CABG: p < 0.001; CTRL vs. MAS: p < 0.001). NETs correlated positively with FIBTEM mean clot firmness (MCF) in septic shock patients  while they correlated negatively in surgical patients . Flow-cytometric quantification of NETs showed a significant increase in free-circulating NETs under inflammatory conditions. Furthermore, this study hints to an association of the number of NETs with hypercoagulation in septic shock patients and hypocoagulation in surgery-induced inflammation.This proof-of-concept study aimed to evaluate a novel method of flow cytometry-based quantification of neutrophil extracellular traps (NETs) in septic shock patients and to identify possible interactions between the number of free-circulating NETs and alterations of the coagulatory system. Patients suffering from septic shock, a matched control group (CTRL), and patients suffering from systemic inflammation after cardiac (CABG) or major abdominal surgery (MAS) were enrolled in this prospective proof-of-concept study. Compared to the matched controls, free-circulating NETs were significantly elevated in septic shock and postsurgical patients (data are presented in median (IQR)); septic shock: (2.7 (1.9–3.9); CABG: 2.7 (2.1–3.7); MAS: 2.7 (2.1–3.9); CTRL: 1.6 (1–2); CTRL vs. septic shock:  Despite tremendous efforts to develop new diagnostic and therapeutic approaches, septic shock still remains associated with high mortality. Particularly, patients suffering from septic coagulopathy are considered to be at high risk for limited outcome ,2. This Neutrophil extracellular traps (NETs) were identified as major players in immunothrombosis ,17. ConsElevated plasma levels of NETs were first identified in autoimmune and cancerous diseases, but current studies proved their crucial role in the development of sepsis ,30,31,32Since NETs may offer an opportunity for novel diagnostic and therapeutic approaches, the need for robust and clinically feasible quantification of NETs arises. Until today, fluorescence microscopy remains the most established method for NET quantification; however, this method has some limitations. First, it only analyzes an abstract of a sample; second, it is not feasibly for daily intensive care unit (ICU) routine; and third, it is highly dependent on expert scientists’ skills and interpretations. In contrast, flow cytometry offers fast and reliable counting of a high number of neutrophils. In 2015, two methods of flow cytometry-based NET quantification were published by two separate research facilities. While Zhao et al. established a combination of high-speed multi-spectral imaging and morphometric image analysis, Gavillet et al. used a direct flow cytometry-based assay for NET identification and quantification ,40. AlthThis proof-of-concept study aimed to investigate the flow cytometry-based quantification of NETs in a clinical intensive care setting. First, we hypothesized that flow cytometry-based quantification of NETs is able to distinguish between matched control and septic shock patients as well as patients suffering from surgery-induced inflammation. Second, we aimed to identify possible interactions between the number of free-circulating NETs and coagulatory dysfunctions using thrombelastography as a practicable solution for use in the clinical routine.This single-center, prospective, observational proof-of-concept study included 80 patients who were enrolled from October 2018 to March 2019 at the University Hospital of Giessen. The study was approved by the local ethics committee  and registered in the German Clinical Trials Register . The methods and results are presented in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines and the Declaration of Helsinki.Patients of legal age were enrolled at the University Hospital of Giessen after signing an informed consent form. If patients were not able to consent to the study, consent was obtained through their legal representatives. General exclusion criteria comprised an age <18 years, current pregnancy or nursing, history of recent severe trauma, autoimmune disease, severe valvular heart disease, active hematological disease, the need for immunomodulatory medication, or having undergone therapy with extracorporeal life support or renal replacement therapy prior to study inclusion. Septic shock patients must have met the Sepsis-3 definition of septic shock . Since nBlood was collected shortly prior to surgery as well as immediately, 24, and 72 h postoperatively in surgical patients. From septic shock patients, blood was first drawn after admission to the ICU and again after diagnosis of septic shock, followed by blood collections after 24 and 72 h. CTRL patients were asked for blood donation at a single time point. Blood was collected in ethylenediaminetetraacetic acid (EDTA) for flow cytometry, fluorescence microscopy, and ELISA, while citrate tubes were used for thrombelastometry and hirudin tubes for whole blood ristocetin-induced platelet impedance aggregometry. Plasma samples were stored at −80 °C and only thawed once for the ELISA analyses. Clinical data were extracted from the local patient data management system .+-  and 10 µL of myeloperoxidase-(MPO)-antibody . The mixture was then incubated again for 30 min at room temperature in darkness. In the next step, 1 mL of lysis buffer  was incubated for 10 min under the same conditions for red blood cell lysis. Next, 1 mL of 2% bovine serum albumin in phosphate-buffered saline (PBS) was applicated followed by centrifugation (200 g for 10 min). Following this, the supernatant was separated and 300 µL of PBS were applied. Impairment of membrane integrity by red blood cell lysis was checked in healthy controls using the application of 7-aminoactinomycin  technique was used to set the gating borders of MPO- and anti-H3-Histone-antibody. Finally, MPO- and anti-H3-Histone-antibody-positive cells were defined as surrogates for NETs  were used for flow cytometric analysis. In order to avoid detection bias, samples were blinded for flow cytometry. The gating strategy involved three steps: First, leucocytes were targeted, and neutrophils were identified as CD15for NETs . In ordefor NETs  43]. Re. Re+-celTM (1:10 dilution) for 10 min). Afterwards, lysis was stopped using 1 mL of PBS, and centrifugation was performed with 200 g at 20 °C for 10 min. Then, the supernatant was decanted and 3 min of Cytospin®  centrifugation was used for fixation on cover slips followed by staining with 5 µL of anti-H3-histone , 5 µL of anti-human-CD15 , and 10 µL of MPO-antibody  was performed. After incubation for 30 min in darkness, samples were washed with PBS and analyzed with fluorescence microscopy   for four hours, followed by red blood cell lysis , MPO , and interleukin-8 ; these analyses were performed according to the manufacturer’s instructions. The optical density of samples was measured with the recommended absorbance  and analyzed using an automated plate reader .®, Matel Medizintechnik, Hausmannstaetten, Austria) and whole blood ristocetin-induced platelet impedance aggregometry , while the results of all other coagulatory tests were derived from the local clinical laboratory routine. Thrombelastographic assays included NATEM®, INTEM®, FIBTEM®, and EXTEM®, while the clotting and clot formation time , mean clot firmness , and lysis index after 60 min  were analyzed. Whole blood ristocetin-induced platelet impedance aggregometry was performed after stimulation by adenosindiphosphate (ADPtest®), thrombin-receptor activator protein 6 (TRAPtest®), and arachidonic acid (ASPItest®). Furthermore, the severity of coagulopathy was scored using the SAC score [Point-of-care devices were used for thrombelastography (ROTEMAC score .www.r-project.org).First, values were tested for normal distribution using the Shapiro–Wilk test. Parametric data were described with mean and standard deviation while the median and interquartile range were used for non-parametric data. For statistical analysis of differences in the amount of free-circulating NETs between the study groups, the Kruskal–Wallis test was performed followed by the Wilcoxon test for analysis of intergroup differences. The analysis of the variation in the number of free-circulating NETs according to the timepoint was accomplished by applying the Friedman test followed by the pairwise Wilcox test for an analysis of the differences between timepoints within each study group. For this purpose, septic shock patients were compared to their matched controls. The correlation of NETs with respective parameters was expressed as a Pearson´s correlation coefficient. Clinical data, laboratory routine data, and experimental data were recorded in an external database . Data were processed and analyzed using R statistical software version 3.4.2 . Preoperative values of both surgical groups were significantly higher compared to those of the matched control group . Septic shock patients showed a significant increase at onset and over three days compared to their matched control patients . In cardiac surgical patients, the amount of free-circulating NETs peaked immediately after the surgery and decreased significantly after 24 and 72 h, respectively . MAS led to the lowest increase of NETs but gained statistical significance immediately after surgery (p = 0.03). The postsurgical levels of free-circulating NETs did not differ compared to septic shock patients . While MPO showed a significant postoperative increase only in MAS patients , no detectable changes were found in CABG patients. With the exception of a significant elevation of IL-8 immediately after CABG, similar results were found for IL-8 expression in CABG patients (p = 0.008), while MAS patients presented a significant postoperative increase in IL-8 . Compared to the control group, changes of HMGB1 levels in septic shock patients almost reached statistical significance (at the onset of septic shock), but showed a significant increase after 24 and 72 h after septic shock onset . The other study groups did not present significant changes in HMGB1 plasma levels . Free-circulating NETs did not correlate to plasma levels of CRP and PCT nor to the leucocyte count . While CTRL patients did not show any alterations of thrombelastographic parameters, septic shock patients showed a statistically significant negative correlation of the FIBTEM CT to free-circulating NETs and a significant positive association with the MCF . Other thrombelastographic assays did not reach statistical significance in septic shock patients . Patients who underwent MAS showed the same tendencies but only reached statistical significance in the association of EXTEM and FIBTEM CFT to NETs . A significant association of NETs to a reduced LI60 could be shown in CABG and septic shock patients; however, the amount of change was minimal . The results of whole blood ristocetin-induced platelet impedance aggregometry did not reveal any correlations with NETs .In the initial analysis of the association of NETs to global coagulatory parameters, no significant correlation between NETs and PTT, INR, and fibrinogen was revealed for any study group. However, thrombocytes were positively correlated with NETs in septic shock patients .Neither the SOFA scores of septic and surgical patients nor the in-hospital death rates of septic shock patients correlated with the number of free-circulating NETs (SOFA: septic shock: This explorative proof-of-concept study evaluated a novel flow cytometry-based approach of quantifying free-circulating NETs in an intensive care setting. Furthermore, to our knowledge, this study is the first study to directly compare NET generation in patients suffering from septic shock to that in those suffering from surgery-induced inflammation. In accordance with other studies that have used various methods to investigate NET release, flow cytometry was able to prove a significant elevation of free-circulating NETs in septic shock and postsurgical patients ,27,31,33Since NETs are associated with sepsis-associated coagulopathy, our study also aimed to investigate whether the amount of free-circulating NETs is associated with clinically relevant coagulopathies in septic shock and postsurgical systemic inflammation ,21,52,53Analogous to the original descriptions of this method, our validation experiments showed a positive proof of PMA-induced NET formation in flow cytometry and fluorescence microscopy ,43. BaseFurthermore, our study has other limitations: First, due to the proof-of-concept study design, we did not perform a sample size calculation. This may offer an explanation as to why this study failed to correlate the number of quantified NETs with outcome parameters. Second, the study does not allow for a conclusion for patients suffering from sepsis without signs of shock. In order to observe a high degree of NETosis, this proof-of-concept study concentrated only on septic shock patients. However, since septic patients lacking shock symptoms are also associated with adverse outcomes, the role of NETs should be further investigated in these patients . Third, This proof-of-concept study investigated the value of flow-cytometric NET quantification in septic shock patients as well as in patients suffering from postsurgical systemic inflammation. The methodology was able to detect NETs in a reliable manner and showed a significant increase of NETs under inflammatory conditions. However, flow cytometry-based NET quantification did not distinguish between septic shock and postsurgical inflammation, casting doubt on the discriminative power of this method. Furthermore, this study showed a clinically apparent procoagulatory shift in septic patients that was associated with the free-circulating NETs. In contrast, NETs deriving from surgical patients were negatively correlated to fibrinogen-associated thrombelastographic assays. Particularly, the association of free-circulating NETs to a procoagulatory shift in septic shock may offer a therapeutic target worthy of further research. In summary, flow cytometry offers a practicable solution for the quantification of NETs in an intensive care setting. Further investigations are necessary to explain the underlying mechanisms leading to the opposing coagulatory reactions in septic and postsurgical inflammation.


Pulmonary embolism (PE) is a commonly encountered clinical entity in patients with coronavirus disease 2019 (COVID-19). Up to 1/3 of patients have been found to have PE in the setting of COVID-19. Given the novelty of the virus causing this pandemic, it has not been easy to address diagnostic and management issues in PE. Ongoing research and publications of the scientific literature have helped in dealing with COVID-19 lately and this applies to PE as well. In this article, we attempt to succinctly yet comprehensively discuss PE in patients with COVID-19 with a review of the prevailing literature.  Coronavirus disease 2019 (COVID-19), which first emerged towards the end of the year 2019, was declared a pandemic by the World Health organization (WHO) on March 11, 2020 . BesidesPatients suffering from COVID-19 have a hypercoagulable state, the pathogenesis of which is not completely understood . VirchowThe effect of COVID-19 on the coagulation system can be assessed by complete blood count, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and d-dimer values. The common abnormalities include elevated D-dimer and fibrinogen level, normal or mildly prolonged PT and aPTT, and normal, mildly reduced, or increased platelet counts . There iThough coagulopathy in COVID-19 can manifest with an array of clinical features, the main clinical finding is related to that of thrombosis. In contrast, acute Disseminated Intravascular Coagulopathy (DIC) leads to bleeding issues predominantly. The DIC scoring system created by the International Society on Thrombosis and Haemostasis (ISTH) in 2009  might hi2, and had underlying comorbidities such as hypertension, diabetes, and cardiovascular diseases [2 and comorbidities such as chronic kidney disease; malignancy and ulcerative colitis were most commonly noted [n = 25) while DVT in 1. All patients were on thromboprophylaxis [Clinical manifestations related to the thromboembolic phenomenon in COVID-19 have a wide spectrum and vary broadly among the patient population affected. Venous thromboembolism (VTE) includes both DVT and PE while arterial thrombosis manifests as stroke or limb ischemia,.As mentioned above, VTE that includes PE can be present in up to 1/3 of patients with COVID-19 admitted to an ICU . In a stdiseases . In a prly noted . 184 seqphylaxis . A multiphylaxis . In a caphylaxis . The incphylaxis . In anotphylaxis  , VTE prophylaxis should be used unless there exists a contraindication such as active bleeding. VTE prophylaxis is also recommended for peri-operative patients with COVID-19 and those who are in the hospital before or after delivery of a child . Low molIf there is definite evidence of PE as determined by CTA chest, then initiation of full-dose anticoagulation (AC) with either LMWH 1 mg/kg subcutaneous injection every 12 h, UH based on thromboembolic nomogram or fondaparinux in case of heparin-induced thrombocytopenia (HIT) can be used unless a major contraindication exists . Full-doCHEST journal in August 2020, patients initiated on therapeutic AC who continue to deteriorate can be considered for catheter-directed thrombolysis if not deemed to be a candidate for systemic thrombolysis [Sub-massive PE or intermediate-risk PE is not associated with hypotension, bradycardia, and does not cause cardiac arrest. However, in sub-massive PE, there are signs of right ventricular (RV) strain as evidenced by increased RV/LV (Left ventricular) ratio > 0.9 in echocardiogram or CT chest, impaired RV systolic function, elevated brain natriuretic peptide (BNP) or pro-BNP, new ST-T wave changes in the electrocardiogram and elevated Troponin I. Massive PE or high-risk PE is used to describe patients with sustained hypotension (systolic blood pressure < 90 mm Hg), bradycardia or cardiac arrest . Discussmbolysis . For patmbolysis . The Ekombolysis . In the mbolysis . There hmbolysis . A summaThere are certain challenges in the diagnosis and management of PE in COVID-19. Firstly, the clinical manifestations of acute PE itself may mimic or have commonality with acute hypoxic respiratory failure due to COVID-19 pneumonia or acute respiratory distress syndrome (ARDS). A large proportion of patients with moderate to severe COVID-19 may have acute kidney injury or worsening of chronic kidney disease making the definitive CTA chest a difficult proposition. Physicians might have to rely on clinical judgment more often than desired. Safe transfer of patients with proper use of protective personnel equipment within the hospital for imaging or catheterization laboratory and out of the hospital for transfer to a higher center can both be challenging. Patients with severe hypoxemia may not have enough reserve to counteract the burden added by a clot in the pulmonary artery ensuing RV failure and thus PE may add to the sudden fatality of COVID-19 patients. Special populations such as pregnant patients might have to endure extra caution due to the management of acute PE and AC during the peripartum period. The elderly population discharged on AC needs extra surveillance for bleeding complications . In termPE in COVID-19 certainly can be a frequently encountered entity for physicians in ICU as well as non-ICU settings. Even the primary care physicians may have to be in loop to carry out the post-discharge follow up plan to prevent a multitude of potential complications. Thus, it is prudent that we understand and participate in an ever-evolving evidence-based discussion about the challenging and dreaded aspect of dealing with PE in COVID-19 patients.


Severe acute respiratory syndrome coronavirus-2 infection has become a pandemic disease (coronavirus disease 2019). The infection has moved from China to the rest of the world and Italy represents one of the most affected countries. Older adults are more susceptible to develop complications with the consequent highest mortality rates.We report a case of a 95-year-old Caucasian woman affected by pneumonia, initially defined as common aspiration pneumonia in a bedridden patient with vascular dementia, which later turned out to be coronavirus disease 2019 pneumonia during the initial spread of severe acute respiratory syndrome coronavirus-2 in our district. Some features of a computed tomography scan of her chest and her clinical history with known dysphagia had led at first to a different diagnosis with a consequent exposure of health professionals to infectious risk in two distinct hospitals. In this case report, we describe the clinical/imaging features of coronavirus disease 2019 pneumonia and the diagnostic process that led to a correct diagnosis in a nonagenarian with multiple comorbidities.This case report highlights both the possible pitfalls in diagnosing coronavirus disease 2019 pneumonia in very old patients with comorbidities and the greater than expected spread of the infection, even in individuals with reduced interpersonal contacts and no defined epidemiological link. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, has spread globally since the first cases were described in November 2019 in Wuhan, China. Since 21 February 2020, when COVID-19 emerged in northern Italy’s Lombardy region, the number of infected patients in Italy closely followed an exponential trend, with the risk of reaching saturation point for beds in intensive care units. The higher risk of complications and death in older patients was immediately evident . Lung inRespiratory diseases and pneumonia per se are among the major causes of hospitalization in the older population with a marked higher mortality rate compared to the adult population . AspiratIn this case report, we describe the clinical/imaging features of a case of COVID-19 pneumonia, initially defined as common aspiration pneumonia, and its diagnostic process in a very old patient with comorbidities, when the SARS-CoV-2 infection was not yet widespread in Italy.A 95-year-old Caucasian woman was admitted to our Internal Medicine and Geriatrics Department on 2 March 2020. Her symptoms started on 26 February 2020 with onset of fever, cough, and vomiting, which led to her hospitalization in the main general hospital of the town in which she lived, where an initial diagnosis of aspiration pneumonia was made. In the diagnostic workup, she had several comorbidities including arterial hypertension, chronic heart failure, paroxysmal atrial fibrillation, dyslipidemia, stage G4 chronic kidney disease, vascular dementia with deconditioning syndrome, sacral pressure ulcers, and known dysphagia. Home medications were the following: furosemide 25 mg, amiodarone 200 mg, warfarin 5 mg, and pantoprazole 20 mg.2) of 93% with fraction of inspired oxygen (FiO2) of 40%, altered mental status, tachycardia with heart rate of 94 beats per minute (bpm), and high blood pressure (160/80 mmHg). Other relevant features on physical examination were bilateral lung crackles and peripheral pitting edema. On day 1 after admission, a chest computed tomography (CT) scan was performed to better characterize the admission chest X-ray findings  and Citrobacter werkmanii , both sensitive to piperacillin-tazobactam. Based on this information, the empiric antimicrobial therapy started on admission with piperacillin-tazobactam was maintained. A bedside swallowing assessment was also performed, confirming dysphagia to both liquids and solids. During the hospitalization, she had two episodes of high-rate atrial fibrillation (160 bpm) treated with metoprolol and amiodarone, with restoration of sinus rhythm. After hemodynamic stabilization, she was transferred to our ward for geriatric management.She lived at home with her son and a caregiver. At initial interview, both of them denied any travel to areas of high transmission for COVID-19 or contact with people coming from these areas . She had been vaccinated for the seasonal influenza virus. She was almost totally dependent on both basic activities of daily living (BADL) and instrumental activities of daily living (IADL). On admission, she had fever (39.1 °C) and acute respiratory failure requiring oxygen supplementation with arterial oxygen saturation SaO of 93% wA real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for SARS-CoV-2 was not performed on admission, due to the lack of previous history of travels or close contact with a confirmed or probable case of COVID-19 in the previous 14 days, according to the epidemiological criteria disclosed by health authorities at that time . However, after her admission at our ward, the persistence of severe hypoxemia, fever (37.6 °C), cough, and high C-reactive protein (14.68 mg/dl) coupled with reduced pro-calcitonin (0.14 ng/ml) after appropriate antibiotic therapy, prompted us to collect nasopharyngeal and oropharyngeal swabs on 3 March 2020 even in the absence of epidemiological criteria. The rRT-PCR assay tested positive for SARS-CoV-2 infection. She was then isolated and managed by a dedicated health care team, according to the protocol of our hospital. After an initial period of clinical stability with high-flow oxygen, she required continuous positive airway pressure in order to maintain adequate oxygenation. On 25 March 2020 she died of cardiac complications.We described a case of a 95-year-old woman with several pre-existing comorbidities who was affected by COVID-19 pneumonia masked by aspiration pneumonia. If we were not in a SARS-CoV-2 pandemic, this would have been a classic case of aspiration pneumonia in a patient with vascular dementia and dysphagia. However, it turned out to be a case of COVID-19 pneumonia after the initial diagnosis. In our opinion, this case report highlights several important aspects of COVID-19.On 2 March 2020, the date of admission to our ward, 1835 individuals had tested positive for SARS-CoV-2 at rRT-PCR assay in Italy, almost all concentrated in the Lombardy region, and only 35 cases had been tested positive in our smaller region (Marche), mainly in the northern part and not in our city , 10. SomRetrospective studies on Chinese patients hospitalized for COVID-19 showed that the disease has different features in older patients. In fact, they had higher disease severity compared to young and middle-aged patients, with higher Pneumonia Severity Index (PSI) score, higher proportion of multiple lobe involvement, higher C-reactive protein, and lower lymphocytes count . SymptomRadiographic findings of aspiration pneumonia include infiltrates in gravity-dependent lung segments  . On the During hospitalization, two paroxysms of high-rate atrial fibrillation occurred, which is a negative prognostic factor in COVID-19, together with both high NT-proBNP and troponin I levels . This teIn conclusion, this case report highlights how the diagnosis of COVID-19 pneumonia could be challenging in comorbid older patients, given the possible atypical presentation and the overlapping of other acute and chronic conditions that may complicate the interpretation of clinical, radiological, and laboratory findings. Not least, this case report shows that probably there were undocumented cases of infections with a wider spread of the virus before we became aware of it.


Introduction: As the population ages, increasing prevalence of neurodegenerative diseases will have profound implications for the health care system. Recognizing visual symptoms from neurodegenerative diseases can be challenging, especially in the presence of co-existing eye diseases.Methods: A seven-question survey was completed by attendees at the “neurodegenerative diseases in neuro-ophthalmology” symposium during the 2017 North American Neuro-ophthalmology Society annual meeting using a web-based audience response system. Content included demographics, patient prevalence, and perceived barriers.Results: Fifty-five practicing neuro-ophthalmologists  participated in the survey. Twenty (36%) had <5 years of experience, and 19 (32%) had >15 years of experience. Forty-one (75%) reported seeing patients more than five half-day/week. Thirty (55%) reported that at least 1 of 10 or 1 of 20 new patients referred have a prior diagnosis of a neurodegenerative disease. Twenty-one (40%) of the respondents reported attributing visual complaints to higher order effects in at least 25% of patients with a prior diagnosis of neurodegenerative disease vs. five (9%) without a prior diagnosis. For those diagnosed with neurodegenerative disease by the neuro-ophthalmologist, reasons for referral were unknown cause of visual symptom (56%), to confirm diagnosis and/or treat visual complaint due to neurodegeneration (29%), and functional disorder (5%). Perceived barriers to diagnosing visual dysfunction due to neurodegenerative disease included difficulty making a referral to neuropsychologists or behavioral neurologists (73%), lack of time for in-depth assessment (62%), lack of tools to assess visual dysfunction due to neurodegenerative disease (40%), and lack of knowledge about presenting signs and symptoms (31%).Conclusion: Visual symptoms from neurodegenerative disease in patients with and without prior diagnoses of neurodegenerative disease are evaluated by neuro-ophthalmologists. Lack of time, resources, and knowledge are barriers to diagnosis. A larger study is warranted to guide programs to improve diagnosis of visual consequences of neurodegenerative disease. As the population ages, the increasing prevalence of age-related neurodegenerative diseases will have profound implications for health care systems . More thVisual symptoms can present as early signs of neurodegenerative disease and, therefore, are relevant to both diagnosis of neurodegenerative disease and symptom management. For example, posterior cortical atrophy (PCA) is a neurodegenerative syndrome with prominent cortical visual dysfunction with early relative sparing of memory and other cognitive domains that is most commonly associated with Alzheimer's disease (AD) pathology .In this issue, Olds et al. describe the largest retrospective dataset regarding symptoms and signs in patients with PCA presenting to neuro-ophthalmology . Data weTo further understand the landscape and challenges faced by neuro-ophthalmologists consulting on patients with neurodegenerative disorders, we conducted a survey among the attendees at the “Neurodegenerative Diseases and Neuro-ophthalmology” symposium during the 2018 North American Neuro-Ophthalmology Society annual meeting. Conferences i/o web-based audience response system was used to conduct the survey. All participants agreed to anonymous extraction of their survey responses for this project. This study was approved by the Institutional Review Board of Stanford University. The seven-question survey included provider demographics, patient prevalence, and perceived barriers.A total of 69 attendees participated in the survey. Among them, 55 were practicing neuro-ophthalmologists and 14 were non-neuro-ophthalmologists or trainees. Responses from 55 practicing neuro-ophthalmologists  were analyzed . Twenty prior diagnosis of a neurodegenerative disease, suggesting that these patients are not uncommon in neuro-ophthalmic practice. Twenty-one (40%) reported attributing visual complaints to higher order effects in more than 25% of patients with a prior diagnosis of neurodegenerative disease. The extrapolated prevalence of higher order visual dysfunction with pre-existing neurodegenerative disorder was 302–6,314/100,000, which would be ~1/3–1/2 of all patients with Alzheimer's disease  if all patients with neurodegenerative disease were to harbor AD. This is similar to the prevalence reported in a very large AD Neuroimaging Initiative Dataset, where Woodward et al. (Thirty (55%) of the respondents reported that at least 5–10% of new patients referred to them had a d et al.  reportedFor new patients without a prior diagnosis of neurodegenerative disease, 10 (19%) respondents reported attributing visual complaints to undiagnosed neurodegenerative disease in more than 5% of patients. For these patients diagnosed with neurodegenerative disease by the neuro-ophthalmologist, reasons for referral included unknown cause of visual symptom (56%), to confirm diagnosis and/or treat visual complaint due to neurodegeneration (29%), and functional neurological symptom disorder (5%). These data reinforce the important role of experts who can distinguish neurodegenerative from non-neurodegenerative-related visual symptoms.In our survey, barriers perceived by neuro-ophthalmologists to diagnosing visual dysfunction due to neurodegenerative disease were multifactorial and included: (1) difficulty making a referral to neuropsychologists or behavioral neurologists (73%), (2) lack of time for in-depth assessment (62%), (3) lack of tools to assess visual dysfunction due to neurodegenerative disease (40%), and (4) lack of knowledge about presenting signs and symptoms (31%). Both neurology-trained and ophthalmology-trained neuro-ophthalmologists perceived lack of time in clinic as a common barrier to diagnosing visual dysfunction due to neurodegenerative disease. More ophthalmology trained neuro-ophthalmologists identified lack of knowledge, difficulty referring to neuropsychologist, and lack of tools in clinic as barriers than neurology-trained neuro-ophthalmologists.Recognizing visual symptoms from neurodegenerative diseases can be challenging, especially in the presence of co-existent eye conditions. Though our survey is limited by selection bias of those who chose to attend a “Neurogenerative Diseases and Neuro-ophthalmology Symposium” and those who agreed to participate, the data collected highlight the role neuro-ophthalmologists are playing in distinguishing visual symptoms from neuro-degenerative and non-neurodegenerative causes for the purposes of diagnosis and treatment as the population continues to age. They also highlight need for education of referring providers and neuro-ophthalmologists to optimize diagnosis and care. Evaluation of visual function in patients with neurodegenerative disorders can be challenging, but familiarity with the type of visual dysfunction most commonly associated with each disorder will allow for identification, focused assessment, and treatment when possible.Though currently no disease-modifying treatment exists for most neurodegenerative diseases that lead to dementia, early diagnosis, and prompt intervention for visual dysfunction, be they related to neurodegenerative disease or not, can improve quality of life. The data published by Olds et al. regarding the presentation of Posterior Cortical Atrophy syndrome to neuro-ophthalmologists  and our YL analyzed the data, prepared the first draft, and edited the manuscript. VP, GS, and HM conceptualized the survey, collected the data, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cannabidiol (CBD) is the non-euphoriant component of cannabis. In 2017, the New Zealand Misuse of Drugs Regulations (1977) were amended, allowing doctors to prescribe CBD. Therapeutic benefit and tolerability of CBD remains unclear.To review the changes in self-reported quality of life measurements, drug tolerability, and dose-dependent relationships in patients prescribed CBD oil for various conditions at a single institution.n = 400) presenting to Cannabis Care, New Zealand, between 7 December 2017 and 7 December 2018 seeking CBD prescriptionsAn audit including all patients  on the EQ-VAS scale describing overall quality of health. Patients with non-cancer pain and mental-health symptoms achieved improvements to patient-reported pain and depression and anxiety symptoms (P<0.05). There were no major adverse effects. Positive side effects included improved sleep and appetite. No associations were found between CBD dose and patient-reported benefit.Four hundred patients were assessed for CBD and 397 received a prescription. Follow-up was completed on 253 patients (63.3%). Patients reported a mean increase of 13.6 points (There may be analgesic and anxiolytic benefits of CBD in patients with non-cancer chronic pain and mental health conditions such as anxiety. CBD is well tolerated, making it safe to trial for non-cancer chronic pain, mental health, neurological, and cancer symptoms. CBD prescription in primary care was legalised in New Zealand in 2017. Previous preclinical trials have shown CBD to have anxiolytic and anti-inflammatory properties but there remains a paucity of studies investigating its therapeutic potential. In this quantitative observational study of the first 400 patients prescribed CBD in New Zealand, CBD was well tolerated amongst patients with a wide range of conditions and symptoms. Quality-of-life benefit was experienced to a greater degree in patients living with non-cancer chronic pain and anxiety-related mental-health conditions, and to a lesser degree in patients with cancer or neurological symptoms.1With the amendment of the New Zealand Misuse of Drugs Regulations 1977 in 2017, CBD has become a legal prescription medicine. The amendment recognises the right of New Zealand doctors to prescribe CBD products that contain no more of 2% of 9-Δ-tetrahydrocannabinol (9-Δ-THC) in the product.Cannabis genus of plants.2 While 9-Δ-THC is the main psychoactive component responsible for euphoria and the ‘high’ associated with marijuana, CBD is the non-euphoriant component.3CBD and 9-Δ-THC are cannabinoids, active compounds found within the 4 Randomised controlled trials (RCTs) have shown that, when CBD is added to existing anti-epileptic medication in patients with these syndromes, seizure frequency decreases.6CBD is currently FDA-approved for the treatment of Dravet and Lennox-Gastaut syndrome, two childhood seizure disorders.7However, CBD shows potential therapeutic use beyond this. Pre-clinical studies demonstrate that CBD has potential anti-inflammatory effects via inhibition of immune cell migration, which may be useful in chronic inflammatory conditions.10 Crippa et al found that in patients with generalised anxiety disorder given 400 mg of oral CBD, there was decreased cerebral blood flow to anxiety processing areas of the brain and a decrease in patient-reported anxiety when compared to placebo.8 CBD in one double-blinded placebo-controlled RCT decreased symptoms of social anxiety disorder patients and fear of public speaking.10 CBD may also reduce psychotic symptoms of schizophrenia.11Moreover, preclinical studies have demonstrated anxiolytic effects of CBD.12 However, due to a paucity of clinical studies, prescribing guidelines are lacking.CBD appears to be safe for patients, with a recent phase I dosage trial showing purified CBD oil is well tolerated up to doses of 6000 mg.The aim of this study was to conduct a clinical audit on the patient population referred to Cannabis Care  for CBD oil. The authors explored the indications for prescribing CBD oil, patient quality of life, patient satisfaction, and self-titrated dosage levels.13The STROBE statement for reporting observational studies was followed.Patients included in this audit were those who were prescribed CBD from 7 December 2017 to 7 December 2018. Patients included in this audit either were referred by their primary care provider or self-referred to the service. Patients were prescribed CBD oil  containing 100 mg CBD/mL in 25 mL bottles administered orally via a dropper. Bottles costed approximately USD300 each, which was self-funded as CBD is not on the New Zealand government-subsidised pharmaceutical schedule (PHARMAC).Patient sex, age, and details of medical condition were recorded at first consultation. Each participant was grouped into one of four broad clinical groups based on their presenting medical symptoms: non-cancer chronic pain symptoms, neurological symptoms, mental health-related symptoms, or cancer symptoms. When a patient presented with symptoms fitting multiple categories, the clinician assigned the patient to the category fitting their primary presenting complaint.14 The descriptive system measures five domains  each with five levels of severity: no problems, slight problems, moderate problems, severe problems, and extreme problems. The EQ-VAS is a 20 cm vertical printed scale from 0 to 100 whereby the upper endpoint of the scale corresponds the ‘best health you can imagine’ and the lower corresponds to the ‘worst health you can imagine’. The patient rated their current overall health on this visual scale to yield a corresponding numerical score.Patients completed an EQ-5D-5L questionnaire at baseline before taking CBD, and again after at least 3 weeks of using the medicine as part of routine clinical assessment. The EQ-5D-5L is a two-part tool consisting of the EQ-5D-5L descriptive system and the EQ Visual Analogue scale (EQ-VAS).Patients also rated their satisfaction with their CBD use at follow-up. They rated their experience as ‘no benefit, good, very good, or excellent’ and reported any side effects. In addition, variations in patient dosage and the duration and frequency of CBD oil intake were recorded where possible.The EQ-5D-5L data was measured on ordinal scales and hence considered as non-parametric data. The EQ-VAS scores were recorded on a continuous scale and treated as a parametric variable. Non-parametric data was presented as median (interquartile range [IQR]) and analysed for differences using the Wilcoxon rank sum test. Change in patient EQ-VAS scores was presented as mean (standard deviation [SD]) and analysed using one-way analysis of variance (ANOVA) to assess any differences between indication categories. Binary logistic regression was used to analyse potential dose-dependent responses, dose of CBD, and patient-reported benefit.P value of ≤0.05 was taken as statistically significant. SPSS software (version 23.0) was used for statistical analysis.Categorical data such as the indications for CBD, patient satisfaction with CBD use, and side effects of use were presented as frequencies. A A total of 400 patients presented to Cannabis Care from 7 December 2017 to 7 December 2018. Three patients did not receive a prescription based on a clinician decision that they would not benefit. Patients receiving CBD prescription consisted of 214 females (53.9%) and 183 males (46.1%), for a total of 397 individuals. The mean age of patients was 51.48 years (SD 19.1). Of those prescribed CBD, 61 patients (15.4%) fit more than one category of indication and were assigned to a group based on their primary condition.Patient indication for CBD prescription is shown in Of the 397 patients initially prescribed the CBD oil, 253 (63.7%) were followed up either through a second appointment with the clinician or by phone. In total, 250 patients (63.0%) reported their satisfaction with CBD use, with three patients (0.8%) refusing to comment. Within these 250 patients, a subset of 110 patients (27.7%) completed before and after EQ-5D-5L questionnaires; 144 patients (36.3%) did not complete follow-up assessment, with 82 patients (20.7%) lost to follow-up and 62 patients (15.6%) choosing not to take the CBD. Reasons for patients not taking the CBD included death, financial barriers preventing purchase of the oil, severe illness, participation in a clinical trial, or consumption of alternative illicit cannabis products.Median follow up duration for patients who completed their CBD prescription was 36 days (IQR 28–65).P = 0.02), ability to complete their usual activities (P = 0.007), self-reported pain (P<0.001), and self-reported anxiety or depression (P = 0.017). Patients with mental-health related symptoms experienced improvements to their ability to carry out their usual activities (P = 0.002), pain (P = 0.039), and anxiety or depression (P = 0.02). Patients with neurological symptoms experienced no statistically significant differences in any of the five domains. Patients with cancer symptoms experienced improvements in pain (P = 0.047). Complete results of EQ-5D-5L questionnaires are shown in Results from Wilcoxon rank sum tests for EQ-5D-5L domains found that patients experiencing non-cancer pain symptoms had a significant improvement of self-reported mobility scores . Overall, EQ-VAS scores represented an improvement in self-reported health. Results of one-way ANOVA tests showed that there were no significant differences between indication groups for the improvement of EQ-VAS scores.Treatment with the CBD in all population groups showed an increase in the EQ-VAS score. There was an overall mean increase of EQ-VAS score of 13.6 points , reported some level of satisfaction with CBD use . Seventy-five patients  reported no benefit from CBD use. There was no statistically significant relationship found between patient age or sex and patient-reported satisfaction.Adverse effects such as sedation and vivid dreams were experienced by 25 out of 253 patients (9.9%). A worsening of a pre-existing condition was reported by 2 of 253 (0.8%) patients upon follow-up. Thirty-eight of followed-up patients (15.0%) reported positive side effects of CBD use, such as improved sleep or improved appetite. Side effects experienced by follow-up patients are summarised in P = 0.145).Amongst those who completed the course of CBD, the dose per day ranged from 40mg/day to 300mg/day. However, dosage information was incomplete, existing for only 110 patients out of the 253 followed-up patients (43.5%). The clinician recommended dosing to at least 100 mg/day. Overall dosage between patients varied widely and was reported inconsistently by patients. Binary logistic regression analysis was conducted for the existing values and there was no significant association between dosage and patient-reported benefit from CBD  reported good to excellent benefit for relatively intractable conditions; 19.0% of those prescribed CBD (or 30.0% of the follow-up group) reported no benefit. CBD is well tolerated in most patients and may be of benefit to patients with various intractable chronic conditions.The strength of this study is that it assesses effects of CBD on a large range of chronic medical conditions in a clinical context. Only three patients were excluded (due to extreme severity of their conditions).15 resulting in an overestimation of treatment effects. However, due to many patients having resistant symptoms, CBD may conversely prove more efficacious in patients with less severe symptoms.There are several limitations to this present audit. There was a large loss to follow-up due to patients not attending follow-up and cost barriers. Hence, the results reported may not be fully representative of the entire patient population. Moreover, patients had to pay USD300 for 2500 mg of the CBD oil, USD150 for an initial consultation, and USD75 for follow-up. Most patients had refractory chronic pain and conditions resistant to conventional treatment uncommon in the wider population, increasing risk of selection bias. These patients are unlikely to be representative of a wider, more generalised cohort of patients. It is difficult to elucidate the effect of selection bias on these findings. The high cost barriers may augment the placebo effect of treatment,The follow-up period was variable, resulting in interviews with some patients who had stopped taking CBD for a period of months. This may have confounded the patient’s recollection of the effects of CBD, likely causing an underestimation of the effect. Some follow-up was completed by the clinician instead of a non-clinical author. This increased the risk of expectation bias by the clinician, likely overestimating the effect. Considering these limitations, results should be interpreted with the appropriate caution.10 and neuro-imaging studies8 have demonstrated the anxiolytic effects of CBD. A recently published case-series in psychiatric patients found a benefit of CBD for anxiety and sleep,16 which is in agreement with the above findings.Numerous preclinical trials19 The apparent benefit of CBD on pain reported by patients in this audit remains difficult to interpret. Current guidelines for the prescription of medicinal cannabis, including both 9-Δ-THC and CBD, recommend it as a third-line treatment for ‘neuropathic pain, palliative and end-of-life pain, chemotherapy-induced nausea and vomiting, and spasticity due to multiple sclerosis or spinal cord injury’.20 However, RCTs investigating the use of CBD alone for the treatment of pain are lacking.On a pharmacological level, preclinical trials suggest CBD’s analgesic action is due to its effect on certain receptors: TRPV1 and a3 GlyRs, involved in nociceptive transmission. However, human studies are still inconclusive.21 This audit did not find a statistically significant dose-response, likely due to missing data and the aforementioned factors regarding financial cost.The ineffectiveness of the CBD oil for 75 of the 250 patients with available data (30.0%) may be due to lack of patient compliance and inadequate dosing. The expense of the pure CBD 100 mg/mL may have influenced patient attitudes towards this matter. Previous studies investigating CBD’s anxiolytic effect have shown a U-shaped dose curve with highest efficacy at 300 mg as a single-dose.CBD’s ineffectiveness in improving self-reported health in patients in the cancer and neurological groups may be due to the high heterogeneity of the clinical presentations within these groups. Additionally, patients within these groups had more severe disease progression compared to the mental health and non-cancer pain symptom groups, which may have limited efficacy.12 Patient-reported sleep benefits are likely related to these sedative effects. While cannabis (containing both 9-Δ-THC and CBD) has been indicated for use as an appetite stimulant in HIV-affected patients with cachexia,23 it remains unclear if CBD alone has significant appetite stimulating effects beyond placebo. Long-term side effects were not analysed in this current audit and future study is still needed to clarify chronic effects of CBD administration.24Overall, CBD treatment was well tolerated with mild adverse effects, most commonly related to sedation. This is consistent with the findings of a phase I clinical trial showing the main side effects of CBD were related to the gastrointestinal and central nervous system.Some urgency on this topic exists due to the increasing worldwide legalisation of cannabis and its related products. A focus should be placed on confirming the anxiolytic effects of CBD in clinical conditions. Future studies should investigate the effectiveness of full spectrum CBD oil with the retention of terpenes, the essential oils found in cannabis plants. Terpenes may enhance the effects of pure CBD because of a synergistic effect known as the entourage effect.Overall, this audit demonstrated the potential benefit of CBD in treating anxiety and pain. The present study shows that it improves quality of life for a diverse range of patients. CBD in this population has been shown to be safe and well tolerated. However, despite potential biases of patients influenced by the high treatment cost, pure CBD is not effective for all. Benefit was more pronounced in patients who had conditions with less severe disease progression . Due to lack of a control group, high drop-out rate, and an extreme patient population, these study results should be interpreted with caution. Future studies should investigate effects of long-term CBD use, which could not be analysed in this present audit.


Dear Editor,2. Home or virtual visits by community midwives should be provided to reduce the number of times women and newborns need to leave their home1. In order to comply with social distancing requirements, community midwives could deliver home or virtual visits and individualized postnatal care, according to the needs of the mother and the newborn1. Midwifery-led postnatal home visiting could raise consciousness in parents on establishing breastfeeding, family planning and contraception after birth, preparation of milk formula, smoke-free environments at home, home hygiene, and care of the neonatal – especially after NICU discharge3. Midwifery-led virtual visits via videoconferencing decrease the number of visits to health facilities and enable mothers to be consulted immediately and from their own home4. Virtual visits have been demonstrated to be as safe as in-person visits5. Therefore, as early as possible, community midwives  should receive all the relevant technology equipment and training regarding remote consultation5.During the COVID-19 pandemic postnatal midwifery-led care remains a key public health intervention for women and their families6. However, a face-to-face consultation is required for physical examination and/or screening of mother and the newborn1. Therefore, home visiting should be prioritized for women with psychosocial vulnerabilities, operative birth, premature or low birthweight baby and other medical or neonatal complications1. Awareness should be raised about exposure to COVID-19 during a home visit, where midwives should abide by strict infection control equipment and procedures when entering and leaving homes1.The World Health Organization (WHO) recommends at least four postnatal visits for all mothers and newborns, on day 1 (first 24 hours), on day 3 (48–72 hours), between days 7–14 and six weeks after birth1. Μothers with COVID-19 should be advised to establish breastfeeding or to express breastmilk, applying appropriate infection prevention and control measures7. All breastfeeding women should be shown how to hand-express their breast milk and be advised on how to correctly store and freeze it. Breastfeeding counseling, neonatal care, family planning counseling, psychosocial parent support, practical feeding support and home hygiene should be provided to all mothers and their families, whether they or their children have suspected or confirmed COVID-197.Midwifery-led services are vital during the postpartum period for women suspected or confirmed with COVID-19. Parenting and breastfeeding support can be offered through face-to-face or virtual visits8. They need adequate support8 to provide quality, holistic, women-centered postnatal care to mothers and newborns9 in these exceptionally complex circumstances in order to promote safe and respectful10 family-centered care, during periods of crisis including the current COVID-19 pandemic.Community midwives are a valuable resource either providing face-to-face visits or virtual visits


To investigate deviations between planned and applied treatment doses for hypofractionated prostate radiotherapy and to quantify dosimetric accuracy in dependence of the image guidance frequency.Daily diagnostic in-room CTs were carried out in 10 patients in treatment position as image guidance for hypofractionated prostate radiotherapy. Fraction doses were mapped to the planning CTs and recalculated, and applied doses were accumulated voxel-wise using deformable registration. Non-daily imaging schedules were simulated by deriving position correction vectors from individual scans and used to rigidly register the following scans until the next repositioning before dose recalculation and accumulation. Planned and applied doses were compared regarding dose-volume indices and TCP and NTCP values in dependence of the imaging and repositioning frequency.Daily image-guided repositioning was associated with only negligible deviations of analyzed dose-volume parameters and conformity/homogeneity indices for the prostate, bladder and rectum. Average CTV T did not significantly deviate from the plan values, and rectum NTCPs were highly comparable, while bladder NTCPs were reduced. For non-daily image-guided repositioning, there were significant deviations in the high-dose range from the planned values. Similarly, CTV dose conformity and homogeneity were reduced. While TCPs and rectal NTCPs did not significantly deteriorate for non-daily repositioning, bladder NTCPs appeared falsely diminished in dependence of the imaging frequency.Using voxel-by-voxel dose accumulation, we showed for the first time that daily image-guided repositioning resulted in only negligible dosimetric deviations for hypofractionated prostate radiotherapy. Regarding dosimetric aberrations for non-daily imaging, daily imaging is required to adequately deliver treatment. Prostate cancer is the most common malignancy in men with an estimated incidence ranging between 100 and 170 per 100 000 people per year –4. ModerWhile the anatomic changes during the course of therapy have been widely studied, the consequences of these changes for the applied radiation doses remain incompletely understood –12. The Here, we analyzed deviations of the applied treatment doses from the treatment plan on a voxel-by-voxel basis using daily planning CT scans in treatment position performed immediately before each treatment fraction. Additionally, the dosimetric impact of different frequencies for positional imaging was quantified. These data will help to define optimal imaging frequencies and to devise adaptive re-planning strategies for hypofractionated prostate radiotherapy.Ten consecutive patients were treated with definitive prostate radiotherapy at the German Cancer Research Center and were included in this analysis. All patients presented with low or intermediate-risk prostate cancer limited to stages T1c to T2b, PSA values ranging below 20 ng/ml and Gleason scores not exceeding 7 . No patient received implanted fiducials prior to radiotherapy. The clinical target volume (CTV) covered the prostate gland for low-risk tumors and also the base of the seminal vesicles for intermediate-risk cancers. The planning target volume (PTV) comprised an additional 7 mm as a setup margin. Treatment plans were generated based on the hypofractionated arm of the CHHiP trial to a total dose of 60 Gy in 20 fractions of 3 Gy  directly adjacent to the linear accelerator. All patients received daily diagnostic CT imaging in treatment position as a means of position verification. The in-room CT scanner had been approved for treatment planning scans, and all scans were taken to the same specifications used for the individual planning examinations.All target volumes and OARs were contoured by a board-certified radiation oncologist according to current guidelines both on the planning CT scan and the daily in-room CT scans , 25. Daimean), doses to x% of total volume (Dx) and volume at x Gy doses (Vx). Dose conformity and homogeneity was quantified and compared using the conformity index (CI), the conformal index (COIN) and the homogeneity index (HI) for the total prescription dose as described previously, and dose uniformity was assessed using gEUD . No significant deviations were observed for the dose conformity indices of the CTV , and a small decrease was only noted for dose homogeneity . As a result, the TCP and P + values for the CTV were comparable for the planned and applied treatment doses  (98 deviating by 20.5% (p = 0.002) compared to the planned D98. In the high-dose region, only the applied V55 deviated from the plan by 8.9% (p = 0.002), while the D5 and D2 values were comparable .Deviations of the applied doses were compared with the treatment plan regarding the CTV, PTV, bladder and rectum . Due to for P +) . For themean and D50 varied by 8.2 and 39.8% from the planned doses, respectively ; however, no statistically significant difference could be obtained for any tested dose-volume parameter. For the rectum, the accumulated average Dmean and D50 values deviated by only 2.2 and 5.0%, respectively , and all tested dose-volume indices did only show non-significant variations from the treatment plan (p = 0.322) or the rectum (p = 0.770)  .To quantify the dosimetric consequences of various non-daily position verification CT schedules in comparison to daily positional imaging, CT-based correction vectors were derived on days 1, 3, 5, 6, 8, 10, 11, 13, 15, 16, 18, and 20 for the thrice weekly schedule, on days 1, 4, 6, 9, 11, 14, 16, and 19 for the twice weekly schedule and on days 1, 6, 11, and 16 for the weekly schedule, and were used to register all consecutive scans up to the next positional imaging onto the planning scan. Resulting applied doses were accumulated and compared to the dose accumulation resulting from daily CT-based repositioning.5 , the D2 , the D1cc  and the V55 .For the CTV, no significant dosimetric deviations of the applied from the planned doses were observed, when positional imaging was simulated thrice weekly. For the twice weekly and weekly schedules, the V55 and D1cc deviated significantly from the treatment plan, while TCP and P + values were comparable . In cont5 in all non-daily imaging schedules , and the dose uniformity was reduced for thrice weekly (p = 0.049) and weekly CT-based positional correction (p = 0.027). All other dose volume parameters for the accumulated doses did not significantly deviate from the plan, likely due to a large inter-individual variability regarding bladder volumes   . For thep = 0.002), 4.9% for twice weekly imaging (p = 0.006) and 7.1% for weekly imaging (p = 0.010)  .Dosimetric deviations in prostate radiotherapy resulting from intra- and interfractional anatomic variability have been incompletely understood, and an in-depth comprehension about the dosimetric impact of position verification imaging becomes more important as individual fraction dose are increasing for hypofractionated or even stereotactic treatment approaches , 19, 30.This dataset provides for the first time a comprehensive and voxel-based analysis of the dose deviations resulting from different imaging and repositioning frequencies for hypofractionated prostate radiotherapy. As daily planning quality CT scans and a state-of-the-art elastic image registration tool were used in this analysis, applied doses to the target volumes and organs-at risk could be precisely accumulated for each treatment fraction and each patient. Our data revealed that daily CT-based position correction did not lead to significant dosimetric deviations of the applied from the prescribed treatment doses irrespective of the daily pelvic anatomy. However, repositioning based on non-daily positional imaging resulted in significant dose differences especially in the high-dose range and diminished dose conformity and homogeneity to the target volume in case of hypofractionation.Previous analyses have employed varying strategies to accumulate fractional doses for prostate cancer radiotherapy on the basis of widely available but low-quality CBCT imaging, including enhanced or iterative CBCT approaches, portal dose measurements or rigid registration strategies, albeit with limitations regarding dose mapping , 31, 32.The available evidence concerning the ideal frequency for positional imaging remains somewhat inconclusive. A previous publication reported improvements in target volume coverage and a reduction in high doses to the rectum for daily CBCT imaging and repositioning based on registration . It has Although our analysis is based on daily diagnostic-quality CT imaging and state-of-the-art elastic image registration and is therefore able to provide comprehensive voxel-wise dosimetric data, it has limitations: Beyond interfractional variability and setup errors, applied doses may also be influenced by intrafractional motion of the prostate that could not be quantified in our dataset. As out chosen methodology did not allow for intrafractional imaging, the impact of intrafractional variability for hypofractionated prostate radiotherapy needs to be addressed separately. All patients analyzed here received pre-treatment coaching about rectal and bladder filling as well as feedback in case of notable deviations of their pelvic anatomy from that of the planning CT. This coaching and a resulting consistency in rectal and bladder filling may have contributed to the relatively moderate dosimetric deviations observed in the simulated non-daily imaging schedules, and patients without coaching may in fact demonstrate significantly higher dosimetric deviations for non-daily imaging-guided repositioning. Additionally, generous PTV margins of 7 mm may at least have partly compensated for the deficiencies of all non-daily imaging schedules and hence to the reasonable TCP values observed. While the seminal CHHiP hypofractionation trial used PTV margins up to 10 mm, several newer clinical trials with daily positional imaging have employed smaller PTV margins ranging at around 5 mm , 43; theThe transferability of our data to high-risk or locally advanced prostate cancers as well as other concepts of hypofractionation warrant further analyses, as differing treatment margins or treatment times may also influence dose-volume parameters.Nevertheless, we provide for the first time a comprehensive and voxel-by-voxel analysis of the dosimetric effects of interfractional variations for hypofractionated prostate radiotherapy and the resulting consequences for the frequency of position verification imaging. These data will also help to devise strategies for adaptive planning of hypofractionated radiation treatments.Using voxel-by-voxel dose accumulation, we showed for the first time that daily image-guided repositioning resulted in only negligible dosimetric deviations for hypofractionated prostate radiotherapy. Regarding the observed dosimetric aberrations for the simulated non-daily imaging algorithms, daily imaging seems to be required to adequately deliver treatment. These data will help to develop adaptive re-planning strategies for hypofractionated prostate radiotherapy.The datasets presented in this article are not readily available because German federal laws protect patient data and prohibit these data from being transferred or shared. Requests to access the datasets should be directed to nils.nicolay@uniklinik-freiburg.de.The studies involving human participants were reviewed and approved by Independent Ethics Committee of the University of Heidelberg. The patients/participants provided their written informed consent to participate in this study.TB, PH, JD, and NN planned and carried out treatment. MS, IS, TB, TF, OJ, DB, and NN analyzed the data. NN wrote the manuscript. IS, CZ, CT, PH, and DB helped with writing the manuscript. JD helped with data discussion. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and objective: Long-term aspirin use for the primary prevention of cancer remains controversial, and variations in the effect of aspirin use on cancer outcomes by aspirin dose, follow-up duration, or study population have never been systematically evaluated. The objective of this study was to evaluate the effect of aspirin on primary cancer prevention and to determine whether the effect differed according to aspirin dose, follow-up duration, or study population.Materials and methods: Seven electronic databases were searched from inception to September 30, 2019. Randomized clinical trials (RCTs) that compared aspirin use versus no aspirin use in participants without pre-existing cancer and reported cancer outcomes were selected. Data were screened and extracted by different investigators. Analyses were performed using Review Manager 5.3 and Comprehensive Meta-Analysis 2.0. Total cancer incidence was defined as the primary clinical endpoint. Total cancer mortality, all-cause mortality, major bleeding, and total bleeding events were the secondary outcomes. Subgroup analyses were conducted based on aspirin dose, follow-up duration, and study populations.Results: Twenty-nine RCTs that randomized 200,679 participants were included. Compared with no aspirin, aspirin use was not associated with significant reductions in total cancer incidence , total cancer mortality , or all-cause mortality ; however, aspirin use was associated with a 44% increase in the risk of major bleeding  and a 52% increase in the risk of total bleeding events . Subgroup analyses demonstrated consistent results.Conclusions: Long-term aspirin use in individuals without pre-existing cancer was not associated with a significant reduction in total cancer incidence, cancer mortality, or all-cause mortality; however, aspirin use was associated with a significant increase in the risk of bleeding. Therefore, aspirin is not an appropriate choice for the primary cancer prevention. There were approximately 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018 and the cancer incidence and deaths have been rapidly increasing Over the last few decades, continuous long-term aspirin intake has been used as a chemopreventive approach for primary cancer prevention A few previous meta-analyses have evaluated the role of aspirin use in primary cancer prevention, but most of them included observational trials or cohort studies which, compared with RCTs, might weaken the strength of the evidence Aspirin's effect on primary cancer prevention has not been clearly established, and subgroup analyses based on aspirin dose, follow-up period, and study population have not been comprehensively conducted This updated meta-analysis included all eligible RCTs to further evaluate the efficacy and safety of aspirin use for primary cancer prevention and to determine whether the effect differs according to aspirin dose, follow-up duration, or study population.We performed this systematic review and subgroup meta-analysis following the Preferred Reported Items for Systematic Review and Meta-analysis (PRISMA) guidelines Two independent reviewers (QB Wu and HW Chen) performed a comprehensive search of the PubMed, Embase, ClinicalTrials.gov, Anzctr.org.au, Cochrane Library, Google Scholar and ScienceDirect databases without restriction on language or publication period. A conventional search was also performed to find potential studies that were not indexed in the electronic databases. Furthermore, the reference lists of all the related articles were reviewed to identify potential RCTs. The last search date was September 30, 2019. No trials were excluded due to their publication status or language.All RCTs comparing aspirin versus no aspirin (defined as placebo or no treatment) and reporting cancer incidence and/or cancer deaths as outcomes were selected and assessed for inclusion in our research. The trials included in this study met the following criteria: (1) RCT study design; (2) participants without known preexisting cancer (primary prevention of cancer); (3) aspirin at any dose compared with no aspirin; (4) follow-up of at least 1 year; and (5) cancer incidence and/or cancer deaths reported as outcomes.Exclusion criteria were as follows: (1) studies on secondary or tertiary prevention of cancer, treatment of cancer, cancer remission, cancer recurrence or cancer metastases; (2) studies in which the participants were nonhuman populations, pregnant women, institutionalized individuals or postsurgical patients; (3) studies of high-incidence familial cancer syndromes ; (4) trials that were not RCTs; and (5) studies where the full-text article was unavailable or the data were unextractable.All the candidate articles were screened by two independent investigators (QB Wu and HW Chen) on the basis of title and abstract. The full texts were retrieved for further evaluation according to the inclusion and exclusion criteria. All inclusion disagreements were resolved by consensus.Two investigators (QB Wu and XJ Yao) independently rated the included RCTs and extracted the data. An intention-to-treat (ITT) analysis was used to analyze the results whenever possible.We summarized the characteristics of all included RCTs in Table Two independent reviewers (QB Wu and HW Chen) appraised the risk of bias in the included trials using the Cochrane Risk of Bias Tool for Randomized Controlled Trials. If there were disagreements, a third reviewer (ELH Leung) independently repeated the extraction, analysis, and interpretation of the data, and disagreements were solved by discussion until a consensus was reached.Total cancer incidence was defined as the primary clinical endpoint. Total cancer mortality, all-cause mortality, major bleeding, and total bleeding events were the secondary outcomes.We performed subgroup analyses of total cancer incidence, total cancer mortality, all-cause mortality, and bleeding events based on aspirin dose, follow-up duration, and study populations.I-squared test. Substantial heterogeneity was indicated by I2 > 50%, and a random-effects model was used  to estimate the summary RR and 95% CI; otherwise, a fixed-effects model was applied All analyses were performed using RM 5.3, CMA 3.0 and Trial Sequential Analysis (TSA) software . Dichotomous data were summarized as risk ratios (RR) with 95% confidence intervals (CIs). Heterogeneity among the studies was assessed using the Egger's test and funnel plots were applied to examine the potential bias in the RCTs included in the meta-analysis when the number of RCTs was ≥ 10 Subgroup analysis, sensitivity analysis, and Trial Sequential Analysis were applied to assess the robustness of the results and calculate the required sample size in the meta-analysis. The risk of bias for each included study was evaluated by two independent reviewers (QB Wu and XJ Yao) using the GRADE approach As shown in Supplementary These 29 RCTs, which comprised 200,679 individuals, were included   and all-cause mortality rate  were not significantly different between the aspirin and no-aspirin groups , and the fixed-effects model was used to pool the trial results.No significant heterogeneity was found for any of the three outcomes (P < 0.00001) (Table P < 0.00001) ; 100 - 300 mg ; > 300 mg ] (Table P = 0.85); 100-300 mg ; > 300 mg ] (Table P = 0.16); 100-300 mg ; > 300 mg ] ] Table A, total )] Table B, or all)] Table C.The meta-regression analysis showed that total cancer incidence, cancer mortality or all-cause mortality did not vary significantly with respect to daily dose of aspirin (from ≤100 mg to >300 mg) (Supplementary  P = 0.65); 5-10 years ; >10 years ] ] Table A. P = 0.20); 5-10 years ; >10 years ] (Table  P = 0.63); 5-10 years ; or >10 years ] ] Table B or all-)] Table C.The meta-regression analysis indicated that total cancer incidence, cancer mortality or all-cause mortality did not vary significantly with respect to follow-up duration (from 1-5 years to >10 years) (Supplementary  P = 0.78), total cancer mortality  or all-cause mortality   for more than five years did not result in a lower total cancer incidence  Table .P = 0.54), patients with diabetes mellitus , participants with CVD or at increased risk of CVD , individuals at increased risk of cancer , or patients with peripheral arterial disease or venous thromboembolism   Table A. P > 0.05)  Table B and S9CP< 0.00001), 100-300 mg , or > 300 mg ] ] Table A.P < 0.00001); 100-300 mg ; or > 300 mg ] ] Table B.P < 0.05)  Table A, S11B.Generally, there was good homogeneity among the included clinical trials. In particular, the above subgroup analysis results based on the daily dose of aspirin, follow-up duration, and study populations confirmed the robustness of the findings.P = 0.96), study sample size  , and publication year (studies published since the year 2000)  and when studies that enrolled participants with increased risk of cancer were excluded    .P = 0.96)], or follow-up duration (from 1-5 years to >10 years)   needed for a conclusive and reliable meta-analysis  of the outcomes, and the quality of evidence was downgraded by one level . According to the GRADE guidelines, the quality of evidence for the outcomes measured was moderate to high, and majority were of high quality (Table P = 0.348) , cancer mortality (P = 0.81), or all-cause mortality (P = 0.27). The results clearly demonstrated that the current practice of prescribing aspirin as a chemopreventive agent for the primary prevention of cancer brought no benefit to the individuals who underwent aspirin therapy. According to the GRADE guidelines, the quality of evidence for the cancer outcomes  in our study was high.Our data indicated that, compared with no aspirin, long-term aspirin use did not result in a significantly lower risk of total cancer incidence  in the control group and that the cumulative sample size of all included RCTs reached the required size for a conclusive and reliable meta-analysis.Many studies have shown that long-term aspirin use  reduced the risk of developing and dying from cancer and that the benefit increased with the duration of treatment In our study, subgroup analyses based on aspirin dose or follow-up duration showed that different daily doses of aspirin  and different follow-up durations  were not associated with a significant reduction in total cancer incidence, cancer mortality or all-cause mortality. The aspirin dose or follow-up duration did not show any impact on the effect of aspirin, which was not greater for those who received low-dose aspirin or who underwent treatment for more than 5 years.P = 0.78), total cancer mortality (P = 0.33) or all-cause mortality (P = 0.16) with aspirin use. The results of the meta-regression analyses confirmed the above findings of the subgroup analyses. Several previous studies reported that daily use of low-dose aspirin for at least five years reduced the risk of cancer and cancer mortality We also performed a subgroup analysis by only including the participants who used low-dose (≤100 mg/d) aspirin for more than five years. The results showed no significant reduction in total cancer incidence P = 0.7, total cOur study stratified the participants by health status and baseline risk of CVD, cancer, or comorbid conditions, etc., to evaluate the impact of the effect of aspirin on different populations Toxic effects are very common in individuals treated with long-term aspirin; bleeding events are the leading side effects. The present meta-analysis showed that long-term aspirin use was associated with a significant increase in the risk of major bleeding and total bleeding events. Even in the individuals who used low-dose aspirin (≤ 100 mg) for a relatively short duration (1-5 years), the bleeding risk was still significantly increased.Our data indicated that long-term aspirin use as a primary cancer prevention measure had no benefit; however, aspirin use was associated with a significantly increased bleeding risk. Therefore, the present evidence does not favor the use of aspirin as a primary prevention strategy in the general population or in the above subgroups.Because we excluded specific populations with familial cancer syndromes  In this study, we only evaluated the effect of long-term aspirin use for overall cancer prevention. We did not evaluate the effect of this intervention on the prevention of specific subtypes of cancer. There seem to be some evidence to support the use of aspirin for the chemoprevention of a few specific cancers, especially colorectal cancer Our study had some limitations: firstly, although the included trials collected data on cancer outcomes, most of them were designed as RCTs to evaluate aspirin's effect on the cardiovascular system or on non-cancer outcomes (outcomes other than primary cancer prevention). Therefore, some potential confounding factors could have affected the outcomes, thus masking the actual association or falsely demonstrating an association between the aspirin treatment and cancer outcomes.Second, the cancer rates were much lower than in the setting of CVD or other comorbidities. The sample size needed for primary cancer prevention trials is larger, and the study duration must be longer; the sample size or follow-up duration of some of the included RCTs might have been insufficient.Third, some of the included trials had heterogeneity and potential risk of bias, and the quality of evidence of some outcomes was moderate, thus weakening the trustworthiness and strength of evidence from this systematic review.Last, individual patient data were not sufficient, and consequently, information for more stratified analyses  was limited.From the available evidence, our data indicated that compared with no aspirin, the long-term use of aspirin in individuals without pre-existing cancer was not associated with a reduction in total cancer incidence, cancer mortality, or all-cause mortality; however, aspirin use was associated with a significant increase in the risk of bleeding in this population. Therefore, aspirin might not be an appropriate choice for the primary prevention of cancer. Prospective RCTs of the role of aspirin in primary cancer prevention are warranted.Supplementary figures and tables.Click here for additional data file.


COMT) Val158Met polymorphism has been associated with working memory (WM) in many studies, but the results have not been consistent. One plausible explanation is sex-specific effects of this polymorphism as reported in several studies. The current study aimed to explore the sex-specific effects of the COMT Val158Met polymorphism on WM-related brain function in an elderly sample. We found that Val homozygotes outperformed Met allele carriers on the backward digit span subtest for both males and females. The triangular part of the left inferior frontal gyrus and the left inferior temporal gyrus exhibited higher activation in Met allele carriers compared with Val homozygotes during the n-back task, while the background functional connectivity (bFC) between the left angular gyrus (ANG) and the right ANG was enhanced in Val homozygotes as compared to Met allele carriers. Finally, the associations between brain activation, bFC (among various regions), and WM performance were identified only in specific genotype groups of the female participants. These findings provide new insights into the role of COMT Val158Met gene polymorphism in brain function, particularly its female-specific nature.The catechol-O-methyltransferase ( COMT is the Val158Met single nucleotide polymorphism (SNP), whose methionine (Met) substitution of a valine  would lead to a four-fold reduction in COMT enzyme activity and hence slower DA degradation [The capacity to temporarily store and process information is conceptualized as working memory (WM). It plays a crucial role in all forms of higher cognition and day-to-day functioning . WM has radation .COMT Val158Met polymorphism has been thought to be of particular relevance to prefrontal functions such as WM [COMT genotype on WM performance has been mixed. Some studies showed that Met allele carriers outperformed Val homozygotes in a dose-dependent fashion on WM-related tests  [COMT effect with the Val allele being linked to better WM performance, and yet another recent meta-analysis [As the cortical expression of DA is regional and might be greatest in the frontal lobe , the COMch as WM . Howeverng Test) –14, but ng Test)  and one ng Test)  identifianalysis  reportedCOMT genotype are age- and sex-dependent. In terms of age dependency, COMT’s effects may be enhanced in old age [COMT genotype, showing sex-specific genotypic effects on a range of cognitive functions, including WM, memory and verbal ability in children and old adults [COMT interactions might have been due to the role of estradiol in regulating COMT activity through its down-regulation gene expression [COMT’s effects, the current study used an elderly sample to examine sex differences in COMT Val158Met polymorphism’s effects on WM and related brain function.One possible reason for the inconsistent findings across genetic association studies is that the effects of  old age  because  old age , 4. In t old age , 21. Humd adults –24. Suchpression . ConsideCOMT and WM, previous neuroimaging studies have shown a relationship between the Val allele and greater prefrontal activation during WM tasks in both healthy adults and schizophrenia patients [COMT polymorphism and WM [Consistent with the hypothesis that DA expressed in the prefrontal cortex serves as the mechanism underlying the link between patients –28, althpatients , 30. In m and WM , which im and WM , 33. TheCOMT Val158Met polymorphism on WM and related brain function in cognitively normal elderly adults. All participants took the digit span test and the imaging subsample performed an n-back WM task while being scanned. Both task-related activation and background functional connectivity (bFC) [In sum, the current study aimed to examine the sex-specific effects of the ty (bFC) , 35 wereCOMT Val158Met genotypes did not deviate from Hardy-Weinberg equilibrium (p > 0.05). In terms of demographic factors for the whole sample, males were older (p < 0.01) and had more years of education (p < 0.01) than were the females (2-test (p = 0.34).The frequencies of  females . For the females . DemograCOMT genotype for either of the digit span tasks .For the whole sample, the Val homozygotes outperformed the Met allele carriers on the backward digit span task (p = 0.01), but the two groups did not differ significantly in terms of their performance on the forward digit span task. Males performed better than females on both forward (p = 0.01), and backward digit span tasks (p = 0.01). There was no significant interaction between sex and COMT genotype for either of the digit span tasks . Males performed better than females on the 2-back WM task in the scanner (p = 0.02). For this sample, there were no significant main effect of genotype or interaction between sex and COMT genotype for the 2-back task (p > 0.05).For the imaging subsample, no significant main or interaction effects of sex and COMT genotype effects were identified in the left inferior temporal gyrus (ITG) and triangular part of the left inferior frontal gyrus (IFGtri), and the activation was higher in the Met allele carriers than the Val homozygotes. Males showed significantly higher activation in the left angular gyrus (ANG) than did females. There were no significant genotype-by-sex interactions in any region.We also conducted analyses within regions that were deactivated during the task. There were no main effects of genotype or sex, but two clusters, the right precuneus (PCUN) and the right superior occipital gyrus (SOG), showed significant interactions between genotype and sex, with significantly reduced deactivation in male Met allele carriers than the other three groups .Partial correlation analyses were conducted for each group between significant brain regions and performance  on the 2-back WM task. As shown in COMT genotype and sex on bFC , right CAL, left superior temporal gyrus (STG) and left rolandic operculum (ROL). The connectivity was stronger for females than for males . No signFor the seed region of the left ANG, there were significant genotype and sex differences in its connectivity with other brain regions . SpecifiFor the seed region of the left ITG, there were no sex or genotype effects in its connectivity with the rest of the brain.The bFCs that showed significant sex and/or genotype effects were extracted and correlated with performance on the 2-back WM task using partial correlation analyses. The results showed that the strength of connectivity between left IFGtr and left CAL was negatively correlated with 2-back AR, especially in female Met allele carriers . This pattern of limited evidence linking COMT genotype to behavioral measures of WM is consistent with the emerging conclusion based on recent meta-analyses [In this study, we examined the sex-specific effects of the analyses  and otheanalyses .COMT gene may affect the function of many regions [COMT genotypic effects in PFC [Although the imaging subsample was much smaller and hence had less statistical power to yield genotype effects, we found significant genotype effects, mostly sex-dependent, on the more proximal measures of WM, namely, task-related brain activity and task-based functional connectivity. In terms of brain activation during performance of the n-back task, the left IFGtri and the left ITG exhibited higher activation in Met allele carriers compared with Val homozygotes. Using positron emission tomography, researchers found that the DA receptors availability in Val homozygotes was higher (lower DA level) across many regions of the brain, suggesting  regions . And dur regions , 38 and  regions  decreases in PFC , as wells in PFC , 41–43, s in PFC , 45 mighs in PFC , 47. This in PFC . Finallys in PFC , leadings in PFC .Our study also found genotype and sex effects on task-based bFC. In terms of sex differences, the bFC between the left IFGtri and four other brain regions  was significantly enhanced in female participants as compared to male participants. In terms of genotype differences, the bFC between the left ANG and the right ANG was enhanced in Val homozygotes as compared to Met allele carriers. To date, only a few studies have examined the genotypic effects on the functional connectivity but they show consistent results regardless whether they examined resting-state or task-related functional connectivity. Resting-state data revealed that the Val allele was linked to higher connectivity among subregions in the frontal lobe  and between frontal regions and subcortical structures  , 50, andCOMT on the association between bFC and WM performance. We found that the association between bFC (among various regions) and WM performance was stronger in female Met allele carriers than other groups. Besides, the correlations between activation and WM performance we found were also specific to a genotype in female participants. These results are consistent with the growing literature on female-specific effects of COMT on cognitive function [COMT genotype and sex on brain activation during an affective processing task, with Val homozygotes showing greater activation in right temporal pole than Met homozygotes in females [COMT interactions for a wide range of measures including behavioral performance on the task, task-related activation, rest-state functional connectivity, network attributes such as global and local efficiency, and the functional connectivity flexibility [COMT genotype on brain activation in Velocardiofacial syndrome has been found to be moderated by sex [COMT genotype effect on a wide range of brain function and structure. Furthermore, the results appeared to be female-specific, suggesting that the COMT genotype was more sensitive and regulatable in female participants. This sex-genotype interaction may reflect sexually dimorphic effects due to estrogen’s regulation of COMT activity as mentioned above.The most interesting finding of our study was the sex-specific effects of function , 52, 53 function –56. Kemp females . Similarxibility . The effd by sex . Taken tCOMT genotypic effect is robust. Finally, connectivity results were obtained through seed-based bFC. Future work could explore FC among additional regions and even the whole brain to further depict the COMT effects on brain function.Several limitations of this study should be acknowledged. First, dose effects of the alleles were not specified in the imaging analyses. Due to the limited number of Met homozygotes , we combined Met homozygotes and heterozygous individuals in the Met allele carrier group. Further studies with larger imaging sample would provide a greater effect size and could address the possible dose effect. Second, the COMT enzyme activity and DA level were not measured in our participants. Such information would have added physiological evidence for our results. Nevertheless, the available evidence suggests that the sex-specific COMT genotype and sex on WM task-related activity and task-based functional connectivity. Some of the genetic effects, especially those on the association between bFC and WM performance were specific to female participants. These findings provide new insights into the role of COMT Val158Met gene polymorphism in brain function and its female-specific nature. Our findings underscore the importance of integrating sex when examining the genetic effect on cognitive function.In summary, we observed significant effects of both Participants were enrolled in the Beijing Aging Brain Rejuvenation Initiative (BABRI) , which iThe inclusion criteria included: a) native Chinese speakers; (b) 50 years of age or older; (c) at least 6 years of education; (d) 24 or higher on the Chinese version of the Mini-Mental State Examination (MMSE) ; (e) no All 659 participants took the digit span subtest (DST) from the Wechsler Adult Intelligence Scale-Revised . Blood sCOMT Val158Met was genotyped using TaqMan allele-specific assays on the 7900HT Fast Real-Time PCR System . Another two SNPs, rs429358 and rs7412, were also genotyped to determine the APOE genotype (ε4 carriers or non-carriers). The sample success rates for all three SNPs were 100% , and the reproducibility of all the genotyping was 100% according to a duplication of at least 10% of the genotypes. Based on the COMT genotyping, participants were divided into two groups, 289 Met allele carriers and 370 Val homozygotes.A block periodic design that incorporated alternating 0-back, 1-back, and 2-back conditions was used during the n-back task in the scanner. Participants were trained prior to scanning to ensure task compliance. During each condition, three blocks of single digits (0-9) were pseudo-randomly shown to the participants. During the 0-back condition, participants were required to press a button when the target digit  appeared on the screen. During the 1- or 2-back condition, participants pressed a button when the digit shown on the screen matched the one presented one or two items back. There were three blocks for each condition, and every block started with a 10-s cue presentation that indicated 0-, 1- or 2-back, followed by 20 consecutive trials of single-digit stimuli .The responses and reaction time were recorded by an MRI-compatible response button box. The stimuli were presented using E-Prime version 1.0 software .2, acquisition matrix = 64 × 64. For each participant, 235 image volumes were obtained. High-resolution T1-weighted structural images were also acquired for co-registration, using the sagittal 3D magnetization prepared rapid gradient echo (MP-RAGE) sequence with the following parameters: 176 sagittal slices, TR = 1900 ms, TE = 3.44 ms, slice thickness = 1 mm, flip angle = 9°, inversion time = 900 ms, FOV = 256 × 256 mm2, acquisition matrix = 256 × 256.MRI data were collected on a 3.0 T Siemens Trio scanner at the Imaging Center for Brain Research at Beijing Normal University. The WM task fMRI images were acquired using an echo-planar imaging (EPI) sequence with the following parameters: 33 axial slices, repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, slice thickness = 3.5 mm, flip angle = 90°, field of view (FOV) = 200 × 200 mmhttp://rfmri.org/DPABI) [3, and smoothed with a 6-mm full-width half-maximum (FWHM) Gaussian kernel. Additionally, 17 participants with excessive head movement (translation > 3 mm or rotation > 3 degree in any direction) were excluded , resulting in the inclusion of 121 participants in the functional image analysis.fMRI data were preprocessed using Data Processing Ȧ Analysis of Brain Imaging  . Brieflyhttp://www.fil.ion.ucl.ac.uk/spm/software/spm8) at the individual level. Four conditions  with onsets and durations were set up, and the realignment parameters were included in the model as confounding factors. Contrast images of interest were created by contrasting the 0-back condition with the 2-back condition. To identify core brain regions activated or deactivated, task-related activation and deactivation masks were separately generated by group-level one-sample t tests, with age, years of education and APOE ε4 status as covariates (uncorrected p < 0.001). Further full factorial analyses were conducted with the COMT genotype  and sex  as independent factors for brain activation and deactivation under the corresponding brain masks, and age, years of education and APOE ε4 status were also included as covariates. Monte Carlo simulation was used for multiple comparison corrections  to achieve a cluster-level false-positive rate of 0.05 [Task fMRI images were first analyzed by general linear models (GLM) using SPM8 . Two-way analysis of covariance (ANCOVA) was conducted to examine performance on the neuropsychological tests and the n-back WM task, with genotype and sex as the grouping factors and age, years of education and APOE ε4 status as covariates. Partial correlations were obtained to examine the relationships between task performances and task-related activation and task-based bFC, with age, years of education, APOE ε4 status, activation of the seed region, and reaction time of the 2-back task as covariates. A statistical significance level of p < 0.05 was used in these analyses.PLINK software was used to assess Hardy-Weinberg equilibrium. SPSS 20.0 was used to conduct two-way analysis of variance (ANOVA) with the Supplementary FiguresSupplementary Tables