P-selectin and vascular endothelial growth factors remained unchanged. Flow-dependent dilatation of the brachial artery , carotid intima-media thickness , and fractional anisotropy of the carotid artery  also did not significantly change. Conclusion: Despite a nominal amelioration for flow-dependent dilatation, significant effects of short-term treatment with alirocumab on vascular function were not detectable. More work would be needed to evaluate, whether fractional anisotropy may be useful in clinical atherosclerosis research.Background: Short-term effects of alirocumab on vascular function have hardly been investigated. Moreover, there is a scarce of reliable non-invasive methods to evaluate atherosclerotic changes of the vasculature. The ALIROCKS trial was performed to address these issues using standard ultrasound-based procedures and a completely novel magnetic resonance-based imaging technique. Methods: A total of 24 patients with an indication for treatment with PCSK9 antibodies were recruited. There were 2 visits to the study site, the first before initiation of treatment with alirocumab and the second after 10 weeks of treatment. The key outcome measures included the change of carotid vessel wall fractional anisotropy, a novel magnetic resonance-based measure of vascular integrity, and the changes of carotid intima-media thickness and flow-dependent dilatation of the brachial artery measured with ultrasound. Results: A total of 19 patients completed the trial, 2 patients stopped treatment, 3 patients did not undergo the second visit due to the COVID pandemic. All of them had atherosclerotic vascular disease. Their mean (standard deviation) LDL-cholesterol concentration was 154 (85) mg/dL at baseline and was reduced by 76 (44) mg/dL in response to alirocumab treatment ( Elevated Low-density lipoprotein (LDL) cholesterol is associated with increased atherosclerotic plaque volume . On the We have introduced a completely novel method based on magnetic resonance diffusion tensor imaging to quantify structural changes of the carotid arteries. Ex-vivo, this method was demonstrated to have a high spatial resolution in visualizing collagen fiber bundles of the atherosclerotic fibrous cap . In addiThe present study was designed to explore the usability of this novel non-invasive magnetic resonance imaging method in patients with atherosclerotic vascular disease and to collect longitudinal clinical data on fractional anisotropy for the first time. Moreover, the objective was to investigate the effects of short-term treatment with alirocumab on brachial artery flow-dependent dilatation as well as inflammatory and vascular biomarkers.The ALIROCKS study (NCT03559309) is a prospective, monocentric, and longitudinal pilot study with 24 patients scheduled for treatment with alirocumab. Treatment with alirocumab was initiated based on the current guidelines for the treatment of dyslipidemia of the European Atherosclerosis Society and the European Society of Cardiology . InclusiThe primary outcome measure was defined as the change of mean carotid vessel wall fractional anisotropy. Secondary outcome measures included the change of flow-dependent dilatation of the brachial artery, change of carotid intima-media thickness, and change of systemic inflammation in response to treatment with alirocumab. The clinical trial was conducted in accordance with the Declaration of Helsinki and was approved by the Austrian Federal Office for Safety in Health Care (EudraCT: 2018-000981-12) and the Ethics Committee of the Medical University of Graz . All participants gave written informed consent.At baseline, demographics, and clinical characteristics such as age, sex, smoking habits, concomitant diseases, and lipid medication were documented. Familial hypercholesterolemia was reported according to medical history. Patients not receiving high-intensity statins defined as ≥40 mg atorvastatin or ≥20 mg rosuvastatin at baseline were categorized as having partial or complete statin intolerance.2, matrix = 346 × 346, slice thickness = 10 mm, TR = 2RR intervals, trigger delay = 50ms to acquire images in the diastolic phase, TE = 93 ms, GRAPPA = 2, number of readout segments = 9, b-values = 0, 200, 400, 600 s/mm2, acquisition time = ~12 min. 18 gradient directions were defined on a hemicycle within a plane oriented perpendicular to the longitudinal axis of the carotid artery. Fractional anisotropy is a scalar value without a unit between one and zero which describes the degree of anisotropy of a diffusion process (High-resolution diffusion tensor imaging was performed with a 2D diffusion acquisition scheme. A readout-segmented Echo-Planar-Imaging (rs-EPI MRI) sequence was used on a 3T whole-body MR scanner  with a 2 × 4 channel multifunctional coil  and FOV = 189 × 189 mm process . A valueBlood samples were taken in the fasting state. LDL-cholesterol and HDL-cholesterol were assessed by lipoprotein-electrophoresis, cholesterol and triglycerides were measured by photometry. Apolipoproteins (Apo) and C-reactive protein (CRP) were measured with immunoturbidimetry, and interleukin-6 (IL-6) was measured with an electrochemiluminescence immunoassay. Monocyte chemoattractant protein-1 , Vascular endothelial growth factor , and P-Selectin/CD62P , were measured by enzyme-linked immunosorbent assays.All vascular ultrasound measurements were performed by a single experienced technician using a linear array transducer, 8–13 MHz . Carotid intima-media thickness was defined as the mean of the left and right intima-media thickness which was each based on three different measurements in a one-centimeter-long segment of the common carotid artery. For the standardized measurement of flow-dependent dilatation of the brachial artery, a blood pressure cuff on the forearm was inflated for 5 min with a constant pressure of 50 mmHg above the systolic brachial artery blood pressure. The post-ischemic brachial arterial diameter was measured 45 s after cuff release. Flow-dependent dilatation was defined as the percent change of the brachial artery diameter in response to ischemia compared to the baseline diameter .t-test and/or related samples Wilcoxon signed-rank test when Shapiro-Wilk analysis suggested non-normal distribution. The SPSS 26.0 statistical program was used .The statistical design of this pilot trial was explorative and descriptive. The study duration and sample size were based on previous studies investigating the effects of short-term lipid-lowering on endothelial function ,22,23,24Source data verification was performed for carotid vessel wall fractional anisotropy assessed by cardiovascular magnetic resonance and for flow-dependent dilatation of the brachial artery measured by ultrasound. In addition, informed consent forms were monitored. The monitoring was conducted by an independent, external clinical research organization .The recruitment phase for the 24 participants lasted from 20 June 2018 to 30 January 2020. About 80% of the participants received 75 mg of alirocumab every two weeks and 20% received 150 mg alirocumab every two weeks. Two patients only injected alirocumab twice, at baseline and week 2. The reasons for their non-adherence were documented as drug access and communication barriers in the clinical routine. Four patients had minor changes in their oral lipid-lowering therapies during the trial. Among these, two patients stopped their red yeast rice product, one stopped ezetimibe, and another one reduced the oral combination therapy from 80 mg atorvastatin plus 10 mg ezetimibe to 20 mg atorvastatin monotherapy. Due to the COVID-19 pandemic and the subsequent implementation of national countermeasures restricting all hospital visits to essential interventions only, the clinical trial had to be terminated before the last 3 participants had completed the study. Hence, 19 patients completed the trial according to the protocol . The meaThe cohort included middle-aged and elderly females and males. All 19 participants had documented coronary heart disease. Among these, 7 patients had poly-vascular disease with an additional diagnosis of cerebral and/or peripheral artery occlusive disease. The vast majority, 16 participants, had received the coronary intervention or bypass surgery. All 3 patients without prior coronary intervention or surgery had poly-vascular disease with coronary stenosis (50%) confirmed by cardiac computed tomography and carotid artery occlusive disease. One of the 3 patients without prior coronary intervention or surgery also had peripheral artery disease. Familial hypercholesterolemia was documented in 2 patients. A total of 16 study participants had partial or complete statin intolerance. Hence, very few participants received high-intensity statins at baseline. However, the proportion of patients taking ezetimibe as concomitant medication was high .p = 0.018). We did not detect changes in the systemic inflammatory biomarkers CRP and MCP-1 in response to treatment with alirocumab. There was only a minor but statistically significant reduction of IL-6. The vascular biomarkers P-selectin/CD62P and VEGF did not change in response to alirocumab treatment . There was a trend towards an inverse relationship between LDL-cholesterol reduction and the change of flow-dependent dilatation , claustrophobia, an outlier value, and the time-consuming measurement regimen, values were only obtained for a subgroup of the ALIROCKS study limiting the current clinical and scientific applicability of the method. During the ALIROCKS study, factional anisotropy did not significantly change, as expected. The short duration of treatment with alirocumab, which resulted in a 49% LDL-cholesterol reduction, and the low sample did not suggest significant changes in vascular structure. Moreover, there are no established cut-off values differentiating normal fractional anisotropy values from pathologic ones. The reproducibility of this magnetic resonance imaging method was analyzed in a previous study of our group in healthy subjects by providing the coefficients of variation. Four repeated measurements in four healthy male volunteers showed coefficients of variation between 2.5% and 5.4%. This previous cross-sectional evaluation in healthy subjects also demonstrated an inverse relationship between age and fractional anisotropy. These participants had fractional anisotropy ranging from around 0.70 at 27 years to 0.56 at 57 years of age . The preThere is a scarce of non-invasive methods to reliably measure atherosclerosis. Using invasive methods such as optical coherence tomography, PCSK9-antibodies have been suggested to stabilize the fibrous cap of coronary atherosclerotic lesions and to reduce the size of lipid-rich plaques. Two single-center trials used optical coherence tomography to investigate the effects of PCSK9-antibodies vascular structure: Firstly, a retrospective study reported that treatment with evolocumab for 12 weeks increased fibrous-cap thickness and reduced the lipid-rich plaque proportion in 18 patients (intervention group) with acute coronary syndromes . Most reIn addition to the completely novel magnetic resonance method, established ultrasound-based methods, namely carotid intima-media thickness and flow-mediated dilatation, were used in this study. Of relevance, intima-media thickness at baseline ranged from 0.42 to 0.77 mm (mean 0.58) and would not meet current recommendations for significant cardiovascular disease recently defined as ≥1.5 mm by the American Society of Echocardiography . This obThe ALIROCKS trial showed a nominal amelioration (+41%) but no significant change of flow-dependent dilatation of the brachial artery in response to alirocumab. In contrast, 8 weeks of treatment with evolocumab increased flow-dependent dilatation in a small Italian cohort of 14 patients. Interestingly, the effect size was similar in the ALIROCKS trial but baseline values in the ALIROCKS study were lower compared with the Italian study. This difference in baseline flow-dependent dilatation values may be due to differences in patient characteristics and/or observer bias between the present study and the Italian study. All participants of the Italian study had a prior myocardial infarction and did not reach LDL-cholesterol goals despite a combination therapy of a high-dose statin and ezetimibe. Another recent study with 25 patients suffering from familial hypercholesterolemia : 69 mg/dL) also showed amelioration of endothelial function in response to 12 weeks of treatment with PCSK9-antibodies. The reason for the difference in the results may be because all ALIROCKS participants had an advanced atherosclerotic cardiovascular disease but considerably lower baseline LDL cholesterol. Of note, the characteristics of the ALIROCKS participants were consistent with recent real-world evidence from Austria .Another objective of the trial was to test the effects of treatment with alirocumab on inflammatory and vascular biomarkers. Whereas a pro-inflammatory role of PCSK9 in atherosclerosis has been suggested, clinical studies did not show a significant reduction of systemic inflammatory biomarkers by PCSK9-antibodies, even in myocardial infarction ,31. ConsIt is a strength of the ALIROCKS study that a novel and innovative magnetic resonance-based method to visualize and evaluate the vascular structure of the carotid arteries was used. We also included high-quality ultrasound-based vascular imaging data acquired by a single well-trained technician. Moreover, the magnetic resonance and ultrasound data were monitored by an independent clinical research organization. Finally, we were able to provide “real world” data of patients with an indication for treatment with PCSK9-antibodies. We recruited patients with diverse vascular complications, concomitant disorders, lipid-lowering premedication, and lipid levels. This approach reduces the risk of selection bias but at the same time leads to a more heterogeneous collective with differences in medication as well as in locations and extent of atherosclerotic lesions. Such differences in baseline characteristics may also have an impact on the results of the ALIROCKS study.It is a limitation of the ALIROCKS study that no control group is available. However, all participants had a medical indication for treatment with PCSK9-antibodies. Hence, it would have been unethical to withhold alirocumab from half of the participants. The magnetic resonance imaging method has not yet been implemented in patients with atherosclerosis and comparisons with healthy individuals have not been made. Therefore, reference values for fractional anisotropy values in atherosclerotic vessels versus those vessels without atherosclerosis are currently not available. The relatively low sample size may also be regarded as a weak point of the ALIROCKS study. Nevertheless, beneficial effects of short-term lipid-lowering therapy on vascular function have previously been documented in studies with similar or smaller sample sizes and with even shorter treatment duration ,22,23,24To conclude, endothelial function measured with flow-dependent dilatation of the brachial artery did not significantly change in response to short-term treatment with alirocumab. More work would be needed to evaluate whether fractional anisotropy may be useful in clinical atherosclerosis research.


Invasive pulmonary aspergillosis (IPA) is increasingly recognized as a complication of severe respiratory viral infections (RVIs), including influenza and COVID-19. However, the incidence and outcomes of IPA following other RVIs is not well-described. We hypothesized that IPA may be an underreported complication of non-influenza RVIs. The objective of this study was to quantify the incidence and associated outcomes of IPA following RVI in hospitalized patients.We conducted a single-center retrospective cohort study of adult hospitalized patients with RVI diagnosed by multiplex PCR-based assay at the University of Kansas Hospital  from September 2018-October 2019. Patients with a diagnosis of proven or probable IPA prior to RVI and those with hospital admission < 24 h were excluded from analysis. Proven or probable IPA was defined according to EORTC/MSGERC consensus definitions. The primary outcome was 1-year all-cause mortality.n=113), parainfluenza , influenza , and respiratory syncytial virus . The cumulative incidence of IPA infection within 6 weeks of RVI was 5.6% (n=11). Excluding patients co-infected with multiple respiratory viruses (n=5), IPA was numerically more likely to occur following influenza compared to non-influenza RVI . Overall, one-year all-cause mortality was 20% (n=39/195) in this cohort. Development of IPA as a complication of RVI was associated with a significant decrease in 1-year survival , and this relationship persisted after adjustment for age . A total of 195 patients met study criteria and were included in the analysis. The most common types of RVI observed were rhinovirus/enterovirus (57.9%, In a cohort of hospitalized patients with RVI, 5.6% of patients developed proven or probable IPA. Although IPA was more likely to occur in patients with influenza, this complication was also observed with other types of RVI. Invasive pulmonary aspergillosis may be an underappreciated complication of non-influenza RVI in hospitalized patients and warrants continued study.All Authors: No reported disclosures


Post-acute COVID-19 syndrome (PACS) is an emerging healthcare burden. The risk factors associated with PACS remain largely unclear. The aim of this study was to evaluate the frequency of new or persistent symptoms in COVID-19 patients post hospital discharge and identify associated risk factors.Our prospective cohort comprised of PCR-confirmed COVID-19 patients admitted to King Fahad Medical City, Riyadh, Saudi Arabia between May and July 2020. The patients were interviewed through phone calls by trained physicians from 6 weeks up to 6 months post hospital discharge. Multivariate Cox proportional hazards and logistic regression models were used to examine for predictors associated with persistence of symptoms and non-return to baseline health.222 COVID-19 patients responded to follow-up phone interviews after a median of 122 days post discharge. The majority of patients were men (77%) with mean age of 52.47 (± 13.95) years. 56.3% of patients complained of persistent symptoms; 66 (29.7%) experiencing them for >21 days and 64 (28.8%) reporting not having returned to their baseline health. Furthermore, 39 patients (17.6%) reported visiting an emergency room post discharge for COVID-19-related symptoms while 16 (7.2%) had required re-hospitalization. Shortness of breath (40.1%), cough (27.5%) and fatigue (29.7%) were the most frequently reported symptoms at follow-up. After multivariable adjustments, female gender, pre-existing hypertension and length of hospital stay were associated with an increased risk of new or persistent symptoms. Age, pre-existing lung disease and emergency room visits increased the likelihood of not fully recovering from acute COVID-19. Patients who were treated with interferon β-1b based triple antiviral therapy during hospital stay were less likely to experience new or persistent symptoms and more likely to return to their baseline health.COVID-19 survivors continued to suffer from dyspnea, cough and fatigue at 4 months post hospital discharge. Several risk factors could predict which patients are more likely to experience PACS and may benefit from individualized follow-up and rehabilitation programs. More than one year into the Coronavirus disease 2019 (COVID-19) pandemic, several parts of the world are still experiencing high level of new cases and hospitalizations. To add to this burden, a sizeable subset of patients who have recovered from acute COVID-19 infection have reported lingering symptoms, leading to disability and impairment of their daily life activities. These patients are considered to suffer from “long” or “chronic” COVID-19 or a form of post-acute COVID-19 syndrome. Patients experiencing this syndrome have been coined COVID-19 long-haulers –5. We reThere is no standardized definition of “long COVID” or “post-acute COVID-19 syndrome (PACS)”. The National Institute for Health and Care Excellence (NICE) defined it as a constellation of “signs and symptoms that develop during or after an infection consistent with COVID-19, continue for more than 12 weeks and are not explained by an alternative diagnosis” . HoweverSymptoms have been reported to persist for as long as 9 months after onset . In addiThis cohort study was conducted at King Fahad Medical City (KFMC), Riyadh, Kingdom of Saudi Arabia. The study included all participants who were 18 years of age or older and were admitted to KFMC with Polymerase Chain Reaction (PCR)-confirmed COVID-19 infection during the months of May until July in 2020. We excluded patients who died, those who continued to be hospitalized at the time of follow-up, those who were unable to participate due to a mental illness, and those who failed to respond to phone calls after discharge.A structured questionnaire was used to extract clinical and demographic characteristics  from the patient’s hospital records. Data collected included presence of medical comorbidities, pre-defined symptoms at presentation and hospital course. Enrolled patients were subdivided into intensive care unit (ICU) or general ward admission, and disease severity  according to the WHO/ISARIC COVID-19 clinical characterization and patient’s utilization of supplemental oxygen need and hemodynamics during admission. In accordance with WHO guidelines, a seven-category ordinal scale was further used to characterize disease severity during hospital stay , 14. AccParticipants were contacted through phone calls by trained physicians from 6 weeks to 6 months after hospital discharge to collect information on unresolved symptoms at follow-up, and duration for resolution of symptoms. In order to ensure that data were collected in a standardized manner, a structured interview process was used to collect information from patients. General interviewer training which covered the basics of interviewing skills, probing, and how to avoid refusals was conducted, in addition to research-specific training including a complete review of the data collection instrument, the time that should be taken to conduct the interview, and how to address questions from patients. All interviewers were trained to follow a set script and protocol of questions to ensure the consistency and structure of questions  at KFMC (IRB No. 20–557). All participants gave their verbal informed consent to participate in the telephone interviews.Demographic, clinical characteristics, and symptoms at presentation and follow-up were presented in means (and standard deviations [SD]) and medians (and interquartile ranges [IQR]) for continuous variables, and expressed as absolute values and percentages for categorical variables. For the comparison of symptoms at follow-up with demographic and clinical characteristics, the Pearson’s Chi-Square (χ2), or Fisher’s exact test were used for categorical variables and the Mann-Whitney-Wilcoxon and Kruskal Wallis tests were used for ranked data, as appropriate. Data were tested for normality visually using histograms and Q-Q plots, and statistically using Kolmogorov-Smirnov test. Homogeneity of variance was tested using Levene’s test.p>0.05) and did not violate the assumption of proportional hazards. The estimates of risk of predictor variables and persistence of symptoms at follow-up (event) were reported as Hazards ratio (HR) with a 95% confidence interval (CI).A multivariate Cox proportional hazards model was constructed to identify factors associated with persistence of symptoms at follow-up with time-dependent days since discharge. Only statistically significant variables in the univariate analysis were incorporated into the multivariate Cox regression model. To assess the proportional hazards assumption, an extended COX regression model with time covariate interactions tested the interaction between time and covariates in the model. Predictor by time interactions for all predictors were non-significant  showed that multicollinearity was not a problem and assumptions of collinearity were met. Variable tolerances ranged between (0.71–0.96) and VIFs between (1.09–1.40). The automatic selection of predictors in the model was performed by a stepwise backward method with an entry threshold of 0.05 and an exit threshold of 0.1. The adequacy of the model was verified by the Hosmer and Lemeshow test, and its specificity by Link Test. The estimates of the strengths of associations for the multivariable logistic regression analyses were reported as odds ratio (OR) with a 95% confidence interval (CI).The percentage of missing values across variables in the dataset varied between 0 and 7%. Multiple imputation was used to create and analyze 5 imputed datasets to account for missing data. All statistical analyses were performed using IBM SPSS version 26,  software. A two-tailed p < 0.05 was considered statistically significant.A total of 461 patients were hospitalized at KFMC with PCR-confirmed SARS-CoV-2 infection between May and July 2020. A total of 222 patients discharged alive from the hospital post-acute COVID-19 infection responded to the follow-up phone survey. The demographic and clinical characteristics of these patients are summarized in More than half of the cohort, 125 patients (56.3%) experienced unresolved symptoms after a month of discharge with 66 (29.7%) experiencing these new or persistent symptoms for >21 days . DespiteApproximately 40% of the patients reported the persistence of shortness of breath, followed by fatigue (30%) and cough (27.5%) . PatientShortness of breath and cough were the two most prevalent symptoms at presentation as well as at follow-up. Among the neurological symptoms, headaches followed by difficulty concentrating were the most prevalent at presentation as well as at follow-up. In terms of evolution of symptoms, we observed that the prevalence of dyspnea, cough and fever decreased over the course of presentation to follow-up. However, other neurological and musculoskeletal symptoms, such as insomnia, fatigue, body/muscle pain, joint pain, memory impairment, concentration issues, headaches, and loss of appetite increased over time .Because patients had different follow-up durations, a COX proportional hazards model was used to examine the risk factors associated with new or persistent symptoms at follow-up . Female Multivariable logistic regression analysis was used to identify risk factors associated with non-return to baseline pre-COVID-19 health state at a median of 4 months follow-up. The logistic regression was adjusted for age, gender, nationality, pre-existing morbidities, BMI, disease severity, severity category, treatment, ICU admission, hospital re-admission, ER visit, hospital stay (LOS) and ICU stay (LOS) . Almost To our knowledge, this is the first report from the MENA region assessing a broad spectrum of functional outcomes in COVID-19 patients following hospital discharge. We observed that 56.3% of our cohort reported new or persistent symptoms at follow-up with most symptoms lasting over 21 days after diagnosis. At a median of 4 months follow-up after hospital discharge, shortness of breath, fatigue and cough were the main symptoms that prevailed in these patients. We also observed that more than one quarter of patients failed to return to their baseline health state after recovery from acute infection and 17.6% of the patients visited the ER after discharge. Female gender, pre-existing hypertension and longer hospital stay were associated with higher risk of new or persistent symptoms. Older age, pre-existing lung disease and post-discharge ER visits were predictors of delayed return to baseline health state. Interestingly, patients with diabetes and on triple antiviral therapy during hospital stay were less likely to experience new or persistent symptoms and more likely to return to their baseline health.Although multiple recent studies have reported the long-term outcomes of COVID-19 survivors in the American , Asian  and EuroA systematic review and meta-analysis of studies assessing long-term effects of COVID-19 identified fatigue (58%), headache (44%), attention disorder (27%), hair loss (25%), and dyspnea (24%) as the five most common persistent symptoms [A recent report analyzing data from over 4000 COVID-19 survivors using the COVID Symptom Study app identified female gender, increasing age, body mass index, and patients who experienced more than five symptoms during the first week of infection at risk of long COVID . Like ouIn addition, patients in the age group of 50–66 years, Saudi nationals, those with pre-existing lung disease and those who visited the ER after discharge were less likely to return to their baseline health state. Similarly, in the large cohort from China, age was positively associated with fatigue and muscle weakness  per 10-year increase of age . PatientSurprisingly, COVID-19 patients with pre-existing diabetes mellitus were less likely to experience lasting symptoms and were more likely to return to their baseline health state. This may be a chance finding or due to selection bias given that patients with diabetes experience severe disease and are more likely to stay longer in the ICU or die. Patients with diabetes who survived and participated in this survey could therefore be a selected sample with controlled diabetes and exhibit the healthy user effect. The observation that triple antiviral therapy was associated with lower risk of PACS may be explained by the effect of this therapy on the viremia level in the acute phase of the disease resulting in less inflammatory consequences at follow-up. Type I interferon (IFN) signaling is crucial particularly in the early phase of viral infection for optimal virus control  and IFNβOur study has multiple strengths. To our knowledge, it is the first report from the MENA region to describe long-term symptoms experienced by COVID-19 survivors after hospitalization and one of very few studies that examined predictors of PACS. Our patients were subjected to a unified COVID-19 treatment protocol which reduced the variations in the clinical management of patients allowing the comparison of clinical outcomes in a uniform cohort. Moreover, we addressed multiple limitations that affected previous studies such as lack of baseline assessment or use of diagnostic codes. Our study also has certain limitations. Firstly, the sample size of our cohort is relatively smaller and is a single center experience. Although we did not observe an association between disease severity and PACS, our analysis could be underpowered. Secondly, the exclusion of non-hospitalized patients affects the generalizability of our findings to patients with mild COVID-19. The risk of PACS in non-hospitalized patients is typically low .At a median of 4 months after hospital discharge, shortness of breath, fatigue and cough were the main symptoms experienced by patients who had recovered from COVID-19. Nearly 30% of these patients did not return to their baseline pre-COVID-19 state and 17.6% had to visit the ER for this reason. We identified multiple risk factors for new or persistent symptoms and lack of return to baseline health state after acute COVID-19 infection. Longer follow-up studies in a larger population are warranted to understand the persistent chronic symptoms experienced by these long-haulers to develop guidelines for their improved clinical management and care.S1 Questionnaire(DOCX)Click here for additional data file.S2 Questionnaire(DOCX)Click here for additional data file.S1 Table*Significant at p<0.05 using Chi-square and T-tests.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableUsing Chi-Square and Fisher’s exact test analysis for categorical variables and T-test and Mann-Whitney U for continuous variables #Seven category scale: Scale 3: admitted to hospital not requiring supplemental oxygen, Scale 4: admitted to hospital requiring supplemental oxygen; Scale 5: admitted to hospital requiring HFNC or non-IMV or both; Scale 6: admitted to hospital requiring ECMO or IMV or both.(DOCX)Click here for additional data file.S4 Table* Using Chi-Square analysis and Fisher’s exact test where appropriate # Seven category scale: Scale 3: admitted to hospital not requiring supplemental oxygen, Scale 4: admitted to hospital requiring supplemental oxygen; Scale 5: admitted to hospital requiring HFNC or non-IMV or both; Scale 6: admitted to hospital requiring ECMO or IMV or both.(DOCX)Click here for additional data file.


Although Latino men have the highest prevalence (45%) of obesity among all men in the United States, traditional weight loss interventions have not effectively engaged this hard-to-reach and diverse group. Offering choices among technology-mediated weight loss interventions may offer advantages.The aim of this study is to examine Latino men’s preferences among 3 weight loss intervention options. We also examined whether attendance in group sessions (videoconference and in person) and weight loss differed according to intervention choice.Latino men  participated in a comparative effectiveness trial based on primary care and were randomized to receive the 1-year HOMBRE  intervention. HOMBRE is a weight loss intervention that offers 3 delivery options. During an orientation session, a trained bilingual coach helped men select 1 of the 3 intervention options that differed in coach, peer support, and available language. We used canonical discriminant analysis to assess multivariate associations of demographic, clinical, employment, cultural, and technology use and access factors with men’s intervention choices. We used generalized linear models to estimate weight loss at 6, 12, and 18 months for men in each intervention option.P<.001) and 18 months  than those who chose web-based videos. Men who chose in-person group sessions also lost significantly more weight at 12 months (P=.008) than those who chose web-based videos.Among Latino men, 28% (56/200) chose videoconference groups, 31% (62/200) chose web-based videos, and 41% (82/200) chose in-person groups. The canonical discriminant analysis identified 1 orthogonal dimension that distinguished between men who chose an in-person group and men who chose web-based videos. Men who were older, spoke Spanish, and did not use a computer frequently had a higher probability of choosing in-person groups versus web-based videos. For men who selected a group delivery option, 86.9% (107/123) attended ≥25% of the sessions, 83.7% (103/123) attended ≥50% of the sessions, and 73.2% (90/123) attended ≥75% of the sessions, with no differences by type of group (videoconference or in person). Men who chose videoconference and in-person group sessions lost significantly more weight at 6 months  lost significantly more weight than those who chose web-based videos. Providing options that accommodate the diversity of Latino men’s preferences is important for increasing engagement in behavioral interventions.ClinicalTrials.gov NCT03092960; https://clinicaltrials.gov/ct2/show/NCT03092960 Obesity is a major contributor to the leading causes of death among all men in the United States, such as heart disease, cancer, and type 2 diabetes ,2. As paTechnology, including web-based and smartphone apps, offers opportunities for extending the reach and engagement of behavioral lifestyle interventions to priority populations such as Latino men. Technology can help overcome prevalent barriers to engaging in interventions because of competing priorities from family, inflexible work schedules, and unreliable transportation. In addition, technology-mediated approaches became essential during the COVID-19 pandemic, when in-person meetings were not allowed or encouraged. Latinos increasingly have access to technology in general and smartphones specifically, which makes this a promising approach to maximize reach and engagement in this population -9. HowevThe HOMBRE  trial was designed to compare a culturally adapted behavioral lifestyle intervention for Latino men with minimal-intensity control. The culturally adapted behavioral lifestyle intervention offered men 3 options for engaging in the intervention sessions: coach-facilitated group sessions using web-based videoconferencing, prerecorded videos of group sessions available on the web, and coach-facilitated group sessions in person. The 3 choices differed in the used technology, the level of coach and peer support, and language options. The goal of this study is to examine Latino men’s preferences among the 3 intervention options according to demographic, clinical, employment, cultural, and technology use and access factors. We also examined whether attendance (for the videoconference and in-person groups) and weight loss differed among the intervention options. Understanding Latino men’s preferences according to key baseline characteristics can inform future implementation of technology-based interventions for this high-priority population.The institutional review board for Sutter Health, Northern California, and Stanford University approved the study. All participants provided written informed consent. The trial protocol has been previously published . Partici2 and ≥1 cardiometabolic risk factor  were enrolled in the HOMBRE trial following a multistep process, as described in the trial protocol [A total of 424 Latino men who had a BMI of ≥27 kg/mprotocol . PatientMyPlate visual in the orientation and in the early intervention sessions, given its effectiveness for communicating the types of food choices recommended by the intervention; and (3) invited family members to sessions 6 and 12, given the cultural importance of family support [The HOMBRE intervention was based on the Group Lifestyle Balance (GLB) intervention, a group-based adaptation of the original Diabetes Prevention Program intervention -14 groun support . The prereal-time coach involvement. Using the tracking data from participants, the health coach provided individualized feedback on diet and physical activity goals during the intensive phase. Individualized feedback provided ample opportunity for tailoring based on cultural and other individual differences. During the maintenance phase, men who chose the videoconferencing and in-person options received monthly phone calls from the coach, which focused on supporting continued goal progress and problem solving for encountered barriers. The web-based video format had the lowest level of coach involvement, according to the protocol, as participants watched prerecorded videos of a coach facilitating the 12 sessions with a multiethnic group of men and women. Men in this option received standardized weekly messages in months 1 to 3, with reminders to watch the videos, use written materials, self-monitor, and reach out to their assigned coach with questions or requests for more individualized feedback. This was in alignment with this intervention choice, which included less proactive coach interaction. In months 4 to 12, men in the web-based video option received monthly standardized messages that included handouts on maintenance topics, reminders to self-monitor, and contact information for the coach.In addition to the differences in delivery, the 3 intervention options differed in level and type of coach feedback . The vidFollowing randomization, all participants attended an in-person group orientation session. Trained health coaches offered the orientation sessions in English and Spanish at different times of the day throughout the 2 weeks following randomization to accommodate all participants. Group sessions were designed for approximately 10 participants but could accommodate varying sizes as needed. Participants were encouraged to bring their partners and other family members to the orientation session with the purpose of increasing understanding and social support among family members. Group orientation sessions followed a standardized protocol that featured a didactic component to provide information on the background and goals of the intervention and a small group discussion component specific to their randomization arm. The goal of the small group discussion for the HOMBRE arm was to support men in making a choice among the 3 intervention options. The small group discussion included 3 components to support men in making a choice: (1) description of the 3 intervention choices provided by a health coach, (2) a worksheet that helped men reflect on each of the 3 options, and (3) a small group discussion on the pros and cons of each option. The worksheet prompted men to think about whether they liked participating in groups, the degree to which they would like support from a coach, their comfort with new technologies, and their availability for attending regular weekly sessions. In the small group, the coach asked men to discuss the pros and cons of each option to assist them in considering the choices. At the end of the session, men were asked to make their choice, given their intervention materials, and assisted with using the activity tracker and other technologies based on their choice.The intervention participants’ initial choices of intervention delivery were used to group them into the 3 options . The participants were allowed to change their choice within 4 weeks. However, none of the participants elected to change. Coaches could transfer patients from the videoconference or in-person groups to the web-based videos if they did not attend the first 4 sessions; 15 men  were transferred to the web-based videos group.Baseline characteristics included demographic  and clinical characteristics , employment , cultural characteristics , and technology use and access. Weight, waist circumference, and blood pressure were measured in duplicate according to standard protocols -19. DeprWe used 2 steps to identify the different profiles of demographic, clinical, employment, cultural, and technology use and access characteristics based on their choice of delivery options. First, we performed a bivariate analysis to choose a set of candidate variables. Second, we conducted multivariate analysis based on the variables identified in the bivariate analysis to derive the baseline characteristic profiles that significantly differentiated the men who chose 1 of the 3 intervention delivery options .P<.05 (2-tailed) from the first step were then further assessed for pairwise comparisons using Student t tests for continuous variables and chi-square tests for categorical variables.Percentages and means and SDs were used to describe the baseline characteristics among HOMBRE intervention participants overall and by intervention delivery option chosen. We used the Fisher least significant difference method  to test P<.15 from the bivariate analyses [Canonical discriminant analysis was used to derive linear combinations of the baseline characteristic profiles that significantly differentiated the men who chose 1 of the 3 intervention delivery options. Canonical discriminant analysis is a multivariate dimension reduction technique that derives a linear combination of explanatory variables that has the highest possible multiple correlation with the groups of a classification variable. The dimension defined by the linear combination is the first canonical dimension. This maximum multiple correlation is called the first canonical correlation. The coefficients of the linear combination are the canonical coefficients. The second canonical dimension is obtained by finding the linear combination with the next highest possible multiple correlation with the groups that is uncorrelated with the first canonical dimension. The process of extracting canonical dimensions can be repeated until the number of canonical dimensions equals the number of original variables or the number of groups minus 1, whichever is smaller. We included only the baseline characteristics with analyses . The catWe used generalized linear models to compare weight loss among men in different intervention options at 6, 12, and 18 months after randomization. We examined weight according to the men’s initial and final intervention choices. Weight was measured by trained study staff at baseline. Weight was measured using a standard calibrated scale at baseline and 18 months at local clinic sites according to standard protocol . Particit tests.Session attendance was calculated for weekly sessions and monthly phone calls combined and separately in the videoconference and in-person groups, excluding the 15 participants who were transferred to the web-based video option. Session attendance was then compared between the videoconference and in-person groups using Student P<.05 (2-sided).All analyses were conducted using SAS version 9.4 (SAS Institute Inc). Statistical significance was defined as As shown in P=.04), have higher income (P=.005) and education (P=.03), have a white-collar job (P=.004), prefer English (P<.001), be more acculturated (P<.001), have higher health literacy (P<.001), have access to a computer (P=.004), and have higher technology skills  compared with men who chose the in-person group. Similarly, men who chose web-based videos were more likely to be younger (P=.02) and more acculturated (P=.001) than those who chose the in-person group. They were also more likely to have a blue-collar job compared with those who chose videoconference (P=.04) and more likely to speak English than men who chose the videoconference (P=.01) or in-person (P<.001) groups. Men who chose web-based videos had intermediate technology skills compared with those who chose either the videoconference or in-person group and were less likely to have (P=.02) and use a computer (P=.02) than those who chose videoconference but as likely to use emails (P=.08) and video chat apps (P=.33) as men who chose videoconference.Participants in the 3 intervention delivery options had similar clinical characteristics  but differed significantly according to demographic, employment, cultural, and technology use factors  and 3. MP<.001) on the canonical dimension 1. This signified that this dimension distinguished most significantly between these 2 intervention choices. According to characteristics with the highest positive or negative correlation coefficients and using 0.25 as a cutoff  and 18 months , and those who chose in-person group sessions also lost significantly more weight at 12 months  in session attendance between the videoconference and in-person groups  of men chose videoconference groups, 31% (62/200) chose web-based videos, and 41% (82/200) chose in-person groups. There were significant differences in demographic, employment, cultural, and technology use and access factors among men who chose 1 of the 3 different options. For example, men who chose web-based videoconference groups were more likely to be younger, have higher income and education, have a white-collar type of job, prefer English, be more acculturated, have higher health literacy, have access to a computer, and have higher technology skills  compared with men who chose in-person groups. Our multivariate analysis distinguished most significantly between those who chose in-person groups versus those who chose web-based videos. Men who were older, spoke Spanish, and did not use a computer frequently had a higher probability of choosing in-person groups versus web-based videos. In terms of weight loss, men who chose the videoconference and in-person group sessions lost more weight than those who chose web-based videos.P<.001). In addition, Piatt et al [Similar to this study, Piatt et al  tested 3tt et al  reportedtt et al , group sThese findings have important implications for translation to practice, especially given the COVID-19 pandemic, which has made technology-mediated options necessary in the short term and possibly more common in the long term. The fact that 59% (118/200) of Latino men in this study preferred a technology-mediated option  suggests that these can be acceptable delivery formats for chronic disease prevention programs for this important high-risk population. This is consistent with previous literature indicating that Latinos are comfortable with technology and open to technology-mediated interventions -36. HoweThese findings demonstrate that Latino men from lower socioeconomic backgrounds may need additional support for accessing and using technology-mediated interventions. Technology use and access differ along socioeconomic lines in the United States, including among Latinos, with lower socioeconomic-level subgroups having less reliable access to technology than their higher socioeconomic-level counterparts ,38. AddiThe findings from the HOMBRE trial may be specific to the type of technology-mediated interventions offered. The 2 technology-mediated options included smartphone apps for tracking diet and physical activity and either videoconferencing or web-based videos for the intervention sessions. Preferences may vary according to the type of technology used in the intervention. Other technologies such as SMS text messaging, social media, and digital voice assistants may be appealing to other groups of Latino men. For example, Latino migrant farm workers have high (81%-97%) access to mobile phones and prefer talking and SMS text messaging using their phones ,39. TherThis study has several limitations. First, all the technology use data were self-reported. Participants may have reported answers regarding their prior and current use of technology that they may have perceived as desirable . Second,In conclusion, this study revealed that among a diverse group of Latino men recruited from primary care, 28% (56/200) chose videoconference groups, 31% (62/200) chose web-based videos, and 41% (82/200) chose in-person groups. Differences in demographic, employment, cultural, and technology use factors distinguished between men who chose each of the options, suggesting that when offering interventions in diverse groups of Latino men, choice of delivery may be recommended. We also found that men attending either of the group-based options (videoconference and in-person) lost more weight than men who chose the web-based videos.


Cancer prevention in the era of precision medicine has to consider integrated therapeutic approaches. Therapeutic cancer prevention should be offered to selected cohorts with increased cancer risk. Undoubtedly, carriers of hereditary cancer syndromes have a well-defined high cancer risk. Lynch Syndrome is one of the most frequent hereditary syndromes; it is mainly associated with colorectal cancer (CRC). Nonsteroidal anti-inflammatory drugs and, in particular, aspirin use, has been associated with reduced CRC risk in several studies, initially with contradictory results; however, longer follow-up confirmed a reduced CRC incidence and mortality. The CAPP2 study recruited 861 Lynch syndrome participants randomly assigned to 600 mg of aspirin versus placebo. Like sporadic CRCs, a significant CRC risk reduction was seen after an extended follow-up, with a median treatment time that was relatively short (2 years). The ongoing CAPP3 will address whether lower doses are equally effective. Based on pharmacology and clinical data on sporadic CRCs, the preventive effect should also be obtained with low-dose aspirin. The leading international guidelines suggest discussing with Lynch syndrome carriers the possibility of using low-dose aspirin for CRC prevention. We aim systematically promote this intervention with all Lynch syndrome carriers.  Cancer prevention for the general population is commonly associated with screening programs with early detection to lower cancer mortality as the primary endpoint. In parallel to this approach, preventive interventions strategies have been investigated to reduce cancer incidence. Therapeutic cancer prevention has great potential, but more research and educational and communication programs for health care providers and the general population are needed.Colorectal cancer (CRC) is still among the more common cancers in terms of incidence and mortality worldwide . A recenScreening programs are designed for the general population, and, especially in countries where there is publicly funded health care, age is the only discriminant to access to the program. In the era of precision medicine, screening programs should be personalized. For instance, in a recent paper by Helsingen et al. , a CRC sProposing a preventive treatment with a drug, which implies possible side effects and a long treatment period, underlines the importance of stratifying people based on their own risk. Higher disease risk may justify a different risk/benefit ratio of a specific drug intervention.Large cohort studies on cancer prevention, particularly on CRC prevention, are hampered by the time length, as they could take at least ten years before they produce evidence of the effect on cancer events. At the same time, the success of cardiovascular prevention at long-term follow-up was based on studies with reliable surrogate biomarkers. This supports the effectiveness of preventive interventions in the short–medium term. Hence the need to find and validate effective biomarkers to identify high-risk individuals better and predict the intervention efficacy in cardiovascular disease and CRC.The primary clinical marker for CRC prevention is adenoma due to its role as a precancerous lesion, and polypectomy reduces the risk of CRC . AdenomaThe systematic review and meta-analysis of randomized clinical trials by Veettil et al.  showed pA recent large cancer screening trial over a median duration of 13 years  showed tp = 0.02) for colon cancer, reaching a 55% reduction (p = 0.001) for proximal colon, but a not significant 10% reduction for rectal cancer was shown. The risk-reducing effect of aspirin on cancer is seen overall in the gastrointestinal tract. Further analyses of cancer events in randomized clinical trials with aspirin [Many lines of clinical evidence support the potential role of aspirin in cancer prevention ; however aspirin ,13,14,15A recent meta-analysis of observational studies  showed aThe risk reduction observed was similar between colon and rectal cancer. The authors showed a dose- and time-dependent linear response for CRC. In contrast to the results of the metanalyses of randomized clinical trials, a dose-response was apparently detected in this study. Out of 11 studies, a significant 10% reduction with 75 mg/day up to 50% with 500 mg per day was shown. Out of 22 studies, a time-dependent risk reduction was found, i.e., 4%, 19%, and 29% reduction were seen at one, five, and ten years of treatment, respectively .Recent observational studies strengthened the positive effect of regular aspirin use to reduce CRC risk and specific mortality ,18. The Overall, pre and post intake showed a significant 30 to 40% mortality reduction. The finding was that long-term aspirin use before a diagnosis of nonmetastatic CRC may be associated with lower CRC-specific mortality after diagnosis, consistent with possible inhibition of micrometastases before diagnosis. Recently, Lau et al.  showed tMechanisms driving aspirin potential in CRC chemoprevention are still under debate and will be discussed below.Overall, there is no doubt that aspirin can play a significant role in cancer prevention, especially for large bowel cancer; moreover, low-dose aspirin is recommended for the management of acute ischemic syndromes (both coronary and cerebrovascular) and for the prevention of their recurrence . Howeverhttps://www.uspreventiveservicestaskforce.org/uspstf/draft-recommendation/aspirin-use-to-prevent-cardiovascular-disease-preventive-medication#bootstrap-panel--4, accessed on 1 December 2021). The decision to initiate low-dose aspirin use for the primary prevention of cardiovascular disease in adults ages 40 to 59 years who have a 10% or greater 10-year cardiovascular disease risk should be an individual one. USPSTF recommends against initiating low-dose aspirin use for the primary prevention of cardiovascular events in adults age 60 years or older. However, persons who are not at increased risk of bleeding and are willing to take low-dose aspirin daily are more likely to benefit. The ASPREE trial put a substantial warning against using aspirin in elderly subjects [Clinical decision-making in this setting is guided by evaluating the risk of cardiovascular disease, CRC, and bleeding . The U.Ssubjects . The triNevertheless, dose and duration remain open issues.Precision medicine by tailoring drug treatment to specific individuals or populations could help to improve efficacy while reducing side effects; however, selecting the appropriate target population requires information on genetic and other biomarkers, together with environment and lifestyle for each person. These detailed patient data will be integrated using structured ontological approaches, analytics, mathematics, and statistics which constitute the tools of quantitative systems pharmacology; this strategy can predict drug efficacy and safety on an individual basis. The selection of patients and the dose for aspirin treatment will be performed using machine learning algorithms based on demographic, clinical, genetic, and biochemical information, such as BMI, diabetes, previous cardiovascular diseases, smoking status, genetic variants, inflammatory status, microenvironment (including microbiota and DNA methylation status), and risk factors for susceptibility to bleeding.The ADD-Aspirin randomized trial  is ongoiAspirin  is an NSAID . At ther2 and PGH2, the common substrate for many downstream isomerases and synthases, catalyzing its conversion to different prostanoids [In contrast to other NSAIDs that rapidly or slowly reverse inhibitors of COX-isozymes, the binding of aspirin to the COX active site is followed by covalent modification of COX-1 and COX-2, i.e., the acetylation of a specific serine residue located at position 529 and 516, respectively . Thus, astanoids .Aspirin has a short half-life (approximately 20 min); thus, the aspirin pharmacological effect duration depends on the rate of COX-2 protein synthesis. In a nucleated cell, COX-2 turnover is fast; thus, aspirin has to be administered every 4–6 h to obtain a persistent pharmacological effect. Once-daily aspirin administration at medium-high doses can produce only a transient inhibition of COX-2 activity due to rapid de novo protein synthesis. Several lines of evidence have shown that some NSAIDs, including aspirin, can inhibit the proliferation and induce apoptosis of colon cancer cells in vitro independently from their inhibitory effect on COX-2-dependent prostanoid biosynthesis . HoweverIn contrast, the irreversible inactivation of COX-1 in platelets (which do not express COX-2) by aspirin is long-lasting due to a low rate of de novo protein synthesis in the anucleated cell. Thus, low doses (75–100 mg) of aspirin given once a day are appropriate to cause a maximal inhibitory effect of platelet COX-1 activity , which persists throughout the interval between doses . AnotherWithin the limitations of post hoc analyses of cancer events that were not pre-specified endpoints, the results of the Thrombosis Prevention Trial are of interest to make a mechanistic interpretation since the anticancer effect of aspirin was apparent in men at high cardiovascular risk treated with a 75-mg controlled-release aspirin formulation associated with very low concentrations (approx. 0.29 μM) measured in the systemic circulation . This as2 in the intestinal mucosa, which occurs in the early stages of tumor development through cyclooxygenase (COX)-1 activity, in association with the suppression of the prostaglandin-degrading enzyme 15-prostaglandin dehydrogenase (15-PGDH) [2 production, thus promoting colorectal adenoma [Platelets are activated in response to environmental factors, atherosclerotic plaque rupture or fissuring, and intestinal mucosa damage. Activated platelets release a vast repertoire of mediators that may evoke numerous signaling pathways associated with a phenotypic switch of the cellular compartment of the stromal environment . These e15-PGDH) . Later,  adenoma  and its  adenoma .2 and its capacity to induce the phosphorylation of ribosomal protein S6 (p-S6) [The development of a novel direct biomarker of drug action via the assessment of the acetylation of COX-1 at serine 529 has allowed showing that low-dose aspirin acetylates normal colorectal mucosal COX-1 . This ef6 (p-S6) ; p-S6 re2 generation.The ratio of p-S6/S6 is associated with tumor progression. Thus, the antitumorigenic effect of low-dose aspirin can also involve the prevention of S6 phosphorylation via the inhibition of colorectal mucosal COX-1-dependent PGE2 production can also disrupt the normal apoptotic processes, allow the affected cells to accumulate genetic mutations, and ultimately lead to loss of proliferative control [Enhanced PGE control ,38.Moreover, it may suppress immune functions and facilitate tumor immune escape.2.Thus, low-dose aspirin can affect early events of intestinal tumorigenesis indirectly via the inhibition of platelet function and in part directly by affecting intestinal mucosa COX-1-dependent PGE2 production, thus promoting the progression of colorectal adenoma to adenocarcinoma [2 biosynthesis remains to be demonstrated. It is still unknown whether higher doses of aspirin can translate into a more profound inhibitory effect COX-2, thus improving the anticancer effect. Mechanistic studies are ongoing in Patrignani’s laboratory to address these issues, using the recently developed aspirin biomarker, which assesses the extent of acetylated COX-2 at serine 516 in CRC tissues together with the assessment of prostanoid biosynthesis in the cancer biopsies of CRC patients treated with aspirin at low and medium doses [The induction of COX-2 in the colorectal mucosa is associated with the aberrant increase of PGEarcinoma . The posarcinoma . Low-dosum doses .Platelets can uptake circulating proteins and RNAs/microRNAs, thus acquiring a different intracellular molecular repertoire specific to the individual clinical condition. The analysis of platelet content (and of platelet-derived microvesicles), as a whole of transcript (transcriptomics) or proteins (proteomics), has the promise of being a novel tool for diagnosis and prognosis of several diseases, including cancer .The analysis of systemic prostanoid biosynthesis (by measuring the urinary levels of prostanoid enzymatic metabolites) in patients at baseline or after aspirin treatment provides important information about the impact of aspirin on platelet activation, vascular and inflammatory state ,39,42.Plasma markers, such as soluble tumor necrosis factor receptor-2, as well as tumor expression levels of genes involved in prostanoid biosynthesis or signaling pathways activated by the aberrant expression of COX-2, such as phosphatidylinositol 3-kinase ,44 have A systems biology approach to the analysis of heterogeneous datasets  would allow dynamic systems modeling of candidate pathways involved in the antineoplastic effect of aspirin. This strategy would also allow identifying susceptibility profiles for CRC and their use to develop new biomarkers to predict its occurrence and recurrence.MLH1 and MSH2, lower for the other syndromic genes) [Lynch syndrome, caused by germline mutations of DNA mismatch repair (MMR) genes, accounts for about 3.0–5.0% of all CRCs; carriers of pathogenic variants face a CRC lifetime risk of approximately 50% (considering c genes) . For thic genes) . This obc genes) ,48: (1) Lynch syndrome carriers are a very high-risk population, appropriate for cancer prevention trials where aspirin may play as an “adjuvant therapy” to improve surveillance results (but not to quit colonoscopy).The CAPP2 trial  was desip = 0.02) with a similar prevention rate also for all other syndromic cancers.A planned 10 year follow-up publication  showed ap = 0.035), and this result was significantly evident in overweight patients. The protective effect for extra-colonic cancers seen in the previous publication was not confirmed even in the compliant subjects . No significant differences of adverse events  in aspirin subjects vs. placebo were observed. This finding is important to support the use of aspirin since adverse events are still the major barrier to its clinical use in primary prevention.With 20 years of follow-up , intentiSimilarly, to the data obtained from the cardiovascular trials ,9, the CAmong cancer prevention guidelines, the Cancer Council Australia (CCA) in 2017 updated its indication for CRC prevention, suggesting that all people aged 50–70 years with an average risk of CRC should consider taking low-dose aspirin to reduce their CRC risk . RegardSince the guideline publication, there has been no widespread modification in CRC prevention in clinical practice. Ongoing studies have been launched to better understand the awareness of physicians and the general population of CRC prevention, particularly the opinions and obstacles to taking aspirin ,55.As reported above, the United States Preventive Services Task Force considers taking aspirin for prevention based on personal cardiovascular risk level (10% or greater at 10 years) with an age range from 50 to 69 years old . Within In the United Kingdom, the National Institute for Health and Care Excellence (NICE) and the British Society of Gastroenterology (BSG), together with the Association of Coloproctology of Great Britain and Ireland (ACPGBI) and the United Kingdom Cancer Genetics Group (UKCGG) guidelines specifically for Lynch syndrome carriers, recommend considering aspirin intervention for at least two years ,57.The NCCN guidelines, probably one the most popular and frequently mentioned publications, recommend in this regard the use of aspirin but in a personalized way, in that the decision should be made on an individual basis (as well as the dose) after a discussion in which all aspects including individual risk profile, benefits, and side effects are fully considered.Same indications for Lynch Syndrome carriers are reported in the European Hereditary Tumor Group (EHTG) guidelines and the European Society of Coloproctology (ESCP). Their indication is to use aspirin at low doses (75–100 mg daily) and consider a higher dose for overweight subjects .MLH1, MSH2, MSH6) could benefit more from a preventive intervention than PMS2; genotype may also influence the starting age: for MLH1 and MSH2 carriers the starting age could be 25 years old since their cumulative risk to develop cancer reaches 10% at 35 years, whereas MSH6 carriers could start aspirin later, approximately at 35/40 years old, since their cancer risk reaches 10% at 60 years old, and even later, if any PMS2 carriers [In the decision process to aspirin use for CRC prevention in LS carriers, several issues have to be considered to balance pros and cons. In carriers ; (c) Forcarriers ; excess carriers , and placarriers ; thus, acarriers ; clinicaWe intend to discuss the information on aspirin as preventive medicine with all LS carriers followed by all the AIFET  adhering members. In line with the major international guidelines, the proposed dose will be 100 mg per day. The results from sporadic CRC prevention frame ,34 togetIn conclusion, we propose a spontaneous program within the AIFET network (former AIFEG), discussing with Lynch Syndrome carriers the possibility of taking aspirin for CRC prevention and, after acceptance, monitoring for treatment adherence and side effects. Our aim is also to seek predictive surrogate biomarkers and to monitor treatment efficacy.


Pain is affecting acceptance of supportive periodontal therapy and primary periodontitis prevention. Our objective was to evaluate the efficacy of a 1-week pre-treatment use of dentinal-hypersensitivity-reducing mouth-rinses (DHM) in periodontal maintenance (SPT) or dental prophylaxis patients.n = 52), arginine (n = 52), or herbal extracts (n = 51). At baseline and reassessment, procedural pain was assessed by visual analogue scale (VAS) and verbal rating scale (VRS). Self-reported efficacy was documented.One hundred fifty-five participants attending for professional mechanical plaque removal (PMPR) were randomly assigned to use a mouth-rinse twice daily for 1 week prior to their next PMPR. Rinses were containing either potassium oxalate (p < 0.05).No inter-group differences were estimated between both test groups and the control for baseline and reassessment means . In the SPT group, VAS reduction and self-reported efficacy were found  contains supplementary material, which is available to authorized users. The medical benefits of professional mechanical plaque removal (PMPR) are well documented for primary and secondary prevention of periodontitis –4. FocusA source of discomfort or pain during PMPR is the mechanical irritation of the dentin-pulp complex. The pain origin is similar to dentine hypersensitivity—the hydrodynamical theory . In the Therefore, the aim of this study was to evaluate the efficacy on procedural pain during PMPR of a 1-week use of two mouth-rinses designed to relieve dentin hypersensitivity prior to PMPR on pain or discomfort in patients ongoing primary or secondary periodontal prevention. Primary outcome was procedural pain during PMPR measured by VAS and VRS, and secondary outcomes were self-reported efficacy and safety.This was a randomized, clinical, single-blind, controlled, parallel-group, investigator-initiated trial conducted in Germany to evaluate the impact of a 1-week pre-operative use of two mouth-rinses designed for dentine hypersensitivity  on pain or discomfort caused by PMPR in maintenance patients . All examinations were carried out at the Department of Operative Dentistry and Periodontology at the University of Cologne. The study was approved by the local ethics review board of the University of Cologne (no. 16-257) and registered (DRKS00010811). All participants gave written informed consent before study-related procedures were carried out.Participants were individually instructed in the use of the mouth-rinses, and written instructions were also provided. The participants were instructed not to use any other kind of mouth-rinse and continue the use of their pre-study dentifrice, toothbrush, and interdental cleaning routine.A study of a continuous response variable from independent control and experimental subjects with one control per experimental subject was planned. In a pre-study, the response within each subject group was normally distributed with standard deviation 1. If the true difference in the experimental and control means is 0.5, 64 experimental and 64 controls are needed to reject the null hypothesis that the population means of the experimental and control groups are equal with the power 0.8. The type I error probability associated with this test of this null hypothesis is 0.05. A priori, an interim analysis was planned after 12 months duration of recruitment and at least 50% of participants included.After the ethical and organizational tasks were fulfilled, the screening began in September 2016. Participants were recruited in the Department of Operative Dentistry and Periodontology at the University Hospital Cologne. Inclusion criteria were (1) risk-based periodontal maintenance or dental prophylaxis patient , (2) recall frequency twice a year (or more), (3) recall appointment third or more, and (4) adult patients (> 18 years old). Exclusion criteria were (1) presence of acute dental pain, pulpitis, or other acute dental infections; (2) caries lesions with invasive treatment need; (3) antibiotic therapy (up to 7 days prior to the study appointments); (4) history of allergic or undesirable reactions to the test products or ingredients; (5) long-term medication with analgetics, (6) diseases or medication influencing inflammation or the immune system (including anti-inflammatory drugs); (7) abuse to alcohol; and (8) pregnant or lactating women.https://prod.tenalea.net/zkskoeln/dm/). Allocation concealment was achieved by having a person not involved in clinical examination (L. V.) distributing the mouth-rinses and giving instructions for use. Examiners were blinded regarding the mouth-rinse used.Block randomization (strata: self-reported pain sensitivity and diagnosis) into three groups was provided by the Institute for Medical Statistics, Informatics and Epidemiology, University of Cologne  and plaque index (PI) were obtained as described in detail elsewhere , 16. PMPParticipants started to use the assigned mouth-rinse 1 week prior to the second PMPR appointment twice daily—in the morning and the evening—for 1 min directly after toothbrushing. The amount of mouth-rinse per use followed the application instructions of the manufacturers . Participants were instructed not to use any other kind of mouth-rinse and continue the use of their pre-study dentifrice, toothbrush, and interdental cleaning routine. They were asked to complete a rinsing diary and document each use of the mouth-rinse including time of use and amount. The diaries were collected and checked for completeness after second study appointment to ensure adherence to the study protocol.Indices and PMPR were proceeded as prescribed above. Additionally, intra-oral soft tissue examination was performed (safety protocol). Again, all participants filled in a questionnaire regarding their self-reported efficacy of the assigned mouth-rinse and the procedural pain via VAS and VRS.p values ≤ 0.05 were considered to indicate statistical significance. Data were typed twice to minimize data entry mistakes. Missing data (drop out) were handled by the last-observation-carried-forward method (LOCF).Between groups differences  were evaluated for baseline, study appointment, and the treatment effect by one-way ANOVA. Within group analysis of the treatment effect was performed by Wilcoxon signed-rank test. Pearson’s chi-square test was used for dichotomous parameters. Additionally, subgroup analyses dividing the groups by initial diagnosis were performed as described above. All analyses were carried out at participant level  using SPSS Statistics 24 Software . A total of 155 participants  completed the trial  in contrast to arginine and control (p > 0.05). Pointing out the responder analysis, dipotassium oxalate showed the highest amount of responders and the lowest amount of non-responders compared to arginine and control group , accompanied by the largest VAS reduction  and a superior intra-group treatment effect (Wilcoxon signed-rank test p = 0.001) in periodontitis patients. None of these effects was found in the gingivitis group . Subgrouping by diagnosis revealed the same results for gingivitis and periodontitis patients . In gingivitis patients, no differences were shown  quit using the assigned mouth-rinse because of bad taste.To our knowledge, this randomized controlled trial is the first to evaluate the efficacy of a 1-week use of mouth-rinses designated for dentinal hypersensitivity as pretreatment to control pain and discomfort during professional mechanical plaque control in patients undergoing primary or secondary periodontal prophylaxis. Even if the dentinal-hypersensitivity-reducing mouth-rinses were not able to achieve a predictable impact on discomfort during PMPR for all patients, dipotassium oxalate proved a pain reduction ability overall and in the periodontitis group but not for gingivitis patients.Focusing VAS responder-analysis, all groups showed around 50% responders, but the dipotassium oxalate users showed noticeable fewer non-responders with 6% than the others . In patients undergoing periodontal supportive therapy, both test groups showed a higher amount of VAS responders which was not found in gingivitis patients.Also, both test groups showed a higher amount of self-reported efficacy, and again the dipotassium group pointed out with the best results. Despite the fact that a predictable impact on pain experience measured by VAS was not found, the subjective efficacy was reported by more than 50% of the participants in the test groups. Thus, our hypothesis was not supported by the primary outcome but proved for self-reported efficacy. Again, this effect was quantifiable for periodontitis patients but limited in the gingivitis group. Even if the measurable pain reduction could not be found, this patient-reported outcome (PRO) is a very valuable information as it reflects the individual perception of the participants. The recent EFP position paper focusing on endpoints of active periodontal therapy demands PROs to be included in studies additional to clinical measurements . PatientThe overall analysis and subgrouping by diagnosis revealed a higher efficacy of the mouth-rinse pretreatment in the periodontitis-group, especially for DPOX. As expected, the occlusion-based active ingredients proved a higher impact on pain reduction in periodontal patients. This may be explained by the already experienced attachment loss accompanied by a higher amount of open dentine tubules. No quantifiable effect could be found for gingivitis patients. For them, procedural pain during PMPR may rather be soft tissue-associated or caused by pain peak sites with dentine hypersensitivity. The prevalence of dentine hypersensitivity is high and affects around one-third of the patients, even in young adults , 21. OneDuring PMPR both—hard and soft tissues—are stimulated and therefore both may be a source of experienced discomfort with individual distribution. As intrapocket anesthesia is a well-documented option to overcome soft tissue pain in a non-invasive manner, hard tissue–related sensations are predominantly controlled by invasive injection anesthesia or in-office use of medical products –25.A meta-analysis focusing the use of desensitizing mouth-rinses to control dentin hypersensitivity was performed in 2017 . Due to We have also carried out a gender-specific subgroup analysis . Both test products belong to well-known brands from the healthcare sector. This may have triggered a positive expectation in patients, which may have been reflected in an overly good evaluation of self-reported efficacy.Based on the sample size calculation, we needed 64 participants per group and included 10% more for drop-outs. During the study, every fifth participant dropped out. That may be attributed to long period between the PMPR appointments with 3 to 6 months depending on risk-based recall frequency ESM 2(DOCX 15 kb)ESM 3(DOCX 11 kb)


COVID-19, caused by SARS-CoV-2, led to an unprecedented global health crisis. As the majority of people infected with SARS-CoV-2 have no or only mild symptoms, many cases aren’t captured by direct testing. However, it is important to establish the true spread of the virus by identifying how many people have been exposed. Detection of anti–SARS-CoV-2–specific antibodies in blood samples can help us understand how the pandemic is evolving over time. We developed a sensitive and specific assay that performs 1,024 measurements in parallel. To enable decentralized blood sample collection, the method can detect antibodies in a small drop of blood obtainable by finger pricking, and the blood can be collected and shipped with a simple, low-cost blood glucose test strip. Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti–SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 μL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general. The emergence of a new coronavirus at the end of 2019, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to an unprecedented global public health crisis . HoweverDetection of anti–SARS-CoV-2 antibodies using highly sensitive and specific assays can help answer these questions. Several seroprevalence studies have already been conducted, demonstrating rather low seroprevalence rates even in areas that were severely affected –9. Such Several assays, such as enzyme-linked immunosorbent assays (ELISA) or chemiluminescent immunoassays (CLIA), are commercially available, but mainly rely on serum drawn by venipuncture. These tests are also rather expensive, with reagent costs on the order of 3 USD to 10 USD per test. Alternatively, in-house ELISAs are difficult to standardize and require high amounts of recombinant antigen, usually around 100 ng per sample . Other r12There is therefore a need for new technologies to supersede existing methods such as ELISA, CLIA, and LFAs. Novel technologies should be capable of high throughput, low reagent consumption, and low cost per test; achieve high sensitivity and specificity; and be compatible with ultralow-volume whole blood samples in the low or even submicroliter range that can be obtained via a simple finger prick. Biomarker detection using dried whole blood on filter paper or other devices would have tremendous advantages, as the sample can be collected by untrained individuals at home. The samples could then be conveniently shipped by regular mail at ambient temperature to a central laboratory for analysis, and test results could be returned electronically via a mobile app or email.® and DBS System SA’s HemaXisTM DB10, and show that it is possible to repurpose low-cost and widely available blood glucose test strips for sample collection and shipment. Samples could be stored up to 6 d at room temperature with minimal sample degradation. All three methods combined with NIA identified more positive samples than a standard ELISA performed on serum samples collected from the same individuals.In this study, we developed and validated a nanoimmunoassay (NIA) that analyzes 1,024 samples in parallel on a single microfluidic device the size of a USB stick. NIA reagent consumption and corresponding costs are roughly 1,000 times lower than a standard ELISA. NIA achieved a specificity of 100% and a sensitivity of 98%, based on the analysis of 134 prepandemic negative sera and 155 positive sera from RT-PCR–confirmed positive individuals. NIA performed well for samples obtained more than 20 d post onset of symptoms, and performed equally well for samples obtained less than 20 d past onset of symptoms. We go on to demonstrate that NIA can be used to detect anti–SARS-CoV-2 antibodies in ultralow-volume dried whole blood samples, eliminating the need for venipuncture blood collection. We tested two commercial blood collection devices: Neoteryx’s MitraWe adapted a mechanically-induced trapping of molecular interactions (MITOMI)-based , 16 1,02SI Appendix, Fig. S1). The use of trimerized full-length spike and spike receptor binding domain both resulted in high signal intensities and low background. We decided on using the trimerized full-length spike protein, as it most closely resembles the natural conformation of the SARS-CoV-2 spike protein. As a technical validation, we added chimeric anti-spike antibody at different concentrations into human serum, spotted each dilution, and showed that NIA could quantify anti-spike antibody concentrations . NIA achieved a maximum specificity and sensitivity of 100% and 98%, respectively, at a serum dilution of 1:8  and a receiver operator characteristic (ROC) curve with an area under the curve (AUC) equal to 0.99 (n = 138) of the same condition-positive COVID-19 patient serum samples tested above. For samples collected after 20 d post onset, these assays returned sensitivity values in the range of 74 to 98% and, for samples collected less than 20 d post onset, returned sensitivity values ranging from 66 to 89% . These results indicate that NIA is a robust method for detecting SARS-CoV-2 antibodies in human serum samples.We validated the high-throughput NIA for the detection of SARS-CoV-2 anti-spike IgG antibodies with 289 serum samples, collected from 155 PCR-confirmed SARS-CoV-2–infected individuals and 134 prepandemic negative samples collected in 2013/2014 and 2018. Different serum sample dilutions, ranging from no dilution to a 1:256 dilution, were spotted to determine the optimal value . We found that using duplicates only slightly reduced sensitivity from 98 to 97%, whereas using a single measurement lowered sensitivity to 95%, all the while retaining a 100% specificity. Given that using duplicate measurements led to only a minimal change in sensitivity, it will be possible to increase throughput to 512 samples analyzed in duplicate per device. Additional gains in throughput are theoretically possible by scaling the device itself, with MITOMI devices containing up to 4,160 unit cells having been demonstrated in the past . We then correlated the measurements from one of those devices to the measurements made on six other devices and again observed a good correlation of SI Appendix, Fig. S5B). In the first case, the same sample solution was used for spotting two devices, whereas, in the second case, the two measurements being compared came from separately prepared patient sample dilutions. Additionally, we compared the anti–IgG-PE signal measured for a reference serum dilution series on three separate devices and computed an average coefficient of variation (CV) equal to 11.7% for the dilutions measured .To assess device-to-device reproducibility, we tested all serum samples (1:8 dilution) on two separate devices, which resulted in a correlation of SI Appendix, Fig. S6). Using the calculated To explore the technical capabilities of NIA, we determine whether quantitative antibody titers could be obtained. We fit data from the full dilution series to a saturation binding curve model, enabling us to extract the dilution equivalent to the half-maximum signal for each serum sample  for whole blood collection and shipping. The glucose test strips collect 0.6 The NIA platform proved to be highly specific and sensitive when applied to serum samples. We recognized that use of serum samples limits the applicability of our and other methods to samples collected by venipuncture, which has to be performed by trained personnel in hospitals or point-of-care settings. Venipuncture requires large volumes of blood in the 5-mL range, considerable downstream sample processing, and a cold chain from point of sample collection to point of analysis. Large-scale seroprevalence studies are thus challenging to conduct, especially when including remote or large geographic areas. Establishing a large-scale, low-cost, and widely accessible serology platform necessitates the development of venipuncture alternatives. For this reason, we developed a sample collection and processing pipeline that enables us to carry out NIA with ultralow-volume, dried whole blood samples obtainable by a simple finger prick. We tested three different methods to collect, ship, process, extract, and analyze dried whole blood samples . We testSI Appendix, Fig. S7).We evaluated the three collection methods by spiking human whole blood with different concentrations of anti-spike IgG and collected the blood with each collection device. We then extracted the dried blood from each device and spotted the samples for NIA analysis. We first tested an aqueous ethylenediaminetetraacetic acid (EDTA) solution and sonication for extraction , but theSI Appendix, Fig. S8 A–C). We calculated the coefficient of variation for the technical repeats for each anti-spike concentration tested and found that it did not exceed 15% for any of the three collection methods, with an average CV of 5.7%, 7.7%, and 9.2% for Mitra®, HemaXis™DB10, and the glucose test strips, respectively . Although, there may be further variability introduced depending on how each individual collects his or her own blood sample, each device uses a hard-coded method to collect a specific volume of blood that leads to low variability for NIA antibody detection.To assess the variability of each sampling method, we collected whole blood spiked with anti-spike IgG three separate times and compared the on-chip antibody signals  in a small research laboratory not dedicated or equipped for high-throughput molecular diagnostics. Due to increases in efficiency, a small team of three can likely achieve a throughput of six devices or 3,072 samples per day . By comparison, the number of RT-PCR tests performed in all of Switzerland ranged from 6,000 to 18,000 tests per day between April and September 2020. Consumables, reagent consumption, and associated costs are negligible with NIA, which is an important consideration when compared to the high reagent cost of ELISAs and when considering potential reagent shortages that may be encountered during critical phases in a global pandemic.To implement the method, laboratories require a commercially available contact microarrayer, and the ability to fabricate masks, molds, and PDMS microfluidic devices. Instruments required for photolithographic fabrication of masks and molds are available at most major research universities, and mask fabrication can be outsourced to service-based companies. PDMS microfluidic device fabrication can be conducted in a standard research laboratory with low-cost equipment: a spin coater, Platform capabilities could be further expanded in the near term. We previously demonstrated that the platform can be used to perform multiplexed analysis, allowing four or more biomarkers to be tested for each sample , 18, 24.® from Neoteryx and HemaXis™DB10 from DBS System SA. We also explored the possibility of repurposing low-cost and readily available blood glucose test strips for blood sample collection and shipment. Mitra and DB10 allow the collection of a minimal volume of 10 To enable large-scale studies, we placed specific emphasis on the development of simple, low-cost, and ultralow-volume sample collection strategies and integration of these workflows with the NIA platform. We characterized three blood sampling devices and tested all of them with patient samples. Two of these are commercially available devices for the express purpose of collecting small-volume capillary blood samples: MitraThe combination of a high-throughput, highly specific and sensitive NIA and the ability to analyze minute volumes of dried blood samples have enormous potential for SARS-CoV-2 serology, epidemiological studies, vaccine trial, and therapeutic development support. Further areas of use could be large-scale seroprevalence studies in low- and middle-income countries  without New technology and method developments such as those reported here will make it possible to overcome the current centralized molecular diagnostics paradigm, which is focused on hospitals and point-of-care settings rather than on patients and individuals seeking simple, affordable, and convenient molecular diagnostics. The need to visit a clinic is an inconvenience for everyone and can be an insurmountable obstacle for many. The requirement for venipuncture blood collection, sample pretreatment, and costly ELISAs prohibits broad testing and contributes to high health care costs.In the future, NIA could make it possible for individuals to purchase a simple blood sampling kit containing a lancet, a blood sampling device, and a return mail envelope, at a local pharmacy or supermarket B. The kiSI Appendix. High-throughput NIA was conducted using a PDMS microfluidic device. Patient serum or extracted whole blood samples were spotted onto an epoxy-coated glass slide using a microarray robot. All samples were analyzed for the presence of anti-spike IgG antibodies on-chip. In parallel, commercial IgG ELISA assays were performed for all samples according to the manufacturer’s instructions. All samples were collected according to the local ethical guidelines, and ethical approval was waived by the ethics committee of the University Hospital of Geneva (HUG), that approves usage of leftover patient serum collected for diagnostic purposes, in accordance with Swiss Regulations on human research. Patients signed the general informed consent form of the HUG, which states that any leftover material can be used for research purposes.Full details are given in Supplementary FileSupplementary FileSupplementary File