyment, respectively. Participants were instructed to position the dial at the 12 o’clock neutral position before the start of each trial. This design allowed the participants to give ratings ranging from negative to positive without the need for visual feedback that would interfere with a visual experience. Before the experiment, observers were given practice with visual feedback on how to use the dial to help them calibrate their responses.During the videos, participants were asked to make continuous evaluations of their aesthetic enjoyment using a rotary dial. They were told to move the dial to the right if they were feeling more enjoyment or pleasure and to the left if they were feeling less enjoyment or pleasure when watching the video clip  and motion (hMT+) localizer. The PFOB localizer contained blocks of places, faces, objects, scrambled objects and bodies without faces. This scan consisted of five blocks of each stimulus type, during which the participant performed a “1-back” task responding each time an exact repeat of an image appeared. Each block contained 16 stimulus images (image size = 20°× 20°) each presented for 800 ms with a 200 ms inter-stimulus-interval. In the hMT+ localizer participants viewed alternating blocks of biological point-light actions, position-scrambled point-light controls, and static frames of the scrambled point-light control condition . Each paData for both continuous and overall ratings were scaled to range between −1 and 1. Continuous data were collected with a sampling rate of 60 Hz and down sampled to 10 Hz. Overall ratings given at the end of each movie were categorized into four bins and were used in the whole-brain activation analysis and ROI activation analysis. The binning was done for each participant separately by discretizing the values of the overall ratings into four bins based on quartiles.Functional magnetic resonance imaging data collection was carried out at University of Frankfurt’s Brain Imaging Center, using a 3-T Siemens Trio scanner and an eight-channel phased array head coil (Siemens). The blood oxygen level dependent (BOLD) signal was measured using 32 3 mm slices (2 mm + 50% distance factor) that were acquired in an oblique orientation of approximately 20° to the anterior commissure-posterior commissure (AC-PC) axis to reduce the signal dropouts in the ventral prefrontal cortex . The standard Siemens sequence was customized to include an additional slice-wise z shimming (−1.3 mT/m). Before every functional scan a short EPI (3 TR) with opposite phase encoding direction was collected for use in phase unwarping during preprocessing.3) anatomical volume (MPRAGE sequence) was obtained after the functional scans. Data were converted from DICOM to NIFTI and structured according to the BIDS standard (1). Neuroimaging data was preprocessed using fMRIPprep 1.1.8  references with opposing phase-encoding directions using 3dQwarp (AFNI)  which implements boundary-based registration (A high-resolution (1 mmstandard  using Heep 1.1.8 . The detp (AFNI) 2. Based stration . Co-registration ]. The BOstration  was perfstration . The thrstration ]. Princistration . The intstration  mostly wr score for each individual indicating how much this person is in agreement with the rest of the participants. We applied a similar leave-one-out framework to calculate a measure of agreement for continuous ratings in which one participant’s rating timecourse was correlated with the average of all others . This procedure results in a participant-by-movie array that was then averaged across movies to obtain one value per participant indicating how much this person was in agreement with the rest of the participants for their moment-to-moment ratings. To obtain average across-observer MM1c scores, we first transformed individual r-values to z-values, computed the mean and 95% confidence intervals, and then transformed those scores back to r-values  correlation measure . To calcr-values .To quantify idiosyncratic and shared contributions to the overall aesthetic ratings we estimated proportions of “shared” and “private” taste with a variance decomposition method  with a mA measure of motion energy for each video clip was computed by applying a Gabor jet simple cell model  to each Participant-specific maps of the DMN were obtained using the rest scan. High-pass filtering at 0.005 Hz and spatial smoothing with 6-mm FWHM Gaussian filter were applied using FSL to the non-aggressively cleaned fmriprep output in MNI space . Then, wData from the PFOB and hMT+ localizers were analyzed with standard random-effects general linear models (FSL FEAT) after high pass filtering with 100 s cut-off. For the PFOB localizer we used separate predictors for face, object, scene, body and scrambled object blocks. Face-selective voxels were identified by contrasting face blocks to the average of the object, scene, and scrambled object blocks and object-selective voxels were identified by contrasting object blocks to the average of the face, scene and scrambled object blocks. Similarly, scene-selective voxels were identified by contrasting scene blocks to the average of the face, object and scrambled object blocks. For the hMT+ localizer we modeled separate predictors for position scrambled point-light controls, static frames of the scrambled point-light control conditions and biological point-light actions. To localize hMT+, position-scrambled point-light control stimuli  were contrasted with static frames of these scrambled actions . The bioz > 2.3, cluster threshold p < 0.05) activation maps and overlaying them on top of each other in a common stereotaxic space (MNI) to create probabilistic overlap maps for each contrast of interest. We created these probabilistic maps for each contrast and transformed them to Freesurfer’s fsaverage surface (mri_vol2surf) to be able to visualize and delineate each target region of interest. The parahippocampal place area (PPA), occipital place area (OPA) and retrosplenial cortex (RSC) were identified as the set of scene-selective voxels in the collateral sulcus, transverse occipital sulcus and medial parietal cortex , medial OFC (mOFC), mid anterior OFC (maOFC)] were identified with the OFC atlas , Caudate, Putamen, Pallidum] were defined anatomically based on Freesurfers “aparc” automatic segmentation and orbitofrontal cortex (OFC) ROIs . A small portion of this cluster also extended into ventral striatum  and anterior insula .We sought to more closely examine the topographical relationship between modulations of cortical activity by stimulus content vs. by aesthetic appeal. Using data from this experiment, recorded as observers judged the aesthetic appeal of movies of natural landscapes, as well as data from a previously published experiment in which a different set of observers made aesthetic judgments about still images of natural landscapes , we compIn comparison to still images of landscapes , movies activated a greater extent of cortex . We then modeled the trialwise FC estimates as a function of each observers’ overall aesthetic ratings using both linear and quadratic regressors, separately for each observer, and tested the resulting t-scores from these regressions for significance at the group level using one-sample t-tests  scores between each pair of a priori ROIs was only weakly related to overall aesthetic appeal, as none of the edges tested in this analysis surpassed this stringent multiple comparisons threshold. However, when we examine the (uncorrected) pattern of average connectivity, several noteworthy relationships do emerge. For the linear effect of aesthetic appeal (t(23) = 2.60, NAc:hMT+ t(23) = 2.14, NAc:OPA t(23) = 2.21], and NAc also showed increasing connectivity with several orbitofrontal regions . PCC, a core DMN region, showed stronger FC with OPA with increasing aesthetic appeal . Increased connectivity was also observed between two nodes of scene-selective cortex, PPA:RSC t(23) = 2.08, and between mOFC:hMT+, t(23) = 2.24. The quadratic effect of aesthetic appeal was included to identify edges with highest FC for extreme values of aesthetic appeal regardless of direction  = 3.10, caudate:OPA t(23) = 3.11, caudate:PPA t(23) = 2.35], as was FC between several DMN and reward regions , and within several nodes of the reward network . Pericalcarine also showed a U-shaped relationship with one DMN [Pericalc:dMPFC t(23) = 2.31] and one reward node [Pericalc:Putamen t(23) = 2.17]. Finally, the connection between IPL and NAc showed a negative quadratic relationship to aesthetic appeal [IPL:NAc t(19) = −2.45].Connectivity between our We performed an additional analysis to test for possible changes in connectivity linked to moment-to-moment changes in aesthetic appeal, using observers’ continuous ratings of enjoyment. Restricting our analysis to 3 key ROIs  we modeled dynamic (time series) estimates of FC computed using the multiplication of temporal derivatives method  with regressors coding for either positive or negative moments of change. None of the edges reached significance.A core component of visual aesthetic experiences is the transformation of information about the contents of perception to a representation of aesthetic appeal. Using functionally defined ROIs, a robust approach more often used in studies of high-level vision, we tested whether independently localized posterior regions of the brain that represent perceptual features  are themselves modulated by aesthetic appeal. Using movies of natural landscapes, we found strong engagement of scene-selective regions in ventral (PPA) and medial (RSC) occipitotemporal cortex, as well as in lateral portions of visual cortex including scene-selective OPA, object-selective LO and motion sensitive hMT+. However, activity in these feature-selective regions was not significantly modulated by rated aesthetic appeal. Instead, we found greater activity for highly appealing movies in regions that were adjacent to and only partially overlapping with the feature selective ROIs, both on the ventral  and lateral (left pMTG and MOS) cortical surfaces. A large cluster spanning several nuclei of the right basal ganglia also showed increased activity for highly appealing movies. These findings suggest that regions representing core visual features such as scene shape and motion are not directly modulated by aesthetic appeal. Rather, adjacent cortex may be involved in the computation of information that is more directly relevant for aesthetic valuation. Additionally, contrary to what has been observed for artworks, we did not find evidence for modulation of prefrontal or default-mode regions correlated with aesthetic appeal. Finally, we did not find strong evidence for modulation of functional connectivity between feature-selective visual regions and nodes of reward or default-mode networks.In contrast to previous studies using static images of scenes  and natuHighly appealing movies of natural landscapes also led to greater activation in two clusters on the lateral surface, pMTG and MOS. Whereas modulation of lateral visual regions by aesthetic appeal has been reported in MOG for indoor built environments , in hMT+Given previous work supporting the direct modulation of feature- or category-selective regions by aesthetic appeal  and arousal may only be explicitly represented in other, more specialized structures  there isIn a related manner, contextual associations, semantic interpretations and imagery triggered by a stimulus can also influence aesthetic appeal . ContextWhen comparing the areas associated with aesthetic appeal of movies vs. images of landscapes, we found greatly increased engagement of lateral and dorsal regions for appealing movies. For the field, this finding points to the need for visual neuroaesthetics to avoid a narrow focus on the ventral visual pathway. On the ventral surface, we found that while appeal-related activations for movies were in the CoS and PHG, appeal for static images appeared to more strongly engage parts of the fusiform gyrus and LO. One possibility for this difference is that the increase in ecological validity afforded by movies improved their capacity to invoke contextual associations compared to scenes. Alternatively, the presence of motion-induced depth cues in movies may have driven attentional focus to scene shape and layout. Static images, lacking such cues, may permit greater attention to flow to individual object representations, and thus engage object-selective cortex such as LO to a greater degree. If this were the case, future research might test the prediction that aesthetic judgments of images are more determined by object content, whereas judgments of movies may be more related to scene shape than to object content. A final explanation to consider for some of the observed differences is that certain activations reported for static images may reflect short-lived effects related to the onset of a stimulus that are not maintained over the course of a 30 s movie.We found a large cluster of activation extending over several nuclei of the right basal ganglia that responded more strongly to highly appealing movies, compared to other movies. This cluster was primarily contained within dorsal striatum structures such as caudate nucleus and putamen and included only a small fraction of ventral striatum (NAc). There is some discrepancy in the neuroaesthetics literature as to which structures of the basal ganglia are more relevant for aesthetic appeal, given that studies have reported activations in different areas see . One stuper se. Studies investigating reward prediction at different time scales have found that a number of areas in the limbic loop, including the ventral striatum, are involved in the prediction of immediate rewards; dorsal striatum, a part of the motor loop, was found to be involved in future reward prediction (potential reward such as the potential for habitat, for exploration, for resource availability, or for monitoring one’s surroundings – environmental conditions favorable to survival (An alternative explanation for the differential roles of ventral and dorsal striatum during aesthetically rewarding moments relates to the time scale of the reward prediction, rather than anticipation or consumption ediction . The actediction  might resurvival .mPFC, OFC or in nodes of the DMN. Many studies have reported modulation by aesthetic appeal in portions of mPFC, particularly in or around the superior rostral sulcus (mPFC, particularly vMPFC, is strongly implicated in the representation of subjective value (mPFC are also part of the DMN (mPFC, nor in independently localized nodes of the DMN, using both whole-brain and ROI-based analyses. One previous study with images of natural landscapes found sensitivity to landscape appeal only when using a small-volume correction in an a priori defined vMPFC ROI (mPFC (mPFC or mOFC by landscapes is sparse.A secondary aim of this study was to evaluate whether aesthetically appealing natural landscapes lead to greater activation in l sulcus . The mPFve value , and por the DMN . This fi the DMN . In the MPFC ROI . On the OI (mPFC  and seveOI (mPFC . HowevermPFC and other DMN nodes at a spatial scale that is too fine to survive an activation-based analysis, which requires a degree of spatial smoothing and averaging across observers. However, this possibility does not address the discrepancies observed for different aesthetic domains: sparse evidence for mPFC activation by appealing landscapes, but more robust evidence for appealing visual artworks.The discrepancy between activation-based and pattern-based analyses may point to the relevance of spatial scale. Aesthetic appeal for landscapes may be represented in mPFC, PCC; One potential avenue for explaining this discrepancy may come from a differential contribution of bottom-up vs. top-down processing to appealing experiences with landscapes vs. artworks. Aesthetic experiences with artworks, more so than those with landscapes, may engage explicit, effortful top-down mechanisms supporting sense-making, imagery and resolution of ambiguities  incorpormoment-to-moment ratings of enjoyment was higher. These findings suggest that moment-to-moment enjoyment of landscape movies may be a more bottom-up process without much need for integration from top-down mechanisms, but that subsequent overall assessments of these movies, performed after watching each clip, may draw on more top-down processes than is typical for images of natural landscape.Interestingly, in this and a previous study  we obserOur third aim was to test whether category-selective visual regions are functionally connected with prefrontal  or rewarHowever, due to several limitations, it may be too soon to completely rule out the presence of such FC modulations. For example, it is possible that the moment-to-moment responses to the clips might not have varied enough to drive detectable fluctuations in functional connectivity; this possibility derives some support from an inspection of the continuous ratings, which were relatively flat for some observers. Additionally, the overall ratings skewed heavily positive, potentially resulting in a relatively weak contrast between the psychological states associated with high-rated and low-rated clips. This may have affected our ability to detect associated changes in functional network modulations. Alternatively, it is also possible that reward and DMN regions integrated information over time in a manner that blurred out such content-related fluctuations in functional connectivity . A furthper se is likely not represented in well-characterized feature- and category-selective regions of the visual system. Rather, we propose that modulations by aesthetic appeal observed in the visual system reflect a transformation from a feature-based visual representation to a representation of elemental affect, computed through information-processing mechanisms that rely on the detection of deviations from an observer’s expectations and activation of richly interpretable semantic and contextual associations. While the exact nature of these transformations remains unclear, we believe that these findings bring a new perspective to the difficult problem of how the brain computes affectively marked appraisals of aesthetic appeal from representations of visual content.Natural landscapes are rich sources of aesthetic appeal, and by virtue of their central role in research on visual perception, can also serve as an important bridge between the fields of scene perception and neuroaesthetics. We conclude that aesthetic appeal Our findings also hint at a potentially important difference between different aesthetic domains. While interactions between bottom-up and top-down processes likely play a role in the appreciation of all aesthetic experiences, the balance of these interactions may differ from one domain to the next. The evidence suggests that aesthetically appealing experiences with natural landscapes, in contrast to those with visual artworks, engage top-down processes supported by prefrontal cortex to a lesser degree.The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Goethe University Frankfurt. The patients/participants provided their written informed consent to participate in this study.AI and EV designed the study, interpreted the results, and wrote the manuscript together. AI collected and analyzed the data. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hemiscrotectomy with en bloc orchidectomy represents a radical primary, completion, or salvage option in men with inguinoscrotal cancers. We describe our surgical technique and peri-operative and oncological outcomes.Retrospective cohort study of 16 men treated at a supra-regional referral centre with open radical hemiscrotectomy with or without en bloc orchidectomy between 2010 and 2020. Peri-operative and survival outcomes were analysed.Radical hemiscrotectomy with or without en bloc orchidectomy was performed on 16 patients comprising 7 well-differentiated liposarcomas, 4 dedifferentiated liposarcomas, 2 leiomyosarcomas, 1 mesothelioma, 1 rhabdomyosarcoma and 1 mammary type myofibroblastoma. Primary hemiscrotectomy was performed in four, completion hemiscrotectomy in nine and salvage hemiscrotectomy in three. The median hospital stay was 2 days [interquartile range (IQR) 2–4]. Four patients (25%) had post-operative complications including wound infection or haematoma. During a median follow-up of 18 months (IQR 2–66), one patient (6%) died following a recurrence in the pelvis and retroperitoneum.and ConclusionsIf careful dissection is performed, radical hemiscrotectomy and en bloc orchidectomy is a radical but safe procedure with a short hospital stay. Haematoma and infection represent the main complications, and within limited follow-up most men showed no recurrence.The online version contains supplementary material available at 10.1245/s10434-021-10315-4. Several rare urogenital cancers arising from tissues of the spermatic cord, epididymis, testis or scrotal skin have a high risk of local recurrence. A radical resection by a hemiscrotectomy with or without en bloc orchidectomy is therefore recommended to try to reduce this complication. Given the limited literature describing this surgery, we summarised our surgical technique and peri-operative and oncological outcomes.All men with a suspected non-germ cell/non-penile primary inguinoscrotal cancer are staged with computerised tomography of the chest and abdomen to assess the most common sites of metastatic disease. If involvement of adjacent structures including the penis, urethra, femoral vessels or pelvic floor is suspected, we recommend further staging with magnetic resonance imaging.Patients are counselled about their diagnosis, management options and prognosis by medical and specialist nurse practitioners. Prior to surgery, expected benefits, likelihood of success and risks of complications and recurrence are discussed. Potential surgical risks pertinent to this particular surgery include bleeding, haematoma, infection, acute and chronic pain, wound breakdown, inguinal hernia, injury to nerves causing neuropathy, urethral/vascular damage, cosmesis, small scrotum, displaced contralateral testis, lypmhoedema of penis/scrotum and inability to resect completely heightening the risk of cancer recurrence locally and distally.The patient is positioned supine on the operating table with legs slightly abducted. Drapes are placed allowing for visualisation of important anatomical landmarks including the anterior superior iliac spine (ASIS), inguinal ligament, pubic tubercle, and the penis and scrotum to the perineum Fig. a. In somA superficial skin incision in a ‘tear drop’ configuration is marked with pen and performed from the the lateral third of the inguinal ligament to the scrotum, including the raphe Fig. b. Any prThe en bloc orchidectomy specimen is lifted, and the external oblique fascia is opened in the direction of its fibres towards the internal inguinal ring Fig. f. The crTwo 14 French suction drains are placed , and the dead space is closed with 2-0 and 3-0 polyglactin sutures, a subcuticular wound closure with a 3-0 monofilament synthetic absorbable suture .Radical hemiscrotectomy with or without en bloc orchidectomy was performed in 16 patients with a mean age of 56 years (range 20–80 years). Race/ethnicity distribution was not reported in the electronical health records. Four men had a primary resection and nine underwent completion surgery, with the remaining three undergoing salvage surgery. In men with primary surgery, two patients had well-differentiated liposarcoma (WDLS), one rhabdomyosarcoma and one mammary type myofibroblastoma. The patient with rhabdomyosarcoma underwent primary surgery after initial biopsy and neoadjuvant chemotherapy with ifosfamid, vincristine and dactinomycin. He subsequently received adjuvant chemotherapy with the same regimen and radiotherapy with 50.4 Gy in 28 fractions to the retroperitoneal lymph nodes and maintenance vinorelbine and cyclophosphamide.Completion surgery was performed within a mean of 3 months (range 1–4 months) after prior testicular surgery . Histological diagnoses included dedifferentiated liposarcoma (DLS) in four, WDLS in two, leiomyosarcoma in two and mesothelioma in one patient. In the subsequent hemiscrotectomy specimens, six out of nine (67%) showed no evidence of tumour, whereas three patients had residual disease including LDS, WDLS and mesothelioma . Four patients (25%) had 90-day post-operative complications. Two (12%) had wound infections requiring oral antibiotics (Clavien–Dindo II), with the remaining two (12%) developing a haematoma (Clavien–Dindo I) which was managed conservatively.During a median follow-up of 18 months (IQR 2–66), one patient (6%) initially presenting with locally advanced dedifferentiated liposarcoma measuring 130 × 50 × 48 mm had recurrence. Metastatic disease in the pelvic and retroperitoneal lymph nodes was diagnosed 2 months after hemiscrotectomy with rapid clinical deterioration. He died 4 months after initial diagnosis despite palliative chemotherapy with doxorubicin and olaratumab.This report summarises important pre-, intra- and post-operative steps and reports peri-operative and oncological outcomes mainly relating to inguinoscrotal sarcoma.In the pre-operative setting, imaging is vital for pre-operative planning and for help with appropriate patient counselling about the extent of resection. Consideration is also given to whether a biopsy is required and whether this may alter management or extent of resection. For example, cancers limited to the scrotal skin itself do not require en bloc orchidectomy, and if there is not sufficient skin cover on the ipsilateral side, a transposition of the testis to the scrotum on the contralateral side can be consideredImportant intraoperative steps include excision of previous scars, resection of the testicular septum and complete exposure of the internal inguinal ring. Excision of previous scars is required owing to the risk of tumour contamination from previous incomplete resection. The scrotal septum should be resected if there is risk of a positive surgical margin , particularly as there is no significant added morbidity associated with this. Similarly, removal of any spermatic cord remnant should be performed as cephalad as possible to the internal inguinal ring. Closure and reconstruction of the inguinal canal represent important steps to prevent inguinal hernias. In tumours invading the inguinal canal structures, resection and reconstruction of the inguinal ligament preferentially with a pedicled fascia lata flapGiven this study is a retrospective single-centre surgical review, it has significant limitations. Inevitably, there was some selection bias in our series, as small inguinoscrotal cancers are often treated in general urology departments with scrotal-sparing surgery with or without orchidectomy. Patients with rare, large or sarcomatous tumours, or with tumour recurrence after primary surgery were referred to our institution. The study also has insufficient statistical power to comment on cancer-related outcome. Hemiscrotectomy with or without en bloc orchidectomy represents an important surgical procedure for several indications including sarcomas of the inguinal cord,6The primary goal of this report is to illustrate the surgical technique, potential complications and early oncological outcomes. Given the rarity of inguinoscrotal cancers requiring hemiscrotectomy, it is unlikely that larger prospective studies will be performed.Sarcomas and other rare inguinoscrotal tumours are rare, usually requiring radical primary resection. Given their rarity, this is best performed in a tertiary referral centre, where there is likely to be greater expertise in the joint multi-disciplinary management of patients of this type. If careful dissection is performed, radical hemiscrotectomy and en bloc orchidectomy is a radical but safe procedure with a short hospital stay. Haematoma and infection represent the main complications, and within limited follow-up most men showed no recurrence.Supplementary file1 (MP4 71096 KB)Below is the link to the electronic supplementary material.


Nucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Nucleic acid (NA) nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Such systems are exploited in biosensing –5 and coCompared to other biomolecules used in biosensing, such as enzymes and antibodies, NA nanostructures exhibit improved stability . MoreoveDNA aptamers are synthetic oligonucleotides able to detect different target molecules  with a good affinity (nanomolar to micromolar) and specificity by folding into secondary and tertiary structures. Aptamers are identified with a combinatorial method called systematic evolution of ligands by exponential enrichment (SELEX) because their function cannot be simply designed based on the primary structure predictable interactions . Since aThe availability of various strategies for the functionalization of NA scaffolds has made it extremely advantageous to use such synthetic sequences as recognition elements and signal probes or as probe linkers/amplifiers. When employed in amplification methods such as hybridization chain reaction and catalytic hairpin assembly as strand-mediated signal amplifiers, NA scaffolds specifically hybridize the target molecule, thus triggering DNA amplicons’ continuous production. The cascade event responsible for the amplification is specific and allows achieving exceptional detection limits . Such amSeveral reactive groups operating as signalling moieties  or anchoring tags  can be easily added to the DNA sequence to identify the specific interaction with the target molecule in sensing applications. In particular, when a DNA sequence is functionalized with fluorophore/quencher terminal groups, its shape transitions caused by the target hybridization can increase the distance between a tagged fluorophore and another fluorophore or a quencher, leading to alterations in the Förster resonance energy transfer (FRET) properties and, consequently, in the produced optical signal. Hairpin-shaped molecular beacon (MB) probes operate based on the above-described mechanism. The MB recognition of a NA target causes the linearization of the hairpin structure and the detection of the hybridization event by fluorophore/quencher pair . Along t2+ H+) [NA nanotechnology has contributed to improving biosensor design and performance by exploiting the conformational polymorphism of DNA sequences, through the self-assembly of multiple NA fragments to fabricate innovative scaffolds called DNA origami . DNA ori2+ H+) .The technical aspects related to the design, assembly, and characterization of DNA origami structures have been already discussed in excellent reviews –36 and aIn 1996, Tyagi and Kramer reported MB technology’s first application for single-strand DNA detection . MB is bMBs’ selectivity depends on the stem sequences designed to ensure interactions keeping a stable close-state arrangement for MB and favour the conformational change after the hybridization with the target. Instead, MBs’ sensitivity depends on the signalling pair (fluorophore and quencher) attached to stems end termini. A fluorescent signal produced in the close-state conformation and/or a low-intensity signal generated in the open-state conformation translate into a reduced detection sensitivity.Criteria for achieving optimal stability for MBs include its length, usually ranging between 25 to 35 nucleotides, sequence, guanine-cytosine (GC) content of both the stem and loop sequence, and the melting temperature of the MB-target duplex. The loop should include a 15 to 30 single-stranded sequence region complementary to the target sequence. The stem should have a melting temperature 7–10 °C higher than the detection temperature, and its sequence should include 5–7 bps .Various signalling labels are available to obtain MBs producing high signal-to-background ratios. These include inorganic materials, organic compounds , nanomatA typical signalling pair comprises a fluorophore and a quencher  offer superior optical properties, such as broad absorption spectra and narrow emission spectra, high quantum yield, and remarkable photostability, which make them suitable for MB-based biosensing  offer low cytotoxicity, water solubility, and photostability. An MB with CQD and Black Hole Quencher 1 (BHQ1) connected to the stem ends , also exhibit fluorescence quenching properties that have been used in combination with QDs to fabricate an MB nanosensor  amplify the fluorescence signal produced after target hybridization. The QD-C60-labelled MB-modified magnetic nanoparticles efficiently captured DNA targets in the sample then the magnetic force was applied to concentrate the MB/MNP complex to amplify the fluorescence signal for target quantification. The assay enabled rapid detection (<10 min) and, thanks to high signal-to-noise ratio produced by the QD-C60 pairs, a 100-fM detection limit in DNA detection.Graphene and graphene oxide (GO) represent emerging nanomaterials in biosensing. They combine exceptional optical, electrochemical, and electronic properties with an excellent fluorescence quenching activity –58. VariMBs modified with a G-quadruplex scaffold (G4MB) combined with duplex-specific nuclease have been used to obtain a highly selective detection of microRNA-141 . The dupSynthetic NAs have been used as engineering materials to create structures and functional devices with nanoscale precision . RothemuAtomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) are used to confirm the optimal folding of components of the mixture .Molecules and nanomaterials such as proteins , enzymesThe envisaged possibility of adopting biotechnology-based methods for the mass production of DNA origami components  can enabTetrahedral DNAs are among the simplest 3D DNA nanostructures . They caDNA origami nanostructures offer the opportunity to fabricate nanosensors that dynamically change their configuration in response to the analyte’s presence. The shuttling between an open and close conformation of the nanostructure can be triggered by modifying the reaction environment or the target detection . The difSmall solid-state nanopores are not easily obtained while biological nanopores are produced with atomic control, but their use for the detection of large molecules including double-stranded DNA and protein is still limited. DNA origami nanopores open new perspectives for the production of nanopores with accurate geometry and chemical functionality . Two difThe ability to modulate hybrid DNA origami nanopores’ size allows extending the potential of nanopore technologies into biosensing and point-of-care diagnostics. The translocation of a single molecule through the nanopore modulates the electric or electro-optical detected signal, enabling selective detection and quantification . ProgramHybrid DNA origami nanopores display peculiar features for single-molecule detection  since thDensely packed 3D DNA nanostructures have also been considered for their stability and resistance to nuclease and high temperatures –91. SeemCaenorhabditis elegans (C. elegans), whereas Kim and co-workers [DNA nanostructures have also been employed for in vivo imaging. Bhatia and co-workers  synthesi-workers  used a fDNA nanostructures can improve the antifouling properties of biosensing surfaces . The minMB and origami scaffolds are here compared to highlight the pros and cons in biosensing of structures resulting from the programmable design of interactions between DNA sequences. In this respect, MBs represent the simplest structure discussed, whereas multidimensional origamis are the most complex systems whose structure and function can be designed mostly based on Watson-Crick base pair.The application of the conventional stem-loop hairpin structure of MB probes, including fluorophore/quencher signalling pair, allows for measuring an instantaneous signal emission directly in solution, without removing unreacted MB molecules, in one single “mix-and-measure” step. MB-based sensing approaches can operate in the open-to signal or close-to signal configurations, depending on the design and the conformation of the MB probe . The opeBesides MB-based homogeneous biosensing methods, stem-loop hairpin structures can also operate as sensing probes after their immobilization on surfaces . In thisMB-based homogeneous biosensing is faster than solid-state biosensing because hybridization reactions are kinetically favoured. MB-based solid-state biosensors’ analytical performances are also negatively affected by steric hindrance and slow kinetics of the diffusion of target molecules to the sensing surface. Moreover, the fabrication of MB-based solid-state biosensors involves labourious protocols for the immobilization of the MB probe, ensuring an optimal spatial orientation and availability of the anchored probe . NeverthDNA origami scaffolds help overcome the limitations of MB-based solid-state biosensors related to the constrained availability of the sensing probe to the target. Such a possibility improves the performance of the biosensing device, particularly when its surface is exposed to complex biological fluids, and optimal control of probe packing density and target hybridization efficiency is required . The senLiu et al.  fabricatThe use of conventional MB scaffolds for the real-time monitoring of targets in living cells represents another challenging application hindered by limitations in cell membrane permeabilization and efficient delivery of the MB probe into the cytoplasm . SeveralAs already mentioned, many attempts have been made to extend the modest half-life of conventional MBs for in vivo applications by using nanomaterial-based signalling and quenching moieties. The modification of the MB backbone or the use of DNA mimics   . MBs’ abThe multiplexed detection of exosomal microRNAs can be used to develop innovative diagnostic approaches based on the analysis of liquid biopsies . In thisThe optical detection of microRNA has also been performed with the DNA “points accumulation for imaging in nanoscale topography” (PAINT) approach using synthetic DNA nanostructures as a programmable microRNA capture array –137. TheThe plasmonic enhancement in the gap between metal nanoparticles or collective chiral plasmonic systems can be exploited to improve the optical detection process’s sensitivity significantly. DNA origami nanostructures help precisely tune the interparticle gap between assembled plasmonic nanoparticles by exhibiting drastic SERS effects allowing single-molecule detection . Funck aMetal nanoparticles can constitute a plasmonic hotspot in the DNA scaffold , 140 to Table Over the past two decades, NA sequences have been used to assemble a plethora of nanostructures exploiting complementary base pairing, straightforward programmability of interactions between NA sequences, and NA structure rigidity and stability. The efforts paid have identified NA nanotechnology as an emerging and fascinating new field with substantial implications for biosensing. Here, we have summarized recent advances in MBs and DNA origami structures by critically evaluating their intrinsic role as biosensing elements. MBs belong to a more conventional class of NA structures used in biosensing, whereas DNA origami structures are fabricated by fully exploiting possibilities offered by NA nanotechnology.MB-based structures detect NAs (including DNA and microRNA) with satisfactory performances. Different strategies have been adopted to fabricate MB devices well-performing in biosensing. These include MB self-assembly or combination with nanomaterials such as AuNPs, QDs, and superquenchers.Pushing the limit of detection of MB platforms to the sub-picomolar range would bring more opportunities to detect targets such as circulating tumour DNA (ctDNAs), viral DNA or RNAs, and small molecules available in biological fluids at low concentrations. Along this way, conventional hairpin structures, acting as strand-mediated signal amplifiers, combined with 3D DNA origami structures, enhance the signal amplification, thus allowing achieving exceptional detection limits through continuous DNA amplification reactions.The challenging label-free single-molecule detection of complex targets, such as viruses or exosomes, in biological fluids (plasma or serum) remain a prerogative of the more recent origami nanostructures. Moreover, these structures’ programmable nature at the nanoscale level enables multiplexed detection of more than one biomarker in a single assay.Origami nanostructures help improve the control of probe spacing and orientation at the biosensing interface, improving detection sensitivity and selectivity. The stability in vivo of origami nanostructures is better compared with MBs. Besides, they exhibit unpredictable properties such as permeability across the cellular membranes, leading to several exciting applications when combined with other nanomaterials.2+ at a concentration of about one order of magnitude higher than its concentration in blood and tissues. Such a difference may cause the denaturation of the nanostructure in biological environments even though various DNA origami nanostructures keeping their integrity in low magnesium buffers have been described [Despite all these examples, some key challenges still need to be addressed before routinely translating DNA origami nanostructures into robust devices for biosensing applications. The folding of DNA origami nanostructures requires thermal annealing in the presence of a minimum concentration of cations to prevail the negative charge-repulsion of the backbone. Origami nanostructures are usually formed in a buffer comprising Mgescribed . Such a escribed .DNA nanostructures are assembled from a scaffold structure held in place by many ssDNA staples. The large number of ssDNA sequences required to assemble such structures hampers the massive production of origami-based biosensors due to production and cost restrictions. However, new possibilities paving the way toward the large-scale production of DNA nanostructures have been recently identified. Praetorius et al. defined an innovative protocol for litre-scale ssDNA production and large-scale DNA origami assembly . The proMBs and DNA origami nanostructures, each with own strengths and weaknesses Table , will he


While the development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines was rapid, time to development and implementation challenges remain that may impact the response to future pandemics. Trained immunity via bacille Calmette-Guerin (BCG) vaccination (an antigen agnostic strategy) offers a potential intervention against future novel pathogens via an existing, safe, and widely distributed vaccine to protect vulnerable populations and preserve health system capacity while targeted vaccines are developed and implemented. Since emerging as a global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 325 million cases of coronavirus disease 2019 (COVID-19) and over 5.5 million deaths globally (as of January 2022) ) versus the unvaccinated group (299 [10.4%]) ) . HoweverDespite the above caveats, recent data suggest the concept of BCG-induced immunity merits further prospective investigation . While iA potential clinical benefit of the BCG vaccine for future pandemics will not be realized without programmatic implementation. Fortunately, widespread BCG vaccine distribution is feasible and economically viable; 100 million doses of BCG are already being manufactured annually, with scope for additional manufacturing capability . FurtherIt is important to consider whether readily available products such as BCG or other live-attenuated vaccines could be utilized in the response to the next pandemic before a targeted vaccine is developed and implemented . BCG hasBased on the data discussed above, we envision BCG as a supplemental strategy in addition to mRNA vaccines for SARS-CoV-2 in immunocompetent adults. The enhanced protection afforded by the BCG vaccine against novel variants or in times of increased community transmission may also have the advantage of preserving the workforce, specifically frontline healthcare professionals and other essential workers. A major limitation of the studies evaluating trained immunity and BCG is that the data are largely retrospective. Another limitation with the use of BCG vaccine as an adjunct strategy is the variability in the strains of BCG used in previous investigations, such as those evaluating childhood mortality. Evidence from a study of vaccinated infants suggests the BCG-Denmark strain (Statens Serum Institute) is more immunogenic than the BCG-Bulgaria or BCG-Russia strains, as measured by both BCG-stimulated and heterologous vaccine-stimulated responses, and that non-target effects may be strain-specific . As a reBCG vaccination induces broad immune effects that may provide protection against other pathogens. While the impact of BCG on COVID-19 remains to be fully characterized, the trained immunity resulting from BCG and other live vaccinations may provide an adjunctive approach to preventing illness from a range of pathogens, both existing and emerging. As COVID-19 continues to demonstrate, the world was not prepared to avoid devastating losses due to a new pathogen, and it is essential that we evaluate all potential tools to be better prepared for the next health threat.


At the beginning of the COVID-19 pandemic, consuming media was critical to identify precautionary behaviors to reduce the spread of the virus, particularly for older adults. Media consumption leads to heightened awareness, but may also negatively affect mental health. We examined whether non-social and social media consumption impacted anxiety and depression relative to pre-COVID-19 symptoms. We conducted an anonymous, cross-sectional survey in May and June 2020. Participants , were asked to estimate their amount of time spent consuming pandemic-related media each day, and to report on anxiety and depressive symptoms both before and after the pandemic onset. We characterized change in anxiety and depression by subtracting scores on current anxiety and depressive symptoms from their recalled symptoms prior to the pandemic. Respondents with high pandemic-related media consumption (>3hrs) were more likely to have increased anxiety, compared to those with low (<1hr) media consumption . Similarly, respondents with increased social media consumption during the pandemic were 64% more likely to have depression, compared to those who did not use social media. This association was bi-directional— those who reduced their social media use were 45% less likely to have depression and 26% less likely to have anxiety, compared to those who never used social media. Older adults consuming more pandemic-related media had increased anxiety. Increased social media consumption was associated with elevated depression symptoms. The potential benefits of media consumption about the COVID-19 pandemic may have unintended negative consequences on mental health.


Although the physiological control of salivary secretion has been well studied, the impact of disease on salivary gland function and how this changes the composition and function of saliva is less well understood and is considered in this review. Secretion of saliva is dependent upon nerve-mediated stimuli, which activate glandular fluid and protein secretory mechanisms. The volume of saliva secreted by salivary glands depends upon the frequency and intensity of nerve-mediated stimuli, which increase dramatically with food intake and are subject to facilitatory or inhibitory influences within the central nervous system. Longer-term changes in saliva secretion have been found to occur in response to dietary change and aging, and these physiological influences can alter the composition and function of saliva in the mouth. Salivary gland dysfunction is associated with different diseases, including Sjögren syndrome, sialadenitis, and iatrogenic disease, due to radiotherapy and medications and is usually reported as a loss of secretory volume, which can range in severity. Defining salivary gland dysfunction by measuring salivary flow rates can be difficult since these vary widely in the healthy population. However, saliva can be sampled noninvasively and repeatedly, which facilitates longitudinal studies of subjects, providing a clearer picture of altered function. The application of omics technologies has revealed changes in saliva composition in many systemic diseases, offering disease biomarkers, but these compositional changes may not be related to salivary gland dysfunction. In Sjögren syndrome, there appears to be a change in the rheology of saliva due to altered mucin glycosylation. Analysis of glandular saliva in diseases or therapeutic interventions causing salivary gland inflammation frequently shows increased electrolyte concentrations and increased presence of innate immune proteins, most notably lactoferrin. Altering nerve-mediated signaling of salivary gland secretion contributes to medication-induced dysfunction and may also contribute to altered saliva composition in neurodegenerative disease. Saliva is secreted by paired parotid, submandibular, and sublingual major salivary glands, along with hundreds of small, minor submucosal salivary glands. A further pair of “tubarial salivary glands” has very recently been identified and may provide a similar function in the oropharynx . Oral he1- and β1-adrenoceptors, and vasoactive intestinal polypeptide receptors play prominent roles in acinar cell stimulation–secretion coupling (Appendix). Acinar cell fluid secretion and ductal cell modification of saliva are driven by electrolyte concentration gradients created by active Na+, K+ ATPase transport in combination with a polarized distribution of cell membrane transport proteins (Appendix). The total protein concentration of saliva is mostly composed of proteins synthesized, stored, and exocytosed by acinar cells (see Appendix). Other non–salivary gland derived proteins in whole-mouth saliva are derived principally from gingival crevicular fluid. IgG is present in whole-mouth saliva at a concentration of approximately 0.016 mg/mL , while serum albumin is present at approximately 0.05 mg/mL  on salivary gland function has been extensively studied. A number of hypotheses have been put forward and evidence accumulated to explain the loss of function in salivary (and other exocrine) glands, and it may well be that a number of disease mechanisms contribute during the prolonged course of SjS . Loss ofSalivary gland involvement is a component of the diagnostic criteria for SjS and is often confirmed by a labial gland biopsy. Measurement of salivary flow rates or assays of saliva components would allow repeated assessments of glandular function to monitor disease activity over time. However, whole-mouth salivary flow rate is not sufficiently specific to confirm salivary gland involvement in SjS. A large study of mixed groups of patients with salivary gland–related disease concluded that measurements of both submandibular/sublingual and parotid gland flow rates were required to provide sufficient specificity in confirming salivary gland involvement in primary SjS . A combiA proportion of patients with SjS complain of xerostomia even though their measured salivary flow rates do not confirm hypofunction, suggesting that the quality and composition of saliva might be changed . The rheRecurrent, chronic sialadenitis of the submandibular or parotid glands is most frequently associated with obstruction of the ductal system due to stones and/or ductal stricture with episodes of microbial infection associated with salivary stasis in the ductal system . ClinicaStudies on animal models have demonstrated that ductal ligation–induced glandular obstruction causes a rapid loss of secretory function that is not dependent on inflammatory cell infiltration of glands . Saliva + counts were seen in Candida-positive compared with Candida-negative HIV-positive subjects, but there was no association between Candida and salivary mucin concentrations. Influenza A (H3N2) is associated with parotitis, and more recently, the presence of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) in salivary glands and association of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with saliva have been described, but it is unclear whether these viruses affect salivary gland function , immunoglobulins, and albumin, which resolved over a 2- to 3-wk period, although secretory function remained lower than the control gland . Severalfunction .Medication and salivary dysfunction were considered in a series of systematic reviews that emerged from the World Workshop on Oral Medicine VI . It is cXerostomia after radiotherapy (RT) for head and neck cancer (HNC) is a very frequent complication that has a major impact on quality of life in HNC survivors. Loss of secretory function in coirradiated salivary glands occurs early following treatment, even before apparent loss of secretory acinar cells, but progresses to approximately 10% of control levels of salivation as secretory cells die and are not replaced . Valdez The severity of xerostomia following therapeutic irradiation can be reduced by using parotid gland–sparing intensity-modulated radiotherapy (IMRT) in treating patients for HNC. In a longitudinal study, at 3 to 6 mo after IMRT, stimulated and unstimulated parotid saliva flow rates were significantly decreased compared with baseline before IMRT but showed some recovery by 12 mo . ComposiA substantial proportion of thyroid cancer sufferers are treated with radioactive iodine (RAI), which is also concentrated in parotid and submandibular glands by expressed sodium iodide membrane symporter proteins. Acute salivary gland hypofunction is seen in the first week following treatment and chronic sialadenitis from 3 mo onward, associated with ductal strictures and obstructive disease, which causes pain and associated salivary gland dysfunction, usually determined by salivary gland scintigraphy .Radioactive actinium (225Ac)– or lutetium (177Lu)–labeled prostate-specific membrane antigen (PSMA)–617 are low molecular weight ligands of prostate-specific membrane antigen and are used in the treatment of prostate cancer. Dry mouth is the main side effect of this therapy owing to uptake of radioligand by PSMA expressing salivary glands causing salivary gland hypofunction as determined by salivary gland scintigraphy . The impAutologous or allogeneic hematopoietic stem cell transplantation (HSCT) is given for the treatment of leukemia, malignant lymphoma, and other blood malignancies and is preceded by a conditioning chemotherapy and/or radiotherapy. An early complication of treatment is oral mucositis due to the conditioning therapy while graft-versus-host reactions may occur acutely or chronically. Salivary flow rates are reduced following HSCT, but the reported time span differs between published studies.A recent review of published studies showed a trend toward decreasing salivary flow rate days and months after HSCT and reported compositional changes in saliva reflecting an inflammatory response directly after HSCT, followed by increases in salivary antimicrobial defense mechanisms at 6 mo after HSCT . In a loLewy pathology, are the pathological hallmark of PD and have been found in the submandibular glands as well as in multiple sites of the autonomic nervous system involved in control of saliva production in patients with PD . Sialorrhea is problematic in PD, but the drooling and swallowing difficulties experienced by sufferers are due to loss of muscular control rather than hypersalivation. To reduce drooling and inhalation of saliva, botulinum toxin is sometimes injected into major salivary glands . Intrane with PD . Paradoxary flow .A further study of salivary flow and protein composition in PD patients compared with age-matched control subjects found similar salivary flow rates in both groups, but total protein, amylase, and albumin concentrations were higher in the PD group, whether on or off levodopa, suggesting altered glandular function and oral epithelial integrity .In Alzheimer disease (AD), submandibular but not parotid gland secretion is reduced . A recenParotid gland enlargement has been varyingly reported in bulimia nervosa. An early study reported enlargement in 25% of patients with binge eating syndrome of bulimia nervosa, and it appeared to be correlated with raised parotid salivary amylase and total protein concentrations and increased serum levels of salivary type amylase . DynesenThe incidence and severity of xerostomia have been found to be correlated with the severity of Crohn disease (CD) . ReducedSalivary gland function is dependent on nerve-mediated stimuli, which exert acute control of saliva secretion and can alter function through longer-term trophic influences. A reduction in nerve-mediated stimuli may be a contributory factor in addition to iatrogenic reductions in salivary flow rates due to medications taken by elderly subjects. Salivary gland dysfunction manifested as reductions in salivary flow rate is observed in different diseases and often accompanied by the perception of dry mouth, particularly where there is widespread involvement of salivary glands as in advanced Sjögren syndrome and following therapeutic irradiation. Relatively few studies have addressed how salivary gland dysfunction alters the composition and function of secreted saliva and the mechanisms underlying such disease-induced changes.G.B. Proctor, contributed to conception, design, and data interpretation, drafted and critically revised the manuscript; A.M. Shaalan, contributed to design and data interpretation, drafted and critically revised the manuscript. Both authors gave final approval and agree to be accountable for all aspects of the work.Click here for additional data file.Supplemental material, sj-pdf-1-jdr-10.1177_00220345211004842 for Disease-Induced Changes in Salivary Gland Function and the Composition of Saliva by G.B. Proctor and A.M. Shaalan in Journal of Dental Research


Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease with mortality driven primarily by respiratory failure. Patients with LAM frequently have respiratory infections, suggestive of a dysregulated microbiome. Here we demonstrate that end-stage LAM patients have a distinct microbiome signature compared to patients with end-stage chronic obstructive pulmonary disease. TSC1 or TSC2 genes leading to hyperactivation of mechanistic target of rapamycin (mTOR) signaling [Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease primarily affecting young women. It can occur sporadically or in association with tuberous sclerosis complex (TSC), and is characterized by cells with mutations in the Patients with LAM have a high burden of respiratory infections. In a meta-analysis of 11 studies, the incidence rate of respiratory infections was 43.4 and 58.8 per 100 patients-years in LAM patients receiving mTOR inhibitors or placebo, respectively, with an incidence-rate ratio for respiratory infection of 0.71 (95% CI 0.50–1.02), suggesting that the use of mTOR inhibitors does not increase the risk of respiratory infections and may even be protective [The frequent respiratory infections irrespective of treatment in LAM patients suggests that a dysregulated microbiome could be contributing to disease heterogeneity . For exaP = 0.058). There was no difference in the number of distinct species among disease states (data not shown). Utilizing alpha-diversity measures that account for both the abundance and richness of the microbial community within a given environment, we found that patients with end-stage LAM had significantly lower alpha diversity , Simpson (P = 0.018) and Inverse Simpson (P = 0.018) indices, suggesting that patients with end-stage LAM have lower microbial species diversity than patients with end-stage COPD.Evaluation of the microbiome from end-stage LAM versus COPD lungs revealed distinct microbial community structures between the two disease states. Evenness, or the relative amounts of each species present in an environment, was calculated using the Pielou’s evenness index Fig. A, demonsity Fig. B by ShanProteobacteria predominated the lung microbiota of both end-stage LAM and COPD patients. This is consistent with previously published reports of proportional increases in Proteobacteria with COPD exacerbation events and Global Initiative for COPD (GOLD) stage, likely reflecting disease severity [Streptococcus (Fig. Staphylococcus and Pseudomonas were relatively more abundant in end-stage COPD lungs, whereas Acinetobacter predominated in LAM patients.Principal component analysis of the taxonomic composition of the lung tissue microbiomes revealed distinct clustering and separation between LAM and COPD patients Fig. C along tseverity , 11. HowComamonadaceae, Brevundimonas and Diaphorobacter species [Firmicutes phylum [Proteobacteria phylum was observed in patients during exacerbations [Pseudomonas [Streptococcus [Staphylococcus [Acinetobacter, which has also been observed in the allograft microbiome of single lung transplant recipients [Acinetobacter in murine models of LAM [Acinetobacter [This is the first study to examine the lung microbiome of end-stage LAM patients as a potential feature of severe disease, which was marked by decreased microbial diversity, and possibly a distinct microbial signature in LAM compared to COPD patients. Both end-stage diseases demonstrated microbiota that were distinct from that found in non-smoker control lungs, which have been associated with  species , with bos phylum . Alterats phylum , 14. In rbations  and afterbations , which irbations . The finrbations  may contudomonas , Streptotococcus  and Staplococcus —common pcipients . This sus of LAM  and studtobacter  on the gtobacter  in vitroThis analysis had several limitations: the small sample size due to the rarity of the LAM patient population, the absence of clinical data, the heterogeneity of lung disease in patients with COPD, and the lack of longitudinal samples limited our ability to determine if the observed dysregulated microbiome was a consequence of immunomodulator therapy, repeated infections, antibiotic use, or a specific feature of severe progressive LAM. The use of end-stage tissues also may not necessarily represent active pathology, although diffuse disease involvement throughout the lungs may minimize sampling bias in small potentially heterogenous tissue samples. Our study was strengthened by using lung tissue as opposed to bronchoalveolar lavage or sputum samples, which can be contaminated by upper respiratory tract or oral flora, although extraction of adequate amounts of microbial DNA can be challenging in samples dominated by host DNA. The results of this exploratory study suggest that further evaluation of the contribution of microbial populations in LAM patients may represent a novel area of research for the field.