High rates of discontinuation undermine the effectiveness of adjuvant endocrine therapy (AET) among hormone-receptive breast cancer patients. Patient prognosis also relies on the successful management of cardiovascular risk, which affects a high proportion of postmenopausal women. As with AET, adherence with cardiovascular drugs is suboptimal. We examined whether patient adherence with cardiovascular drugs was associated with the rate of AET discontinuation in a French nationwide claims database linked with hospitalisation data.We identified postmenopausal women starting AET between 01/01/2016 and 31/12/2020 and taking at least two drugs for the primary prevention of cardiovascular disease  before AET initiation. Adherence was assessed for each drug class by computing the proportion of days covered. Women were categorised as fully adherent, partially adherent or fully non-adherent with their cardiovascular drug regimen based on whether they adhered with all, part or none of their drugs. AET discontinuation was defined as a 90-day gap in AET availability. Time to AET discontinuation according to levels of cardiovascular drug adherence was estimated using cumulative incidence curves, accounting for the competing risks of death and cancer recurrence. Multivariate cause-specific Cox regressions and Fine-and-Gray regressions were used to assess the relative hazards of AET discontinuation.In total, 32,075 women fit the inclusion criteria. Women who were fully adherent with their cardiovascular drugs had the lowest cumulative incidence of AET discontinuation at any point over the 5-year follow-up period. At 5 years, 40.2% of fully non-adherent women had discontinued AET compared with 33.5% of partially adherent women and 28.8% of fully adherent women. Both partial adherence and full non-adherence with cardiovascular drugs were predictors of AET discontinuation in the two models .Clinicians should be aware that patients who do not adhere with their entire cardiovascular drug regimen are also more likely to discontinue AET. This stresses the importance of integrated care, as suboptimal adherence with both treatment components poses a threat to achieving ideal patient outcomes.The online version contains supplementary material available at 10.1186/s12916-023-03156-3. Robust evidence has demonstrated the benefits of adjuvant endocrine therapy (AET) on the health outcomes of patients with oestrogen-receptor positive breast cancer (BC). Early trials showed that tamoxifen reduced the rate of BC recurrence by almost 50% in the first 5 years and 30% in the following five. For the first 15 years from treatment initiation, the annual BC death rate was around 30% lower in the treatment groups compared with the control groups . Later tImprovement of cancer treatments have resulted in BC patients being increasingly affected by competing morbidity and mortality risks, one of which is cardiovascular disease (CVD). In women with BC aged over 65, cardiovascular mortality actually exceeds deaths from breast cancer . This caSuboptimal medication adherence with AET and cardiovascular medication are well documented and affect patient outcomes. Adherence behaviours are often divided into three components: treatment initiation, implementation and persistence. They indicate whether a patient starts the treatment, respects the dosage schedule and persists with treatment during the intended duration of the prescription. A considerable number of patients discontinue AET before the end of the required 5 years, which negatively impacts disease-free survival –13. StudMuch effort has been put into understanding which factors contribute to medication-related behaviours. Nevertheless, the question of overall drug adherence in patients with multiple medications has received little attention. In a sample of Medicare beneficiaries in the USA initiating statins after a hospitalisation for coronary heart disease, patients with low antihypertensive adherence were more likely to discontinue treatment within one year . This beWe used the French National Health Data System , which merges the national claims database with the national hospital-discharge summaries database and the national death registry through a unique pseudonymised patient identifier. It includes 98.8% of the French population. Individual-level data is collected on all areas of healthcare use. This includes outpatient visits, dispensed medication, procedures, long-term disease (LTD) status, hospital procedures and admission diagnoses using the International Classification of Diseases, 10th revision (ICD-10) codes. A morbidity mapping tool based on algorithms that combine information on LTD status, inpatient diagnoses and pharmacy reimbursement claims allow for the identification of 56 health conditions including CVD and BC. Patient status for each health condition is defined per calendar year. Demographic information such as month and year of birth, date of death and municipality of residence are also recorded .We identified all women aged over 50 initiating AET between 01/01/2016 and 31/12/2020 with an aromatase inhibitor . This was defined as the index date. Women were considered to be initiating treatment if they had no reimbursement for any kind of AET in the previous 2 years. We only kept patients who had undergone lumpectomy or mastectomy in the 18 months prior to the index date or in the 6 following months and had no ICD-10 codes for metastasis  over the same period.Women were considered to be under multiple cardiovascular drugs in the year prior to AET initiation if they were reimbursed for at least two antihypertensive and/or lipid-lowering drug classes in the year before that date, with at least 3 reimbursements per drug class. The following therapeutic drug classes were considered: diuretics, beta blocking agents, calcium channel blockers, renin-angiotensin system (RAS) inhibitors (angiotensin-converting enzyme inhibitors [ACEI] or angiotensin-receptor blockers [ARB]), platelet aggregation inhibitors and lipid-modifying agents. Many patients with cardiovascular risk factors are prescribed a multiple drug regimen, which is why we focused on patients with at least two medications. Using the morbidity mapping tool, we excluded women with a CVD diagnosis  or with end-stage renal disease from the start of the calendar year preceding AET initiation up to the initiation date. Our cohort therefore consisted of women treated for the prevention of CVD. We further excluded women who died within 6 months of AET initiation, did not reside in metropolitan France or for whom information on the French Index of Social Deprivation (FDEP) was missing. The FDEP is a composite measure defined at the municipal level that summarises median household income, percentage of high school graduates in the population aged over 15, percentage of blue-collar workers in the active population and unemployment rate .The primary endpoint was discontinuation of AET, defined as the first day of a period of at least 90 days without any available medication. This is a standard threshold used in the literature . While oThe dispensing dates and number of pills supplied were used to create individual 5-year supply diaries starting from the index date and indicating whether a patient had a pill available during each day of follow-up. Patients could stockpile pills for a maximum of 180 days. When a patient refilled their medication before the end of the previous fill, the days of overlap were credited at the end of the next supply. Time-to-discontinuation was measured in days.The predictor of interest was global cardiovascular treatment adherence in the year before AET initiation. We used a time-invariant measure because it was difficult to make reliable hypotheses about treatment prescriptions over several years without prescription data. Changes in cardiovascular drug regimens over time, such as drug switches and adding or removing therapeutic classes, occur for some patients.We based patients’ prescriptions off the therapeutic drug classes they were dispensed in the second year before the index date. Supply diaries were created for each drug class in a 1-year look-back period before AET initiation, using the same process as with AET. We additionally considered that patients were supplied their cardiovascular drugs by the hospital staff during hospitalisations requiring an overnight stay. Supplies of all drugs within a given class were considered for creating class-specific diaries. Daily dosage was ruled as one pill a day, which is in line with the usual drug dosage for the primary prevention of CVD.From the diaries, we estimated patient adherence for each drug class by computing the proportion of days covered (PDC). The numerator was the number of days in the year during which the patient had an available pill of that class, and the denominator was 365. Patients were considered as adherent with a drug class if the corresponding PDC was greater than 80% .We created a 3-level indicator of global cardiovascular treatment adherence in the year before AET initiation by categorising women as follows : fully aDescriptive statistics were computed at baseline for clinically relevant predictors of AET discontinuation identified in the literature: age, type of BC surgery, adjuvant chemotherapy, number of comorbidities besides BC  AET discontinuation defined as 60- and 120-day gaps in treatment availability and (2) the adherence threshold for cardiovascular drugs set at a PDC of at least 70 and 90%.Analyses were performed using SAS Enterprise Guide 8.3.Details of the population selection process are presented in Fig. The number of patients supplied with each cardiovascular drug class and proportion of adherent patients by drug class are presented in Table By the end of the first year of treatment, 11.3% of the cohort had discontinued AET. Discontinuation reached 17.7% by the end of the second year, 23.8% by the end of the third year, 28.0% by the end of the fourth year and 32.4% at 5 years  and this increase reached 49% among patients who were fully non-adherent (CSHR 1.49 [95% CI 1.39–1.58]). The rate of occurrence of the competing events was not impacted by cardiovascular adherence. Being partially adherent rather than fully adherent did not significantly change the rate of death  or cancer recurrence . Being fully non-adherent did not change the rate of recurrence  and non-significantly increased the rate of death .Log-minus-log plots for each predictor and unadjusted estimates are available in additional Figure St. The Fine-and-Gray model therefore considers how a covariate is related to the competing events, since the value of the SDHRs may result from the direct effect of the covariate on the rate of AET discontinuation or from its indirect effect on the rate of death or cancer recurrence ) and a higher cumulative incidence of the event . Adjuvant chemotherapy before starting AET had a slightly negative effect on the rate of treatment discontinuation  but was associated with a high rate of cancer recurrence  and had an important impact on the cumulative incidence of AET discontinuation . Women who had been taking cardiovascular treatment for over 5 years had a much lower rate of discontinuation . The impact on the cumulative incidence was identical.These main results were supported by the several sensitivity analyses that we conducted. Lowering the threshold for drug adherence from 80 to 70% increased the size of the main effect, as did lowering the definition of discontinuation from a 90- to a 60-day gap in treatment availability. Increasing these thresholds tended to lower the size of the effect but did not change any qualitative conclusions. Details are available Table We used pharmacy claims and hospitalisation data covering 98.8% of the French population to examine whether adherence with cardiovascular drugs was associated with AET discontinuation in women with non-metastatic oestrogen-receptor positive BC and with a multiple drug regimen for the primary prevention of CVD. At each point over the 5-year period, women who were not adherent with any of their CV drugs in the year prior to AET initiation had a higher cumulative incidence of AET discontinuation. Cause-specific hazard ratios showed that CV adherence was a strong predictor of AET discontinuation when adjusting for frequently associated factors. We observed an increasing gradient between this variable and AET discontinuation. Fully non-adherent women had a 49% higher rate of AET discontinuation over the 5-year follow-up period compared with fully adherent women, and partially adherent women a 16% higher rate of discontinuation. The subdistribution hazard ratios yielded by the Fine-and-Gray model showed an almost identical impact of our exposure of interest on the cumulative incidence of AET discontinuation. This is because cardiovascular treatment adherence was not associated with the rate of occurrence of the competing events. Our results were supported by the sensitivity analyses in which we tested more or less lenient definitions of AET discontinuation (minimum 120- and 60-day gaps in treatment) and of the adherence threshold for the proportion of days covered (70 and 90%). We can therefore consider that our conclusions were not biased by our choice of outcome definitions.In our study, 17% of the population either died or had cancer recurrence before discontinuing AET. Competing risks are largely prevalent in studies that include older adults with multimorbidity. Failure to account for them correctly can lead to adverse consequences, including overestimating the cumulative incidence of the event of interest and false interpretations of the hazard ratios , 30. TheWhile the number of prescribed medications and of coexisting health conditions are commonly included in models aiming to understand which patients are more likely to discontinue AET, studies have not focused on patients with multimorbidity and their adherence to other medication classes. Our finding of an association between low adherence with cardiovascular treatment and increased hazard of AET discontinuation is consistent with previous studies demonstrating consistent behaviours across different drug classes, mainly between statins and antihypertensive drugs, antidiabetics and other lipid-lowering drugs , 33–35. Understanding global patient adherence to prescribed medication is all the more relevant given the high prevalence of multimorbidity in breast cancer patients. There is currently no standard definition of multimorbidity which makes it challenging to provide accurate prevalence estimates. Nevertheless, studies are in agreement to say that over 50% of patients with BC have at least one co-occurring chronic condition , 39. HypNon-adherence with preventive medication is a major driver of multimorbidity, and our results show consistent adherence behaviours across cardiovascular treatment and AET. A step towards limiting the burden of multimorbidity in BC cancer patients will therefore require identifying and supporting women with low adherence to their cardiovascular drug regimen early on in the course of AET, as they may be at heightened risk of both CVD and BC-related events. This is an additional argument in favour of “cluster medicine”, a term coined by a BMJ editorial in 2020 to shift the consideration of multimorbidity from a “random assortment of individual conditions to recognising it as a series of largely predictable clusters of disease in the same person”  and encoThere are limitations to our study, mainly linked to our use of an administrative claims database. We used drug refill dates and number of pills supplied to estimate discontinuation and adherence behaviours. While this provides a good indication of whether patients comply with treatment, it gives no indication of whether supplied pills have indeed been ingested. There is currently no gold standard for measuring medication adherence, and claims data are a reliable and non-invasive data source that provide insights into patient behaviour among large community samples . An addiConsequences of high blood pressure and hyperlipidaemia are a leading cause of premature death worldwide, and many affected patients are treated with a multi-pill regimen. One of the leading causes of uncontrolled blood pressure and CVD is non-adherence, and postmenopausal BC patients are especially vulnerable with regard to cardiovascular health. Understanding how multimorbid patients with BC behave towards their entire treatment contributes to identifying patients that may be concurrently at increased risk of negative outcomes for different diseases. Our study suggests that specialist clinicians should pay particular attention to patients if they notice low adherence to a particular drug. This may in fact reveal suboptimal adherence across other medication classes.Additional file 1: Tables S1. List of AET drugs with a dosing regimen of 2 pills per day. Table S2. List of health conditions considered for the comorbidity count included in multivariate models. Table S3. Population characteristics stratified by level of cardiovascular treatment adherence. Table S4. Unadjusted predictors of AET discontinuation using Cox and Fine-and-Gray regressions. Figures S1. Log-minus-log plots for each time-invariant predictor.


Background: It was hypothesized that the time-appropriate return to a resting heart rate (HR) after cessation of exercise could be a marker for predicting outcomes in patients with heart failure (HF). We aimed to evaluate the prognostic value of HR recovery in functional improvement among adults with severe aortic stenosis undergoing percutaneous aortic valve implantation (TAVI). Methods: We performed a 6 min walk test (6MWT) in 93 individuals before TAVI and 3 months after the procedure. The change in walking distance was calculated. During the pre-TAVI 6MWT, we analyzed the differences between baseline HR, HR at the end of the test, and HR at the 1st, 2nd, and 3rd minute of recovery. Results: After 3 months, 6MWT distances improved by 39 ± 63 m and reached a total of 322 ± 117 m. Multiple linear regression proved the differences between HR after 2 min of recovery and baseline HR in pre-TAVI after a 6MWT was the only significant predictor of waking distance improvement during follow-up. Conclusions: Our study suggests that analysis of HR recovery after a 6MWT may be a helpful and easy parameter to assess improvements in exercise capacity after TAVI. This simple method can help to identify patients in whom no significant benefit in functional improvement can be expected despite successful valve implantation. Aortic stenosis (AS) caused by calcification and fibrosis of the valve leaflets leads to left ventricular outflow tract obstruction, heart failure progression and finally poor outcome. Its prevalence in developed countries is between 2–7% of patients over 65 years and its incidence increases with age. Once symptoms occur in severe AS, the prognosis is very poor without invasive treatment, with a 5-year mortality rate of 50%. According to the European Society of Cardiology and American Heart Association guidelines, transcatheter aortic valve implantation (TAVI) has become an established, less invasive treatment method than traditional surgery for severe symptomatic aortic stenosis not only in older, inoperable, or high-risk adults but also in lower-risk populations. The main randomized controlled trials exploring TAVI included patients with typical tricuspid valve anatomy; however, many real-world registries have shown also the feasibility of TAVI in bicuspid aortic valve anatomy. Parallel to native TAVI development, first transcatheter valve-in-valve TAVI procedures were introduced in 2007 and showed to be a feasible alternative to re-do surgery in symptomatic patients.Despite the unquestionable clinical benefit, intermediate and long-term mortality rates and a lack of functional status improvement among patients undergoing TAVI are still high ,2. Thus,The 6 min walk test (6MWT) is a simple, reproducible, and easily carried out method that requires no specialized equipment or advanced training and can provide objective assessments of the functional status in a clinically impaired population. The 6 min walk test distance (6MWTD) is a widely accepted measurement of functional capacity and symptom assessment . It corrIt was hypothesized that the time for peak exercise HR to return to the resting HR after cessation of exercise could be a new marker for predicting outcomes in patients with HF. Accordingly, we sought to evaluate the prognostic value of HR changes during a baseline 6MWT in adults undergoing TAVI. Our primary objective was to evaluate the association between HR changes and functional improvements after the procedure.From February 2021 to December 2021, the Institute of Heart Diseases in Wroclaw, Poland, performed a TAVI procedure in 93 patients. Patients with severe aortic stenosis were enrolled for treatment by the multidisciplinary team (Heart Team). Before TAVI, a 6MWT was performed according to the guidelines of the American Thoracic Society which describes the methodology, indications and contraindications of the test as well as practical recommendations to ensure its quality and reproducibility [Patients were instructed to walk quickly for a period of 6 min or until dyspnea or muscular fatigue appeared. Prior to the start of the study, the patient was resting in a sitting position for 10 min. The walking course was 30 m in length with marks every 2 m and posts at the starting and turning places. The test was performed in a four meter wide corridor with a hard, level floor and total walking distance was recorded after 6 min HR and blood pressure was recorded with blood pressure monitor ) before, at the end of the test, and during the first, second, and third minutes of recovery. During the test the participants had to walk at a rate suitable to their condition and they were allowed to stop or slow down if necessary and asked to resume walking as soon as possible. The difference between baseline HR and HR at the end of the test was calculated (ΔHRpeak), and between baseline HR and the HR at the 1st, 2nd, and 3rd minutes of recovery .The 6MWT was repeated at the 3 month follow-up and the improvement in walking distance was calculated as the difference from baseline measurements (Δ6MWTD).t-tests and chi2 analysis was used to compare categorical variables. The relationship between Δ6MWT and baseline parameters was tested with Pearson correlation and linear regression analysis (backward method). The study group was divided according to the presence of improvement in walking distance on the follow-up 6MWT. To test the value of baseline parameters in predicting 6MWTD improvements at 3 months post-TAVI a multivariate logistic regression analysis was performed. For all statistical tests, a p-value of 0.05 was considered significant. All statistical analyses were performed using SPSS version 17.0 .The distribution of continuous data was tested using the Kolmogorov–Smirnov one-sample test and the Shapiro–Wilk test. The continuous variables were presented as mean ± standard deviation, whereas the categorical variables were presented as numbers and percentages. Comparisons of continuous measurements were performed using Student’s p > 0.05), but a statistically significant correlation was found between Δ6MWTD and baseline 6MWTD  there was no improvement in 6MWTD at the 3-month follow-up after TAVI. In fact, we observed a decrease of 6MWTD above 30 m in this subgroup. The comparison of clinical and 6MWT parameters in patients with and without improvement in 6MWTD is presented in p = 0.017, χ2 of the model = 7.21, p = 0.007.Only the creatinine level before TAVI, HR at peak exercise and HR at 1st, 2nd and 3rd minute of recovery differed significantly between patients with and without improvements in 6MWTD. All parameters were evaluated using a multivariate logistic regression analysis (backward method) to check their relationship with the 6MWTD improvement at 3 months after TAVI. It was found that only ΔHRrec_2 remained in the model with an odds ratio of 1.068 , Receiver operating characteristic analysis of the difference in HR at 2nd minute of recovery and baseline (ΔHRrec_2) for 6MWTD after transcatheter aortic valve replacement . The optPrevious studies have shown that not all patients after a successful TAVI procedure will improve in the 6MWT during follow-up . Our stuThe impaired chronotropic response to exercise was shown to be a predictor of mortality among patients with known or suspected coronary disease. Lauer et al. showed that in patients referred for stress testing with thallium imaging, chronotropic incompetence was predictive of all-cause mortality in 2 years of follow-up. Authors concluded that incorporation of chronotropic response into the interpretation of stress thallium studies may improve significantly its prognostic power . AndrareThe results of the current study show that HR recovery after the 6MWT may be a predictor of improved exercise capacity in TAVI patients at 3 months. We observed that patients with appropriate HR recovery benefited from the procedure in terms of improved walking distance during the follow-up period. The optimal cut-off value to predict post-TAVI 6MWTD improvement was −2 beats/minute after 2 min of recovery concerning the baseline HR in the pre-TAVI test.Altisent et al. showed that the absence of an improvement in physical performance at 6 months post-TAVI is an independent predictor of mortality and adverse cardiovascular outcomes . Mok et.Thus, our results may have important clinical significance and suggest that systematic implementation of exercise capacity and HR recovery assessments in pre-TAVI patients may help to improve patient risk stratification. Close to 30% of patients who underwent TAVI in our study did not improve their exercise capacity after 3 months despite a successful procedure. This result is very similar to previous observations ,24. AlsoThe mean 6MWTD at baseline and follow-up in our cohort was relatively higher compared to other studies like the PARTNER trial where distances achieved by study population were considerably below the normal limits . This caIn conclusion, our study suggests that HR recovery after 6MWT may be a helpful and clinically easy-to-assess parameter to predict exercise capacity improvement after TAVI. This simple method may help to identify patients in whom no significant benefit can be expected and those with a worse prognosis requiring more personalized medical care.There are important limitations to our study. First, though this is a prospective observational study, a small number of patients were included in the analysis. The lack of randomization and the absence of data-gathering monitoring—including self-reported 6MWT outcomes—constitute significant limitations of our research. Consequently, our findings should be considered hypothesis-generating only. Second, the proposed analysis can be performed only in patients able to walk and undergo a 6MWT. Finally, different markers of frailty were not analyzed in our study, as such the incremental prognostic value of HR recovery after 6MWT over that of frailty markers cannot be assessed.


The aim of this review is to identify the common characteristics and prognoses of different subtypes of neovascular age-related macular degeneration (nAMD). We also propose recommendations on how to tailor treatments to the subtype of neovessels to optimise patient outcomes. The authors, selected members of the Vision Academy, met to discuss treatment outcomes in nAMD according to macular neovascularisation (MNV) subtypes, using evidence from a literature search conducted on the PubMed database (cut-off date: March 2019). This review article summarises the recommendations of the Vision Academy on how the characterisation of MNV subtypes can optimise treatment outcomes in nAMD. The identification of MNV subtypes has been facilitated by the advent of multimodal imaging. Findings from fluorescein angiography, indocyanine green angiography and spectral-domain optical coherence tomography collectively help refine and standardise the determination of the MNV subtype. To date, three subtypes have been described in the literature and have specific characteristics, as identified by imaging. Type 1 MNV is associated with better long-term outcomes but usually requires more intense anti-vascular endothelial growth factor dosing. Type 2 MNV typically responds quickly to treatment but is more prone to the development of fibrotic scars, which may be associated with poorer outcomes. Type 3 MNV tends to be highly sensitive to anti-vascular endothelial growth factor treatment but may be associated with a higher incidence of outer retinal atrophy, compared with other subtypes. Accurately assessing the MNV subtype provides information on prognosis and helps to optimise the management of patients with nAMD. Age-related macular degeneration (AMD) is a leading cause of blindness in elderly people, primarily due to the macular neovascularisation (MNV) and atrophy that can occur during the disease , 2. SincSince AMD was first described, many efforts have been made to classify its pathology and the different types of MNV –7. ClassThe aims of this review article are to identify the common characteristics of different MNV subtypes, describe typical nAMD treatment outcomes in each case and propose recommendations on tailoring treatments to the different subtypes.www.visionacademy.org).This article is based on a review of the literature and consensus among retinal experts from the Vision Academy. The Vision Academy comprises an international group of retinal physicians who work together to share skills and knowledge and to provide recommendations based on their collective clinical expertise on clinical challenges in areas where there is a lack of conclusive evidence in the literature  were included. Polypoidal choroidal vasculopathy (PCV) was excluded from this search due to the specific treatment requirements of this neovascular abnormality. A total of 416 publications were initially identified and 75 publications were ultimately reviewed to identify key studies related to MNV subtypes. Additional details on the literature search algorithm are provided in the χ2. Endorsement was established if 50% or more of respondents indicated that they agreed or strongly agreed. The list of Vision Academy members who have contributed to the recommendations is provided at the end of the article.The objectives of this review are to define and describe MNV subtypes in nAMD and to provide treatment recommendations based on disease characteristics. The recommendations were developed by the authors and subsequently reviewed, commented upon and endorsed by a majority of the Vision Academy membership. Vision Academy members were asked to rate their agreement with the proposed recommendations using the options ‘strongly agree’, ‘agree’, ‘neither agree nor disagree’, ‘disagree’ and ‘strongly disagree’. Responses from more than 50% of members were required for the survey to be valid. Respondents were also asked for the reimbursement status of treatment in their country of practice  to determine if this may have influenced their responses. Biases were assessed using The identification of MNV subtypes is facilitated by multimodal imaging. Adding spectral-domain optical coherence tomography (SD-OCT) and indocyanine green angiography (ICGA) to colour fundus photography and FA has been reported to decrease the inter-observer disagreement for neovascular subtype characterisation from 30% to 10% . AlthougThree subtypes of MNV have been described and are characterised through multimodal imaging , 14. TypType 2 MNV, previously known as ‘classic’ neovascularisation, is characterised by the presence of MNV of choroidal origin in the neuroretina, having broken through the RPE layer. FA shows a well-defined neovascular membrane, with intense leakage that increases over time. While the contribution of ICGA is less important to the diagnosis of type 2 MNV, it can reveal a sub-RPE part of the neovascular membrane, defining the MNV as a mixed lesion with both type 1 and type 2 components. SD-OCT shows a disruption of the RPE–Bruch’s membrane complex and localisation of the neovessels above the RPE layer  Type 3 MNV, also known as retinal angiomatous proliferation (RAP), is characterised by anomalous vascular complexes originating in the neuroretinal layers . FA showThe role of OCT-A in distinguishing the different MNV subtypes has yet to be clarified –21. OCT-The various subtypes of nAMD are known to be associated with variable visual outcomes. Occult (type 1) MNV may have better outcomes if left untreated, and the lesion can be stable for months or years , 27. In A drawback of these studies is that they used only FA to classify eyes as having occult MNV, classic MNV or RAP. As such, some patients might have been diagnosed incorrectly . Indeed, in both interventional clinical trials and observational studies, the reported rates of each neovascular subtype vary. For example, in a Brazilian prospective epidemiology study, 62.6% of eyes with nAMD had type 1 or 2 MNV, 12.8% had type 3 MNV and 24.5% had PCV . In the For these reasons, MNV subtypes need to be carefully characterised so that study data can be correctly interpreted.Precise characterisation of MNV is important, as treatment outcomes may depend on the subtype. Compared with other subtypes, eyes with type 1 MNV have been reported to be more likely to maintain vision over time, despite requiring more frequent anti-VEGF injections in a treat-and-extend (T&E) regimen (approximately nine injections per year in a 5-year follow-up) , 35. Morpost hoc analysis of the CATT study, type 2 lesions had a 4.5-fold higher risk of developing a fibrotic scar compared with type 1 lesions [Type 2 MNV is associated with more fibrotic scarring than other MNV types , 10, whi lesions . In addi lesions . However lesions , and pat lesions .Type 3 lesions are more prone to responding to anti-VEGF therapy, with the small size of the neovascularisation and its intraretinal localisation likely to lead to better exposure to treatment. In a recent study, VA and VA gains at 1 and 2 years were better in type 3 MNV compared with other nAMD subtypes . In the The risk of relapse and the involvement of the fellow eye vary between MNV subtypes. Type 3 lesions have a higher risk of being associated with pathology in the fellow eye, so more aggressive follow-up should be implemented in the presence of this lesion type , 52. In It is not clear from the literature whether the rate of recurrence is higher in eyes with type 3 lesions, due to the variation in published results. In the PrONTO study, RAP lesions required more injections than the other MNV subtypes, indicating a higher rate of recurrence . ConversTo optimise functional outcomes in patients with nAMD, the treatment regimen should be individualised for each patient, according to the type of MNV. In all cases, treatment should be initiated promptly, as early as possible. Characterising the neovascular subtype can provide information on the expected prognosis, response to treatment and involvement of the fellow eye; therefore, multimodal imaging, ideally including FA, ICGA and OCT , should be used to accurately classify the lesion as type 1, 2 or 3 MNV. While OCT-A can help in detecting neovessels when they are not clearly visible on classic examinations, it cannot characterise the MNV subtype on its own. Correct multimodal assessment of the MNV subtype can help guide practitioner decisions on choosing an adapted treatment regimen. The following recommendations for treatment according to MNV subtype have been developed and endorsed by the Vision Academy members Fig. ; Table 1Type 1 MNV often requires more anti-VEGF injections than other types of MNV, as large mature vessels may be present in the neovascular complex and may produce recalcitrant or persistent subretinal exudation. However, long-term outcomes are typically better. As such, an individualised regimen such as T&E should be proposed as a priority to reduce patient burden. Alternatively, a fixed-dose regimen could be proposed depending on the observed time to recurrence, where T&E is not feasible due to resource or organisational constraints. Long-term treatment and follow-up are necessary, as the large vessels forming type 1 MNV are prone to developing PCV and its related complications such as subretinal and choroidal haemorrhage.pro re nata (as needed) treatment . Mixed lesions, with both type 1 and type 2 components, can be monitored using an intense T&E regimen in the first 2 years. After the first 2 years, mixed lesions can be managed on a case-by-case basis with T&E or careful and frequently monitored pro re nata treatment .Type 2 MNV usually responds quickly to anti-VEGF therapy but is prone to the development of fibrotic scars. Type 2 lesions may be treated using an intense T&E regimen in the first 2 years, extending beyond once every 12 weeks (q12w) if possible. After the first 2 years, purely type 2 lesions can be managed with careful and frequently monitored pro re nata regimen. At present, patients with type 3 stage 1 lesions, having reached stability after three initial treatment doses, can be kept on a strict (monthly) pro re nata regimen with contralateral eye checks. Patients with non-stable or relapsing stage 1 lesions should be switched to a proactive regimen (T&E or fixed). Patients with type 3 stage 2/3 lesions should be treated with a proactive regimen.Type 3 lesions tend to be very sensitive to anti-VEGF therapy, and treating lesions early leads to better visual outcomes with fewer recurrences and injections. However, the incidence of GA appears to be higher than in other MNV subtypes, and the fellow eye frequently develops neovascular complications. Further studies are needed to determine whether eyes with type 3 lesions at high risk of GA might be safely managed with a In this review, we have excluded PCV from the literature search as this neovascular abnormality generally requires more intensive anti-VEGF treatment than other MNV types, and often needs additional treatment modalities such as photodynamic therapy. Due to the evolving treatment paradigms for PCV, this vascular abnormality should be considered, especially when the initial imaging is not typical of other MNV subtypes or when treatment outcomes are not as expected.Correct assessment of the MNV subtype provides information on a patient’s prognosis and helps to determine the preferred treatment regimen. Additional biomarkers, perhaps as found on OCT-A, are needed to better optimise treatment outcomes.Supplemental methods


SMNgene. As it is the leading inherited cause of infant mortality, identification ofSMNgene pathogenic variant carriers is important for diagnostic purposes with effective genetic counseling. Multiple ligation probe analysis (MLPA), a probe-based method, is considered as the gold standard for SMA carrier analysis. However, MLPA might give false-negative results in cases with variations in the probe-binding regions. Here, we present a case born to consanguineous SMA carrier parents. Prenatal diagnosis with MLPA failed to detect the compound heterozygous mutant state of the proband and she was born unfortunately with SMA phenotype. Further analysis with a real-time polymerase chain reaction kit was able to detect the compound heterozygous state of the patient and was confirmed with targeted next-generation sequencing technology.Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder caused by the presence of pathogenic variants in the Spinal muscular atrophy (SMA) is a rare, recessively inherited neurodegenerative disorder arising from deterioration of motor neurons in the anterior horn of the spinal cord that in turn affect voluntary muscle movements resulting in muscle weakness and atrophy.SMNgene, referred asSMN1, located on chromosome 5q13. Encoded survival motor neuron (SMN) protein is a part of a multiprotein complex found in the cytoplasm and nucleus of various cell types including the motor neurons with significant roles in ribonuclease protein biogenesis and pre-ribonucleic acid splicing.SMN1gene are the deletion in exon 7 or deletion in both exons 7 and 8, which generally results in the synthesis of a nonfunctional or a truncated protein with insufficient function. Reduced amounts of functional SMN protein leads to the deterioration of motor neurons causing muscle weakness and atrophy.SMN1gene are point mutations. Absence ofSMN1can partially be compensated bySMN2, which is the centromeric copy ofSMN1, with 99% nucleotide identity created by an inverted duplication on the chromosome. However, as a large majority ofSMN2transcripts lack exon 7 due to the c.840C > T substitution, only a small amount of normal, functional SMN proteins can be produced.SMN1gene production, thereby reliant on theirSMN2gene production. They also clarified that higher copy numbers inSMN2gene is directly associated with mild symptoms of the disease. As without them loss of function inSMN1gene is extremely fatal.SMA is caused by pathogenic variations in the telomeric copy of theSMN2, or replace theSMN1gene. The first successful genetic therapy was conformed in 2010 to 2011 through the replacement ofSMN1gene in murine mouse models and the use of AVXS-101  that was approved by the Food and Drug Administration for SMA patients under the age of 2 in May 2019.Previous investigation on the treatment of SMA was mainly focused on treating the symptoms and reused pharmaceutical compounds with positive outcomes on other illnesses to address primary and secondary symptoms of SMA.Currently, SMA screening tests are done with multiple ligation probe analysis (MLPA) and are widely considered as the gold standard. However, in some cases MLPA analysis can be inconclusive and/or insufficient for diagnosis and other techniques are required for confirmation. Among these, targeted gene sequencing has been a powerful alternative. In recent years, on the other hand, faster and cheaper alternatives such as multiplex real-time polymerase chain reaction (PCR) and digital droplet PCR (ddPCR) kits were developed and are in use for SMA screening.Here, we present a case where the compound heterozygote state of variations could not be detected with prenatal MLPA screening which was purchased from another laboratory by the gynecologist. Unfortunately, diagnosis was possible after the birth with targeted gene sequencing and real-time PCR (SNP Biotechnology) which has been done from bench to bedside diagnosis in our laboratory.A 3-month-old female patient was admitted to the Near East University Department of Child Neurology Outpatient Clinic with complaints of muscle weakness that was recognized from birth, difficulty in moving her foot, inability to hold her head up, and accompanying respiratory problems. The diminished movements were mostly recognized on her hands and arms. She was born term,3,600 g of weight, and she had a newborn intensive care admission of 1-week period because of respiratory distress. The parents also dedicated a progressively increasing difficulty in feeding from birth.SMN1gene variations. The mother has a known heterozygote deletion inSMN1exon 7 and the father has a heterozygote deletion inSMN1exons 7 and 8. Prenatal genetic analysis with MLPA to detect smallSMN1andSMN2exon insertion-deletion variations reported heterozygousSMN1exon 7 and 8 deletion in the proband. No deletion or duplication was reported in theSMN2gene.She was the third live birth of seventh pregnancy of the mother with five abortus and twin healthy girl siblings. Parents were second-degree relatives with a familial history of two exitus with the diagnosis of SMA (mothers' siblings). Family pedigree is shown inIn her physical examination vital signs were within normal limits. Her weight, height, and head circumference were 4.920 g (10–25 percentile), 58 cm (75–90 percentile), and 38.8 cm (50 percentile), respectively. She had eye contact and social smiling. The head control was poor, and she had fasciculation on her tongue. Deep tendon reflexes were absent. The rest of the examination was unremarkable. The electromyelography revealed a subacute/chronic cervical  and lumbosacral (L3-S1) involvement of spinal anterior horn cells.When the patient was admitted to our hospital with these symptoms associated with SMA type I, MLPA analysis was repeated with SALSA MLPA Probemix P060 SMA Carrier assay (MRC-Holland), using deoxyribonucleic acid (DNA) isolated from peripheric blood samples of the proband and the parents. MLPA results showed intact exon 8 and heterozygote deletion of exon 7 in the mother, and heterozygote exon 7 and 8 deletion in the father. However, the results were inconclusive for the proband (SMN1gene sequencing was performed in Illumina MiSeq platform. Sequencing results indicated a heterozygous c.835-5_835-9delTCCTTinsTG (IVS7-5_IVS7-9delTCCTTinsTG) variation in the mother, which was inherited to the proband  from blood. Compound heterozygote state of the patient, which was missed by MLPA analysis, could also successfully be detected by the qPCR  was used to test the proband's sample. SNP SMA Screening Kit detects the exon 7 deletion with C/T substitution at nucleotide 840 and exon 8 deletion in theSMN1gene in more than 90% of patients establish the SMA diagnosis.SMN1exon 7 leading to the production of a nonfunctional protein product and changes in the centromericSMN2gene copy number is known to modify the phenotype of patients as a small amount of functional SMN protein is produced by theSMN2to compensate the pathogenic loss of the protein bySMN1deletion mutations.SMN1andSMN2genes can be problematic in disease and carrier screening tests.SMA is an inherited neuromuscular disorder that is characterized by muscle weakness and atrophy due to progressive degeneration of the anterior horn cells of the spinal cord. History of motor difficulties, reduced deep tendon reflexes, and muscle weakness together with the presence of biallelic pathogenic variants in theSMNgene mutations is important for diagnostic purposes and for effective genetic counseling. Molecular diagnostic methods that are used for SMA patient and carrier testing include restriction fragment length polymorphism (RFLP) test, MLPA, qPCR, and DNA sequencing.As one of the leading inherited causes of infant mortality, identification of carriers for theSMNgenes to create a reading in presence of a mutation. However, point mutations or less frequent mutations in the probe-hybridization loci such as the deletion/insertion mutation detected in our patient (c.835-5_835-9delTCCTTinsTG (IVS7-5_IVS7-9delTCCTTinsTG)) can cause inconclusive results as seen in our MLPA analysis. In a study by Sharifi et al, where 150 unrelated families with at least one suspected SMA patient in their family were surveyed forSMNgene, they showed false-negative carrier results where individuals had point mutations in the gene and also that heterozygous healthy carriers with two copies ofSMN1in one chromosome may not be identified by the MLPA analysis.In the current case, prenatal and postnatal tests were performed using the MLPA technique, which is often considered as the gold standard for SMA diagnosis, providing an easy, fast, and high-throughput system for analyzing the SMA critical region both in affected patients and in healthy carriers.SMN1targeted sequencing results of our proband's sample clearly revealed the mutation present in a heterozygous state with exon 7 and 8 deletion inherited from the father. In a previous study, a SMA patient with c.835-5T > G variation was reported, which cause the exclusion of exon 7.In this context, it is important to consider additional and alternative testing methods that can also reduce cost and have increased availability in the laboratory, including in-house designed real-time PCR technology.Overall, it is clear that SMA carrier screening tests are of great importance to reduce the burden of having an affected child for families. Even though MLPA is the most frequently used technique for testing and is considered as the gold standard, it can be insufficient in cases where the individual carries nonfrequent or point mutations in probe-binding regions. In this context, we argue that application of prenatal testing with two methods especially in cases with a family history and also in data with inconclusive results is beyond critical. We also would like to point out the importance of in-house genetic testing from bench to bedside diagnosis instead of purchasing genetic tests from external genetic test providers without seeing the results and analysis.


Based on a dosing sub-study of the Odyssey trial supporting the use of the DTG 50 mg tablet in children over 20 kg,9 South Africa’s 2019 ART guidelines expanded DTG access to children from 20 kg,3 which greatly improved treatment in these children. Further Odyssey sub-studies and the P1093 study also showed good data for DTG in children from 3 kg to 20 kg;11 however, for infants and children weighing less than 20 kg, access to DTG is dependent on availability of a new dispersible child-friendly formulation. First-line ART for infants and children < 20 kg has been a backbone of the protease inhibitor lopinavir/ritonavir and the two nucleoside-reverse-transcriptase inhibitors, abacavir and lamivudine. Lopinavir/ritonavir syrup needs to be given twice daily, should ideally be refrigerated, and is often poorly tolerated because of its awful taste. Abacavir and lamivudine were previously only available as relatively high-volume syrups for children unable to swallow tablets. Improvements came in the form of a 60 mg dispersible abacavir tablet and, finally, a fixed-dose combination scored dispersible tablet of abacavir and lamivudine (120/60 mg) which was approved in South Africa in 2021. The fixed-dose combination of abacavir and lamivudine can be administered as a once-daily dose and is easily dissolved in liquid for use in infants from 3 kg of weight.12 It is well tolerated and is an improvement on the large volumes of syrups that were previously required. Stock-outs and supply chain issues have hampered the roll-out of these formulations to all provinces and clinics.Since 2018, dolutegravir (DTG)-based antiretroviral treatment (ART) has been the preferred first-line treatment regimen in the World Health Organization guidelines for adults and children with HIV and has been adopted into South African guidelines for adults since November 2019.2 come with many welcome improvements for infants and children living with HIV. The good news is that there are two new DTG formulations available: a scored 10 mg dispersible tablet that can be offered to infants from 4 weeks of age if they weigh at least 3 kg, and a single fixed-dose combination tablet of abacavir, lamivudine and DTG that can be given to children who weigh 25 kg or more.12 This aligns treatment with adults and will mean that a child will be able to be on DTG from diagnosis through infancy and childhood to adulthood. All infants and children who are currently on a non-DTG regimen, including second-line regimens, should now be evaluated for a switch to a DTG-based regimen.South Africa’s recently released 2023 ART Clinical Guidelines for the Management of HIV2 This means that adolescents over 10 years who weigh more than 30 kg can be transitioned to the fixed-dose combination of tenofovir/lamivudine/DTG in alignment with adult recommendations.Another change in the guidelines is that tenofovir can now be given to adolescents over 10 years who weigh at least 30 kg (previously 35 kg) and have normal kidney function.13 Once-daily dosing of a more easily tolerated, effective medication will hopefully improve adherence and outcomes in our vulnerable paediatric population, including those < 20 kg, and support South Africa’s goal of achieving the last indicator in the Joint United Nations Programme on HIV/AIDS 95-95-95 initiative . Current Joint United Nations Programme on HIV/AIDS treatment cascade figures in South African adults are 94-74-67, with children lagging far behind.14 In the paediatric population, only 83% of children living with HIV are known to be infected. Of these, only 48% are on treatment, and a mere 33% of those on treatment are virally suppressed.14 It is likely that the suboptimal regimens used in children in South Africa have played a large role in the poor viral suppression in this group.Countries in sub-Saharan Africa that have adopted the transition to paediatric DTG have demonstrated rapid uptake with improved viral suppression in children > 20 kg.The availability of more child-friendly formulations will undoubtedly be a relief to many parents and clinicians who have struggled with getting infants and children to tolerate existing formulations. The goal of a successful paediatric ART programme is to enable all children living with HIV to live a normal life at their full potential with a suppressed HIV viral load throughout their childhood and continued into adulthood and we hopefully will be moving closer to this goal with the switch to optimised regimens.


Many approaches in systems biology have been applied in drug repositioning due to the increased availability of the omics data and computational biology tools. Using a multi-omics integrated network, which contains information of various biological interactions, could offer a more comprehensive inspective and interpretation for the drug mechanism of action (MoA).de novo targets for developing effective treatments.We developed a computational pipeline for dissecting the hidden MoAs of drugs (Open MoA). Our pipeline computes confidence scores to edges that represent connections between genes/proteins in the integrated network. The interactions showing the highest confidence score could indicate potential drug targets and infer the underlying molecular MoAs. Open MoA was also validated by testing some well-established targets. Additionally, we applied Open MoA to reveal the MoA of a repositioned drug (JNK-IN-5A) that modulates the PKLR expression in HepG2 cells and found STAT1 is the key transcription factor. Overall, Open MoA represents a first-generation tool that could be utilized for predicting the potential MoA of repurposed drugs and dissecting https://github.com/XinmengLiao/Open_MoA.Source code is available at  Drug repurposing, especially computational drug repurposing, is a novel approach for exploring new medical uses of existing drugs or drug candidates . To dateSystems biology-based network analysis can assist in elucidating the global drug/protein relationships and MoA of the drugs. By depicting entities as nodes and relationships as edges, a comprehensive network enables the incorporation of multiple types of data, modeling their correlations, and generation of putative subnetworks. Integrated networks (INs) combine diverse levels of desired biological information into an intensive network with known or predicted relationship. INs could help to better interpret complicated biological associations and address new opportunities for drug repurposing. Several types of INs, such as drug–disease , it coulIn this study, we presented the Open MoA: for revealing the MoAs using IN. Essentially, the method identifies MoAs of targeted interventions  by searching the interactions that are mostly likely activated within the context-specific IN. We demonstrated that Open MoA could reconstruct well-known mechanisms of TGF-b1, WNT1, and metformin. In addition, we revealed and experimentally validated the previously unknown MoA of a repurposed drug (JNK-IN-5A) for non-alcoholic fatty liver disease via Open MoA.Homo sapiens; (ii) all edges in the IN are regarded as independent biological events in the calculation; and (iii) the dysregulated genes are most likely to be modulated via the shortest path. The first assumption is grounded with existing knowledge of biological interactions, while the second and third ones are used by many previous studies to simplify the calculations and infer the functional molecules (We made three important assumptions in Open MoA: (i) the multi-omics generic IN is comprehensive and it includes all potential interactions in cells of H.sapiens interactions from these three databases are kept where applicable. For PPIs from the STRING database, we only selected the ones with high confidence scores (combined score >700)  of the reference IN and relationships (edges) between them. Specifically, drug–targeted gene interactions, PPIs, and TF–gene regulatory interactions with experimental evidence were retrieved from DrugBank v5.1.9  would modulate the end points (the genes that changed their expression) through the HepG2-specific IN. In order to do so, the shortest path between the starting point and the endpoints are identified using igraph v1.3.5 in R. Notably, since gene expression is probably influenced by TFs, the last edge in every shortest path is set as the regulatory interaction that starts from a TF.Next, we would like to calculate the probability of perturbation for each edge by the confidence scores from all shortest paths. Here, the false discovery rates (FDRs) of transcriptomic changes  on each target gene (endpoint) were defined as the Penalty Score (PScore) of the endpoint. It could be interpreted as the probability that their expression has no change. Therefore, we could use this PScore as an indicator to estimate the chance of a perturbation existence. Based on this, we assigned weights step-by-step to all edges from the starting point (drug-binding site or gene modulation target).In case of edges that involved in multiple paths going to different endpoints, we apply the fundamental statistical independency theory  and calcm shortest topological pathways between a starting point and an endpoint of a drug action, the PScore of each single edge within a drug action will be assigned evenly with nth root of the FDR value, which is expressed as following:m refer to the PScores of an endpoint, a single edge, and the total number of shortest topological pathways, respectively. The assumption here is that if the endpoint is perturbed, at least one of the pathways is activated.Therefore, we applied n represents the total number of drug actions that a common edge is involved.An edge involved in more than one drug action is defined as a common edge. The PScore of a common edge will be the product of all the PScores it obtained according to Eventually, all edges in the HepG2-specific IN will be calculated via Using the perturbation-specific weighted network, the step-by-step MoA with the highest confidence score between the starting point and a certain endpoint could be identified by looking for the shortest topological pathway with the highest confidence score by igraph v v1.3.5 in R.In-depth explanations concerning the methodologies adopted, including data collection processes, computational analysis, the development and utilization of the Open MoA script, and the experimental validation procedures are available in We developed Open MoA for discovering the underlying MoAs of the repurposed drugs. As shown in Z-score) of HepG2 cell line after the treatment of TGF-β1 shRNA were converted into two-tailed P-values via pnorm function in R, followed by p.adjust function to calculate FDR values. Out of the 12 205 signature genes, 7524 genes could be mapped to nodes in the HepG2 reference IN (−12), followed by thyroid hormone signaling pathway (FDR<10−7), the FOXO signaling pathway (FDR<10−7), the AGE-RAGE signaling pathway in diabetic complications (FDR<10−6), and the Hippo signaling pathway (FDR<10−3)  . Further complex .To demonstrate the capacity of Open MoA to uncover and dissect the core mechanisms underlying the observed biological processes, we reconstructed the exact TGF-β signaling pathway by measuring the most probable pathway between TGF-β and an ensemble of well-established downstream targets in the weighted TGF-β1 subnetwork. Open MoA predicted SMAD3 as the key intermediate regulator from TGF-β1 to EP300 , both reWe also restricted the transcriptomic data from CMap database to all Best Inferred Genes only, and performed the analysis for TGF-β1 with same study design. Similar results are obtained and shown in To show the predictive capability of the Open MoA, we ran our pipeline for revealing the MoA of a recently purposed drug based on network-based drug repositioning. We have previously established a computational pipeline for drug repurposing and identified JNK-IN-5A could modulate the expression of PKLR for effective treatment of non-alcoholic fatty liver disease  with associated escribed . Only 74escribed .JUN is a classical JNK substrate that can be phosphorylated by MAPK10 at its transactivation domain, especially at the serine residues S63 and S73  predicted by Open MoA, we treated HepG2 cells with JNK-IN-5A and the expression levels of MAPK10 and measured the level of the proteins using western blot analyses .t-test, P<.01) and PKLR  expression levels in JNK-IN-5A-treated HepG2 cells after 2 days of the drug treatment  and PKLR  decreased even more dramatically (We first confirmed the efficacy of JNK-IN-5A by demonstrating that the expression levels of MAPK10 were significantly reduced after 2 days of JNK-IN-5A treatment . We alsoreatment . To examatically . These rAccordingly, we also treated HepG2 cells with three different siRNA sequences targeting STAT1 to examine the effects of STAT1 silencing on PKLR expression and other related proteins predicted by Open MoA. While there was a significant decrease in the PKLR protein levels in parallel with the decrease in STAT1 protein levels, siRNA treatment did not significantly affect the expression levels of MAPK10, PKM2, and JUN, as predicted . In addiin vitro experiments.Finally, we treated HepG2 cells with two different overexpression plasmids of STAT1 to test the effects of STAT1 overexpression on PKLR expression, and other related proteins identified through our MoA predictions. While we observed an increase in PKLR levels in parallel with increases in STAT1 levels, plasmids treatment for overexpression of STAT1 did not affect MAPK10 and PKM2 expression levels . In summIn this study, we proposed a computational pipeline based on the weighted IN that allows the users to predict a possible underlying MoA for the repurposed drugs and to identify potential drug targets. We successfully reconstructed the TGF-β signaling pathway , the WNTOur computational pipeline can predict the drug MoAs at both molecular and genetic levels. The “Drug-Protein-Gene” motifs it portrays could also infer the key regulatory interactions, such as the one presented in our study. Although a growing number of drug repurposing approaches provide insights into the potential MoAs (Although we demonstrated that Open MoA could provide practical suggestions for the step-by-step mechanism of drugs or gene modulation, the key assumptions the method built on are not always true which could lead to some limitation of its application. First, the reference IN is not exhaustive, so the current version of the method may not be able to identify unknown MoAs or the ones which are not included in the scope of the IN, such as metabolic interactions or miRNA regulation. Therefore, one of the future directions of Open MoA could be enlarging the reference IN by increasing the quantity and diversity of biological data, such as metabolomics and miRNA (Moreover, our assumption about the shortest path within the IN can be contentious given that biological processes might not always follow the most direct routes. Yet, when exploring previously undiscovered MoA within vast biological networks, this approach proves relevant. Shortest path analysis has already been a common method for biological research (In summary, Open MoA that emphasizes the regulatory relationships is a powerful network-based method for unveiling the potential fundamental MoA of the repurposed drugs and inferring the key drug candidates. By inputting original FDR values, Open MoA could give out the significant confidence score for each interaction and calculate the most possible pathway of drug action. When further discovering the underlying MoA of PKLR modulation by JNK-IN-5A from our previous study, we found that STAT1 is the essential upstream TF, which regulates PKLR expression. Moreover, the applications of Open MoA are not limited in the drug discovery, since it could also facilitate other mechanistic studies, which searches potential pathways of specific gene modulations as exemplified in the WNT1 and TGF-β1 cases in this study. Collectively, Open MoA is a promising tool for shedding light on drug repurposing by providing holistic understanding of the MoA and identifying novel drug targets.btad666_Supplementary_DataClick here for additional data file.


To determine whether health literacy is associated with an index diabetes-related foot ulcer (DFU).The SHELLED Study is a 4-year prospective study of people with diabetes aged over 40 with no history of DFU. The primary outcome was development of a first foot ulcer. Health Literacy was measured using the short form Test of Functional Health Literacy in Adults (s-TOFHLA) and nine domains of the Health Literacy Questionnaire (HLQ).actively managing my health  and understanding health information well enough to know what to do . This was independent of baseline risk for foot disease.Of 222 participants, 191 (86.0%) completed the study, of whom 13 (5.9%) developed an incident ulcer. In multivariable models, every unit increase in S-TOFHLA was associated with a reduced odds of foot ulcer development by 6% . Better scores on two HLQ domains reduced the odds of foot ulcer (These data provide novel evidence that health literacy is an important clinical risk factor for index foot ulceration. This is an area of potential focus for research and development of educational programs or policy aimed at reducing development of incident foot ulceration.The online version contains supplementary material available at 10.1186/s13047-023-00644-w. Diabetes-related foot disease is one of the most devastating end-stage complications of diabetes. It is a leading cause of diabetes related disability burden  and affeIdentification of people at risk and taking appropriate preventative interventions as part of integrated foot care are considered cornerstones to preventing index or recurrent diabetes-related foot ulcers , 7. ThesA critical component underpinning the interactions between healthcare consumers and providers and the wider health system is health literacy. One way of defining health literacy is as ‘the cognitive and social skills which determine the motivation and ability of individuals to gain access to, understand, and use information in ways which promote and maintain good health’ . HoweverHealth literacy deficits are a major barrier to self-care in people with diabetes . An indiThe Southern Tasmanian Health Literacy and Foot Ulcer Development in Diabetes (SHELLED) study is a 4-year longitudinal study aiming to determine the associations of health literacy with incident foot ulceration in people with diabetes.Participants were recruited from the Royal Hobart Hospital’s Diabetes Centre Outpatient clinics between January 2015 and July 2016. Details on recruitment, data collection and questionnaires used have been described elsewhere . In brieThis was a 4-year prospective longitudinal study aimed at determining whether health literacy predicted occurrence of an index diabetes-related foot ulcer. The primary outcome was whether a participant developed an index diabetes-related foot ulcer (defined as a full thickness lesion below the ankle that was present for > 2 weeks) during the 4-year follow-up period. This was assessed by self-report during annual phone follow-ups conducted by a research assistant, with confirmation either via hospital medical records or by contacting the participant’s general practitioner.The exposure of interest, health literacy was measured using the short form Test of Functional Health Literacy in Adults (S-TOFHLA) and the Health Literacy Questionnaire (HLQ). The S-TOFHLA is a timed, 36-item test of comprehension using a modified cloze procedure. Participants are required to complete two passages, one containing instructions to have an x-ray and the other, from the “patients’ rights and responsibilities” section of an American Medicaid application form . The AusThe HLQ is a more holistic assessment of health literacy, consisting of 9 scales assessing the following domains: 1. Feeling understood and supported by healthcare professionals (4 items); 2. Having sufficient information to manage my health (4 items); 3. Actively managing my health (5 items); 4. Social support for health (5 items); 5. Appraisal of health information (5 items); 6. Ability to actively engage with healthcare providers (5 items); 7. Navigating the health system (5 items); 8. Ability to find good health information (5 items); and 9. Understanding health information well enough to know what to do (5 items). Scales 1 to 5 are scored out of 4 . Scales 6 to 9 measure difficulty of health-related tasks by the individual, and are scored out of 5 . Composite reliability of the HLQ ranges between 0.77 and 0.89 .Risk factors for diabetes-related foot disease were assessed by a podiatrist according to the most current Australian guidelines  at the tDemographic characteristics , medical history , years of educational attainment and household income bracket were assessed by questionnaire. Other covariates identified as potentially influencing health literacy as well as foot ulcer development according to biopsychosocial models of care were also assessed included diabetes self-efficacy , foot cThe sample size of 220 was calculated as the number of participants needed to detect differences in associations of S-TOFHLA categories  with foot ulcer incidence over 4 years. We projected that 60% of our study sample would have inadequate health literacy based on estimates by the Australian Bureau of Statistics . Based oth year of follow up and had not had an incident ulcer, or if they were noted as deceased on their hospital medical records without a known incident ulcer. Those with incident ulceration who died before the end of follow-up were considered to have completed the study as they had attained the outcome of interest.Participants were considered lost to follow up if they were unable to be contacted at the completion of the 4Logistic regression was used to estimate the associations of health literacy with development of incident foot ulceration over 4 years. Ten models were performed, one for S-TOFHLA scores and one for each of the nine health literacy domains measured by the HLQ . Models All analyses were performed in R V 1.10.44  using the package VGAM .This study was approved by the University of Tasmania Human Research Ethics Committee (H0014284).No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for recruitment, design, or implementation of the study. No patients were asked to advise on interpretation or writing up of results. All participants were provided education on foot ulcer prevention and where concerns of depression (PHQ-9) were identified these were communicated to participants’ treating medical practitioner for follow up.Figure Table Table actively managing my health and understanding health information well enough to know what to do were also associated with a first foot ulcer. For the former, each unit increase in mean HLQ domain score  was associated with a 77% reduction in odds of incident foot ulcer . For the latter, each unit increase  was associated with a 61% reduction in odds of a first foot ulcer .After adjustment for age, gender, BMI and education level, better health literacy scores on the S-TOFHLA and two HLQ domains were protective against foot ulcer development. Every unit increase in S-TOFHLA score reduced the odds of foot ulcer development by 6%  Table . Of the actively managing my health  persisted. Scores on the MOCA were statistically significantly associated with index foot ulceration in all models, with reductions in odds ratios ranging for 17–23% per unit increase in MOCA score. Odds ratios ranged from 0.83 (95%CI (0.72 to 0.96) for domain 9, to 0.77 (95%CI 0.65 to 0.88) and 0.77 (95%CI 0.63 to 0.94) for domain 3 and the S-TOFHLA respectively. Further adjustment for duration of diabetes, insulin therapy, baseline category of risk for foot disease at study enrolment, diabetes distress and depression or any other covariate measured did not materially change the magnitude or statistical inference of results (data not shown).After further adjustment of the models for cognition (MOCA score), only the protective effect of the HLQ domain understanding health information well enough to know what to do and actively managing my health as well as functional health literacy measured by the S-TOFHLA were associated with potentially clinically important reductions of up to 77% in the odds of developing a foot ulcer at 4 years. Additionally, cognitive impairment was independently associated with foot ulceration in all models, with every unit increase in MOCA score reducing the odds of incident foot ulcer development by between 17 and 23%. Consideration should be given as to whether policymakers and health care providers should identify people with health literacy and cognitive deficits so as to target them for interventions to improve health literacy, and tailor educational programs and other interventions for diabetes-related foot disease prevention to meet their needs.This longitudinal study provides novel data that functional and multiple domains of health literacy may be crucial in preventing a first foot ulcer. It is the first to prospectively investigate the relationship between health literacy and incident foot ulcer development in people with diabetes, enhancing previous evidence from cross-sectional data on this relationship . Two domunderstanding health information well enough to know what to do performed similarly in regression models and better scores were protective for incident foot ulceration. This is unsurprising. The HLQ domain understanding health information well enough to know what to do broadly matches the Nutbeam schema of functional health literacy [Health literacy could have a key role to play in incident foot ulcer prevention. In this study, both S-TOFHLA and HLQ domain of literacy  so indivliteracy . By exteliteracy . Evidencliteracy . There aliteracy .actively managing my health had the greatest protective effect on incident foot ulceration, with a 83% reduction in odds of first foot ulcer in the model with MOCA, and 77% reduction in the model without, independent of baseline risk of foot disease in our study. Better scores have also recently been shown to have the strongest protective effects amongst all nine HLQ domains for admission and mortality [The tenets of diabetes-related foot prevention are heavily reliant on an individual (or their carer) routinely undertaking actions to minimize risk of developing foot disease. Basic requirements include, but are not limited to adhering to recommended footwear, performing regular foot inspections (including daily self-monitoring of skin temperatures) and maintaining optimal foot and skin hygiene  in additortality . Clearlyortality . They arortality . AddressBased on the key aspects of health literacy that may impact on incident foot disease development identified in this study, we propose a range of strategies to address poor health literacy which could be implemented or tested. First, assume all patients may have difficulties comprehending health information and simplify communication and confirm comprehension to minimize the risk of miscommunication, otherwise known as taking “universal precautions” in health literacy . This apactively managing my health with foot ulceration, but weakened the associations of functional health literacy and HLQ domain 9  to the extent that these were no longer statistically significant. The logical explanation is that functional health literacy assessed by both domains  is most dependent on cognitive skills. We hypothesize that the HLQ domains not affected by confounding by the MOCA score may indeed reflect compensation for these cognitive deficits as previously described in the literature [Cognitive decline is a crucial issue afflicting people with diabetes with significant implications. People with diabetes are at a 50% increased risk of having two or more cognitive deficits that interfere with daily activities , and cogterature . Indeed,terature . A “healterature . It becoThe key strength of our study is that we investigated development of an index foot ulcer, rather than recurrent foot ulcers, which has been highlighted as a key gap in diabetes-related foot ulcer prevention research , 7. AnotTo conclude, this is the first study to identify inadequate health literacy and cognitive impairment as important risk factors for incident foot disease development. Better implementation of current strategies and trials to test new interventions to address the impacts of these deficits on diabetes-related foot disease is essential.Additional file 1: Supplementary Table S1. Baseline characteristics of participants who did and did not complete the study.