Triple-negative breast cancer (TNBC) is one of the most common malignancies worldwide and shows maximum invasiveness and a high risk of metastasis. Recently, many natural compounds have been highlighted as a valuable source of new and less toxic drugs to enhance breast cancer therapy. Among them, S-adenosyl-L-methionine (AdoMet) has emerged as a promising anti-cancer agent. MicroRNA (miRNA or miR)-based gene therapy provides an interesting antitumor approach to integrated cancer therapy. In this study, we evaluated AdoMet-induced modulation of miRNA-34c and miRNA-449a expression in MDA-MB-231 and MDA-MB-468 TNBC cells. We demonstrated that AdoMet upregulates miR-34c and miR-449a expression in both cell lines. We found that the combination of AdoMet with miR-34c or miR-449a mimic strongly potentiated the pro-apoptotic effect of the sulfonium compound by a caspase-dependent mechanism. For the first time, by video time-lapse microscopy, we showed that AdoMet inhibited the in vitro migration of MDA-MB-231 and MDA-MB-468 cells and that the combination with miR-34c or miR-449a mimic strengthened the effect of the sulfonium compound through the modulation of β-catenin and Small Mother Against Decapentaplegic (SMAD) signaling pathways. Our results furnished the first evidence that AdoMet exerts its antitumor effects in TNBC cells through upregulating the expression of miR-34c and miR-449a. Breast cancer is the most commonly diagnosed invasive cancer and the second-leading cause of mortality among women worldwide ,2. The iTumor metastasis is responsible for up to 90% of cancer-related deaths so, prevention and treatment of metastasis are key to improving clinical outcomes . MetastaEMT has been implicated in breast cancer progression and metastasis and is also involved in cancer stem cell expansion and chemoresistance to cancer treatment. For this reason, finding a natural molecule without significant toxic side effects and capable of reducing the spread of cancer cell is a major clinical challenge and recently intensive efforts have been focused on understanding the molecular mechanisms, as well as new targets for suppressing these processes ,9,10. MaAdoMet, the second most extensively-used enzyme cofactor after ATP, performs a wide of well-documented biological functions in all living cells and it is the linking of three primary metabolic pathways: transmethylation, polyamines biosynthesis, and transsulfuration ,12. GiveExperimental evidence has been reported showing that AdoMet is able to regulate, through an epigenetic mechanism, the expression of genes playing a crucial role in cell migration, invasion, and metastasis ,29,30,31Recent experimental and clinical studies have improved our knowledge of tumor metastasis formation, a dynamic program triggered by complex regulatory networks involving transcription factors, non-coding RNAs, epigenetic modulators, and exogenous inducers by the tumor microenvironment ,9,10. AmOur research group has thoroughly investigated the antiproliferative and proapoptotic role exerted by AdoMet in breast cancer ,41,42 anHere, we reported that in MDA-MB-231 and MDA-MB-468 TNBC cells AdoMet upregulated the expression of miR-34c and miR-449a, well-known regulators of specific oncogenes and modulators of tumor growth and cancer metastasis in breast cancer ,46,47. WEmerging evidence suggests that miRNAs play important roles in the pathogenesis of many types of human cancers by modulating different genes in the context of signaling pathways involved in tumor promotion or suppression ,36,37,38MiRNA-34c and miRNA-449a, that belong to miRNA-34/449 superfamily, share several target genes and a very similar seed sequence, a conserved heptameric sequence comprising nucleotides 2–7 at the 5′ end of the miRNA, essential for binding to target mRNA ,46,47. MTo obtain insight into the functional mechanism underlying AdoMet’s anticancer effects in TNBC cells, we evaluated its ability to induce the expression of miR-34c and miR-449a by performing quantitative real-time PCR (qRT-PCR) analysis with pre-designed probe-primer sets after 24- and 48 h treatment of MDA-MB-231 and MDA-MB-468 cells with 500 µM AdoMet. The results obtained showed that the relative expression of the two miRNAs was up-regulated by AdoMet in both TNBC cell lines. As shown in To explore the antitumor activity of AdoMet in TNBC cells, we first tested the ability of the sulfonium compound to induce apoptosis in MDA-MB-231 and MDA-MB-468 cells. The cells were treated with AdoMet 500 µM and the apoptotic process was evaluated after 72 h by flow cytometry. As shown in Firstly, by qRT-PCR we demonstrated that, the transfection of cells with miR-34c or miR-449a mimics effectively up-regulates miR-34c and miR-449a transcriptional levels in the two TNBC cell lines reaching a value approximately double (data not shown). Next, to analyze the antitumor activity of miR-34c and miR-449a we assessed apoptosis induction. Our results showed that in both MDA-MB-231 A and in Altogether, these findings indicated that miR-34c and miR-449a played a tumor suppressive role in TNBC cells and that upregulation of miR-34c and miR-449a by AdoMet mediated AdoMet-induced apoptotic cell death.In order to evaluate the effect of AdoMet on TNBC cell migration, we performed transfection experiments with miR-34c or miR-449a mimics in highly aggressive and invasive mesenchymal-like MDA-MB-231, and in the basal-like MDA-MB-468, characterized by a relatively low invasive phenotype and potential  and thenFirst, we have assessed that in a short time, such as 24 h, after AdoMet and/or miRNAs treatment the apoptotic cell death evaluated by FACS analyses did not interfere with the migration process evaluated at 24 h in both cell lines because it was not significant and reached, as a maximum, a value 10% more than the control (data not shown).Cell migration was detected in real-time by using video time-lapse microscopy (TLVM), and representative images of the wound closure process are shown in In Cancer metastasis begins with detachment of metastatic cells from the primary tumor, followed by an increase in cellular motility and invasion, proteolysis, and resistance to apoptosis. These four essential steps are correlated and influenced by multi-biochemical events and parameters.N-cadherin and vimentin, a protein overexpressed in most epithelial cancers, whose levels are correlated with tumor migration, invasion, and poor prognosis  expressed as mm/hour. Moreover, the fields of view selected and used to build up the overall averaged curves all had a similar scratch width, ranging from 0.7 to 0.9 mm, corresponding to a wound area of 16–20 mm2. The statistical significance of the experiment was ensured by the possibility to visualize contemporarily several fields of view (up to 30–36 h) of the same sample (depending on the delay time chosen by the operator) in the staged incubator. Furthermore, a single field of view (~10 × 105 μm2) represented 5% of the total scratch area (~20 × 106 μm2) of each well. Because we captured at least five field-views in three repetitions per well, this ensured us that we analyzed 25–30% of the scratch in each specific well. Triplicates were performed for each scratch assay.The wound closure measurements were calculated by the software as Area t /Area tg for 30 min a 4 °C and the supernatant was recovered. Protein concentration was performed by Bradford method as previously reported [MDA-MB 468 and MDA-MB-231 cells were transfected with 100 nM miR-34c or miR-449a mimic, treated or not with AdoMet 500 µM and after 48 and 72 h, collected by centrifugation, washed twice with ice-cold PBS, and the pellet was lysed using 100 µL of RIPA buffer. After incubation on ice for 30 min, the samples were centrifuged at 18,000× reported .Western blotting analysis was performed as previously reported . All priStatistical analysis was performed as previously reported .Overall, our results provided the first evidence that AdoMet exerts its antitumor effects in TNBC cells by regulating miRNA expression and gave new information for a better and deeper understanding of the molecular mechanisms underlying the anticancer properties of this naturally-occurring multifunctional sulfonium compound suggesting the use of AdoMet as an attractive chemopreventive and therapeutic strategy miRNA-mediated in TNBC.Our study also provided significant contribution to the knowledge of miR-34/449 biological functions furnishing the first evidence that in MDA-MB-231 and DA-MB-468 cells miR-34c and miR-449a act as tumor suppressors and inhibitors of the metastatic potential of cancer cells by targeting TGF-β/SMAD and β-catenin signaling pathways.Taken together, these data suggested the advantage of using natural compounds with pleiotropic effects, such as AdoMet and miRNAs, which can regulate many target genes achieving more efficient modulation than single-target drugs. These discoveries offer another approach for the scientific community in cancer therapy using only natural compounds, and highlight a new tool to fight cancer giving the possibility to recover health by avoiding typical side-effects, improving prognosis, and increasing the percentage of remission. However, it appears that miRNAs are very suitable in combination with AdoMet in cancer therapy and more data are expected in the coming years to deepen the knowledge of the regulation of non-coding RNAs by AdoMet in order to improve currently applied anticancer therapies.


This online structured survey has demonstrated the global impact of the COVID-19 pandemic on vascular services. The majority of centres have documented marked reductions in operating and services provided to vascular patients. In the months during recovery from the resource restrictions imposed during the pandemic peaks, there will be a significant vascular disease burden awaiting surgeons.One of the most affected specialties Coronavirus disease 2019 (COVID-19) has had a profound effect on the availability of surgical resourceshttps://www.medrxiv.org/content/10.1101/2020.05.27.20114322v1; ISRCTN 80453162).International guidelines on designing and reporting of surveys were usedS2, supporting information). Results reported here are for the period 23 March to 3 May 2020, divided into three 2-week periods for comparison. Duplicate responses were removed.A remote digital survey was developed by a global team of vascular healthcare professionals. Questions related to all aspects of vascular care, including staff availability, multidisciplinary team input, and personal protective equipment (PPE) .International/continental comparisons were performed, where possible, to describe relative change in practice. A score of 0–3 was allocated to each answer based on perceived relative service reduction by 12 VERN healthcare professionals (0 represents no change and 3 the most significant change) (S3 (supporting information).Overall, 465 completed survey responses were collected from 249 different units in 53 countries across six continents  maintained their pathways for acute aortic syndromes . A small proportion (5·9 per cent) moved to conservative management only, 4·5 per cent were offering early endovascular surgery, and 26·6 per cent limited surgery to ruptures.Thresholds for abdominal aortic aneurysm (AAA) repair were raised in the majority of centres; 11·7 per cent of vascular units limited surgery to AAA more than 6·5 cm in maximal diameter, 16·4 per cent to those above 7 cm, 25·1 per cent to symptomatic or ruptured AAA, and 2·3 per cent to AAA suitable for endovascular AAA repair (EVAR) only. Despite this, 25·1 per cent reported no change in practice. Access to EVAR out of hours was initially available to 8·5 per cent of responding units, increasing to 21·2 per cent in the following 4 weeks. Overall, only 14·2 per cent of units maintained a 24/7 EVAR service, 26·3 per cent maintained an ‘in hours’ service, 31·5 per cent offered EVAR for urgent cases only, and 18·5 per cent were able to run their service on an Changes to the management of lower-limb pathology are shown in Some 27·5 per cent of units moved to a triage clinic system, and 29·0 per cent cancelled all planned outpatient clinics. Use of technology permitted 14·9 per cent of units to move to video or telephone clinics, with 18·7 per cent including subsequent triage for attendance if required. The use of ‘hot’ clinics (reserved for acute/urgent patients) increased during the pandemic, and 79·1 per cent of units reported using some form of hot clinic to accommodate vascular patients.Overall, 32·2 per cent of units that normally participated in a multidisciplinary team (MDT) continued with face-to-face meetings; 59·5 per cent stopped regular face-to-face meetings and, of those, 39·1 per cent did not replace them. Overall, 36·8 per cent moved to remote conferencing.Globally, 5·5 per cent of senior surgeons were redeployed to support other specialties, compared with 53·5 per cent of junior vascular surgical staff.The majority (80·5 per cent) of units had PPE guidance in place. Some 26·2 per cent of units did not have access to adequate PPE at the start, compared with 17·5 per cent at the end of the period.The COVER study is the first international prospective study of unit-level vascular surgical practice during the COVID-19 pandemic. Findings from tier 1 suggest radical changes in practice in a range of services.One notable change across participating vascular units is the reduction in AAA screening activity. The benefit of AAA screening, and the likelihood of finding a new AAA (less than 1·5 per cent)14. For EVAR, a paradigm has been created where, potentially, more EVAR is performed during the pandemic, but with a reduction in post-EVAR surveillance. There are important implications relating to the financial resources, operating time and staffing that will be required to catch up with missed scans and scheduled operations as services begin to resume. Vascular patients will be competing with the estimated 28 million operations cancelled or postponed during the peak of the pandemicAnother common finding is the reported preference for endovascular strategies to address aortic and peripheral arterial disease; this is thought to be based on a drive to minimize hospital stay and reduce demand on ICU bedsMDT meetings support individual clinician decision-making by navigating complex decisions through a multifaceted approach. COVID guidelinesDespite the large number of units taking part, correlating individual country or regional data with dates of lockdown is challenging. Dates of lockdown were, however, similar for countries providing the majority of responses . All participating units entered lockdown in March 2020, and were in lockdown when the survey began. If there are any subsequent COVID-19 ‘waves’ in areas that are ‘past the peak’bjs11961-sup-0001-SupinfoAppendix S1: Supporting informationClick here for additional data file.


This study (a) assessed quality of life (QoL) in a patient sample with severe mental illness in an integrated psychiatric care (IC) programme in selected regions in Germany, (b) compared QoL among diagnostic groups and (c) identified socio-demographic, psychiatric anamnestic and clinical characteristics associated with QoL.This cross-sectional study included severely mentally ill outpatients with substantial impairments in social functioning. Separate dimensions of QoL were assessed with the World Health Organisation’s generic 26-item quality of life (WHOQOL-BREF) instrument. Descriptive analyses and analyses of variance (ANOVAs) were conducted for the overall sample as well as for diagnostic group.A total of 953 patients fully completed the WHOQOL-BREF questionnaire. QoL in this sample was lower than in the general population  32.8 to 35.5), with the lowest QoL in unipolar depression patients  and the highest in dementia patients . Main psychiatric diagnosis, living situation , number of disease episodes, source of income, age and clinical global impression (CGI) scores were identified as potential predictors of QoL, but explained only a small part of the variation.Aspects of health care that increase QoL despite the presence of a mental disorder are essential for severely mentally ill patients, as complete freedom from the disorder cannot be expected. QoL as a patient-centred outcome should be used as only one component among the recovery measures evaluating treatment outcomes in mental health care.The online version of this article (10.1007/s11136-020-02470-0) contains supplementary material, which is available to authorized users. Quality of life (QoL) has been reported to be reduced in patients with severe mental illnesses. Awareness of the topic in psychiatric research is rising , 2. BecaSeveral studies have shown a clinically relevant reduction in QoL in patients with unipolar depression. Socio-demographic factors such as age, sex, relationship status and living situation explained only a small proportion of the variance in QoL , 5. SelfFor patients with bipolar depression, scientific reviews have reported a reduced QoL compared to healthy people even in euthymic phases , 13. No Subjective QoL in patients with schizophrenia is usually lower than in the general population , 23 but Studies on QoL in patients with schizoaffective disorder are limited but suggest a reduced QoL compared to the general population . No assoPatients with anxiety disorders have reported a reduced QoL in previous research, especially when diagnosed with a generalised anxiety disorder . SubjectPatients with dementia have reported a subjective QoL level similar to elderly persons without dementia , 38. A lIn patients with alcohol addiction, QoL has been shown to be lower than in the general population , 52. HigThe present study on QoL was embedded in an integrated psychiatric care (IC) programme for patients with severe mental disorders. Since 2008, this programme has been offered in several federal states within the framework of selective contracts between providers and statutory health insurance funds .The aim of this study was to (a) assess the QoL in patients with severe mental illness participating in an IC programme in Germany, (b) compare the QoL among different diagnostic groups, and (c) identify socio-demographic, psychiatric anamnestic, and clinical characteristics associated with different QoL domains in this population.This cross-sectional observational study was performed within a research project at the Charité—Universitätsmedizin Berlin (Germany) for the evaluation of a model of IC. The model was implemented in the regions of Berlin/Brandenburg and Lower Saxony/Bremen to strengthen the network of therapeutic care providers by allowing complex outpatient care for patients with severe mental illnesses –61.aged 18 years or older;resided in the participating regions;insured with one of the participating statutory health insurances ;diagnosed with an F0.X to F8.X ICD-10 code;needed hospital admission requiring care;entitled to receive complex outpatient care instead of inpatient treatment according to the assessment of the attending physician;impaired social functioning level  );assessed with illness severity of ≥ 5 on the clinical global impression scale  (CGI);provided written consent .The IC model included patients who met the following inclusion criteria:Patients with acute suicidality could not be included in the IC programme.The present study was based on a subsample of IC patients who were selected between 01 January 2008 and 31 March 2010 and were categorised into seven diagnostic groups based on the following main diagnoses: affective disorders , schizophrenia (F20), schizoaffective disorder (F25), neurotic disorders , dementia (F00–03), and alcohol-related disorder (F10). The patients with specific personality disorders (F60), other psychoactive substance-related disorders (F19), organic mental disorders other than dementia (F06–07) and other acute or chronic psychotic disorders  were excluded from the analysis due to insufficient numbers.Patients were asked to complete the World Health Organisation QoL-BREF (WHOQOL-BREF) questionnaire , 65. ThePatients were consecutively included by the attending physicians. On a quarterly basis, physicians assessed socio-demographic , psychiatric anamnestic  and clinical data  according to a standardised manual. The main diagnosis was the one that led to the acute need for psychiatric therapy and admission to the IC. The patients completed the WHOQOL-BREF at inclusion in the IC and at the beginning of every quarter year. The physicians’ documentations and the patients’ questionnaires were checked by trained study personnel according to standard operating procedures (SOPs), and continuous quality circles with the participating physicians were implemented .The present analysis includes only cross-sectional data collected at the time of inclusion in the IC. Statistical analysis was conducted with SPSS 19.0 for Microsoft Windows. Socio-demographic, psychiatric anamnestic and clinical patient characteristics were described for the entire IC sample and stratified by main diagnostic group by mean and standard deviation (SD) or frequencies and percentages.In the first step, the association between age, sex and main diagnosis was assessed for each of the diagnostic groups of interest to identify potentially confounding factors. In a second step, QoL mean values were compared among diagnostic groups separately for each WHOQOL-BREF domain and for the overall QoL scale using analysis of variance (ANOVA). The reference values for QoL of the German general population were taken from Angermeyer et al. .2 and adjusted R2 values). Results were checked for multicollinearity and heteroscedasticity. All results are considered exploratory .Due to differences in the QoL values among the diagnostic groups, means were adjusted for diagnosis but not for age and sex (no relevant differences). To identify the influence of patient characteristics on the QoL, adjusted mean values for each of the WHOQOL-BREF domains and the overall QoL scale are presented by socio-demographic, psychiatric anamnestic and clinical characteristics, based on domain-specific models of multivariable ANOVA .Overall, 1433 patients were included in the IC programme, of which 1347 patients had one of the study diagnoses. Among those, 953 completed the WHOQOL-BREF questionnaire. The calculation of scores for the subdomains of the WHOQOL-BREF was possible for all 953 patients, while the assessment of the global score was possible for 941 patients.The largest groups were patients with unipolar depression 58.1%) and psychotic disorders (13.7%), followed by neurotic disorders (11.7%)  and those with bipolar depression , schizophrenia  and dementia . Psychological health-related QoL was impaired mainly in the patients with unipolar depression and with neurotic disorders, who showed markedly lower values than the patients with schizophrenia and dementia. In the domain of social relationships, the patients with dementia showed a notably higher QoL level than the patients in four out of the remaining six diagnostic groups. Regarding the environmental domain, QoL was higher in the dementia patients than in the patients with unipolar depression and those with neurotic disorders. Additionally, on the WHOQOL-BREF global scale, the patients with dementia were the least impaired group, which was shown by higher values than most of the other diagnostic groups . Furthermore, the schizophrenia patients reported a markedly higher QoL than those with unipolar depression or neurotic disorders.To estimate the association between socio-demographic, psychiatric anamnestic and clinical factors and QoL, domain-specific multivariate models were fit. All potential confounding socio-demographic, psychiatric anamnestic and clinical factors , while a high CGI score was associated  with a lower QoL in the global scale section  and the environmental domain .The largest adjusted mean differences were observed among the main diagnostic groups in the global scale model  was related to a lower QoL in these domains, which was also observed by Henkel et al. .Independent of socio-demographic, psychiatric anamnestic, and clinical characteristics, the main diagnostic group remained the factor with the most distinct differences in all models. This suggests QoL as a diagnosis-specific aspect that should be taken into account in evaluation studies in different patient groups.Overall, these exploratory results indicate potential clinical relevance of some of the factors investigated in the present study. They should, however, be cautiously interpreted against the background of a limited precision due to a small number of cases in some categories, which resulted in rather large CIs. The selection of factors potentially influencing the patients’ QoL in this study was limited to variables that were assessed in the context of the research project for the IC model evaluation. Therefore, factors that had been identified as potential determinants in other studies, such as self-esteem, individual expectations, personality traits, self-efficacy , illnessp values of one predictor variable (i.e. reduction in power) when another predictor is included in the model. However, since this was an exploratory study without adjustments for multiple testing, p values were not the focus of our analyses. In addition, dropping important variables from the model might have introduced bias. In our view, our data might not be suitable for disentangling all of the combination effects which are common in socio-demographic and lifestyle factors. A graphical check did not find relevant evidence for heteroscedasticity. Hence, no methods to account for heteroscedasticity (e.g. robust standard errors) were used.The statistical examination for multicollinearity revealed indications of dependencies between a few of the variables (e.g. living situation and subsistence). However, we chose to keep all of the factors in our analyses. One reason is that collinearity mostly is a concern to result in essential shifts in the Despite the controversial discussion about the assessment of QoL in psychiatric patients , the cond between 0.10 and 0.37) [F scores and df to describe differences between people with varying diagnoses and clinical as well as socio-demographic characteristics [The interpretation of the results is hampered by the lack of a definition of MCID of different QoL measures as patient-reported outcomes. The MCID is the smallest difference in a score that a patient would identify as important. In our study, no control group was available neither did we test the effects of an intervention. Responsiveness of the WHOQOL-BREF instrument has been shown in a variety of settings and conditions. The instrument is able to detect even small changes induced by treatments as shown by effect sizes in more than 20 studies being highly significant even with low or moderate values (Cohen’s nd 0.37) . Howevernd 0.37) , 100, thnd 0.37) . In the nd 0.37) , we usederistics .The study population exclusively consisted of outpatient and mostly female, unipolar depressive patients from selected regions in Germany that were enrolled in an IC project, which limits generalisation of the results. In addition, due to a low number of patients in some of the diagnostic groups, differentiation into clinically relevant subgroups was not possible; e.g. ‘dementia’ included patients with different forms of dementia (mainly Alzheimer’s disease), while ‘neurotic disorders’ comprised mainly but not only anxiety patients.It also needs to be considered that analyses were performed only for patients who completely answered the WHOQOL-BREF, which might selectively exclude more severely ill patients. Especially for dementia patients, non-declared support by family members for filling in the questionnaire cannot be excluded. Additionally, physicians might have graded patients as more severely ill on the psychopathological scales to allow for the patients’ inclusion in the IC programme. Because no causal conclusions can be drawn due to the cross-sectional study design, further prospective longitudinal studies would be desirable.A notable strength of the study was that it allowed an analysis and a comparison of the QoL among different diagnostic groups based on a large psychiatric outpatient sample. It thereby differs from studies conducted in inpatient settings and provides results that are highly transferable to clinical practice and care.In summary, the results provide further indication that socio-demographic and clinical variables have little impact on the QoL of people with mental illness. However, if factors that can be improved by psychiatric or psychosocial interventions have only a limited influence on QoL in the short to medium term, the question arises whether QoL is an appropriate outcome in mental health care research. Severely mentally ill patients are often affected by recurrent disease episodes or chronic disease courses, and only a minority can expect to stay completely free from symptoms for the remainder of their life. Especially for those patients, health care that improves QoL despite the presence of the illness is essential.A recent review emphasised that there is an association between low social functioning and negative QoL in psychotic disorders . The resScepticism about the increasingly widespread use of QoL as an outcome in psychiatric health care research was expressed more than 10 years ago , 106, agQoL is a markedly subjective measure, and the individual rating depends on the underlying type of mental disorder. QoL can also be seen within a personal frame of reference. This frame of reference is formed by the level of social functioning and the degree of integration into society. When estimating QoL values, this subjective frame of reference must be taken into account. If the change in QoL is used as an outcome after an intervention or after an observation period, the typical characteristics such as measurability, change sensitivity, reliability and validity must also be considered.As a conclusion of the study, symptoms of mental disorder, clinical impression, and social functioning alone are not sufficient as outcome measures because they do not reflect the subjective patient’s perspective. However, QoL as a patient-centred outcome measure is not unproblematic either. Therefore, the effects of health care should not be measured by the change in QoL alone, but QoL should only be used as one component alongside other recovery measures. Future research should look for alternative patient-related outcomes that better reflect the success of long-term psychiatric care.Supplementary file1 (PDF 22 kb)Below is the link to the electronic supplementary material.


Leishmania growth and survival is dependent on polyamine biosynthesis; therefore, inhibition of Leishmania arginase may be a promising therapeutic strategy. Here, we evaluated a series of thirty-six chalcone derivatives as potential inhibitors of Leishmania infantum arginase (LiARG). In addition, the activity of selected inhibitors against L. infantum parasites was assessed in vitro. Seven compounds exhibited LiARG inhibition above 50% at 100 μM. Among them, compounds LC41, LC39, and LC32 displayed the greatest inhibition values . Molecular docking studies predicted hydrogen bonds and hydrophobic interactions between the most active chalcones  and specific residues from LiARG's active site, such as His140, Asn153, His155, and Ala193. Compound LC32 showed the highest activity against L. infantum promastigotes (IC50 of 74.1 ± 10.0 μM), whereas compounds LC39 and LC41 displayed the best results against intracellular amastigotes . Moreover, compound LC39 showed more selectivity against parasites than host cells (macrophages), with a selectivity index (SI) of 107.1, even greater than that of the reference drug Fungizone®. Computational pharmacokinetic and toxicological evaluations showed high oral bioavailability and low toxicity for the most active compounds. The results presented here support the use of substituted chalcone skeletons as promising LiARG inhibitors and antileishmanial drug candidates.Arginase catalyzes the hydrolysis of  Leishmania infantum is the etiological agent of visceral leishmaniasis (VL), a lethal infectious disease that afflicts neglected populations mainly distributed in Africa, Asia and Latin America. Recently, the World Health Organization estimated that 30,000 new cases of VL occur worldly  has been proposed as a potential target for new drug candidates of natural  and in vivo  contexts; this finding reinforces that ARG is essential for Leishmania pathophysiology and is therefore an intriguing target for new therapeutics.Arginase plays a pivotal role for the survival of Trypanosoma cruzi and Leishmania  by naturally occurring phenolic substances  cells were transformed with RP1B-LiARG. Cells were grown at 37°C until mid-exponential phase (Abs600 ~ 0.6). Protein expression was induced with 1 mM IPTG and cells were grown for 16 h at 30°C. Cells were harvested by centrifugation, resuspended in lysis buffer  and lysed by sonication . The cell lysate was centrifuged , and the clarified supernatant was loaded onto a His-Trap HP column . A wash step with 100 mM manganese chloride was carried out for enzyme activation. LiARG was eluted with an imidazole gradient ranging from 5 to 500 mM. Fractions containing LiARG were pooled and dialyzed in the presence of His6-TEV protease for removal of the N-terminal His6 tag. A second nickel-affinity chromatography step was used to remove the His6 tag and His6-TEV. Finally, purified LiARG was dialyzed against , concentrated and stored at −80°C.LiARG expression and purification was performed as described previously  at 37°C for 5 min. A set of thirty-six synthetic substituted chalcones was used to screen for potential LiARG inhibitors. Synthesis of chalcone derivatives were described previously by Ventura et al. . Urea concentration was determined spectrophotometrically  by hydrolyzing urea into ammonia and then converting ammonia into indophenol blue, which absorbs light at 600 nm  into the active site of LiARG, we used LiARG three-dimensional model and docking parameters from our previous work   required to kill 50% of the rats tested.The two-dimensional structures were drawn with ACD/ChemSketch  were added and the cultures were incubated at 37°C in 5% CO2 for 48 h. Subsequently, cell viability was measured by a colorimetric assay using 3--2,5-diphenyl tetrazolium bromide (MTT) . MTT was added to the culture medium at a final concentration of 1.0 mg/mL, and cultures were incubated at 37°C in 5% CO2 for 3 h. MTT was transformed by the living cells in formazan crystals that were dissolved in DMSO. The concentration of formazan was measured spectrophotometrically at 570 nm . The 50% cytotoxic concentration (CC50) values were determined by the nonlinear regression fits of the dose-response curves using GraphPad Prism 8.0. All measurements were performed in triplicates, twice independently.RAW 264.7 macrophages were cultured in DMEM medium  supplemented with 10% fetal bovine serum at 37°C in 5% COLeishmania infantum strain MHOM/BR/1974/PP75 were cultured in Schneider's medium supplemented with 10% fetal bovine serum at 26°C. First, the selected LiARG inhibitors were diluted in culture medium at concentrations ranging from 2.9 to 1,723.0 μM. Then, late-exponential phase (after 96 h growth) L. infantum promastigotes, at a final density of 105 parasites/mL, were incubated with previously diluted inhibitors at 26°C for 96 h. Subsequently, parasite viability was determined by a colorimetric assay using resazurin . Resazurin was added to the culture medium at a final concentration of 0.001% (w/v), and cultures were further incubated at 26°C for 4 h  values were determined by the nonlinear regression fits of the dose-response curves using GraphPad Prism 8.0. All measurements were performed in triplicates, twice independently.Promastigotes of 5 cells/well and incubated at 37°C in 5% CO2 for 2 h. After this period, adherent cells were washed twice with phosphate buffered saline  and infected with stationary-phase L. infantum promastigotes at a 10:1 parasite/macrophage ratio. After 4 h of infection at 37°C in 5% CO2, free parasites were removed by a wash step with PBS and infected macrophages were incubated for an additional 24 h to allow parasite differentiation into amastigotes. Then, infected cells were treated with increasing concentrations of selected LiARG inhibitors (11.0–54.0 μM) for 48 h. After this period, the culture supernatant was collected for the evaluation of NO production by the Griess reaction  , as described earlier. Fungizone® (130–540 nM) was used as the reference drug. Results were expressed as percentages of viability in relation to the control (100% viability). The 50% inhibitory concentration (IC50) values were determined by the nonlinear regression fits of the dose-response curves using GraphPad Prism 8.0. All measurements were performed in triplicates, twice independently.Intracellular anti-amastigote activity was determined using the promastigote recovery assay with minor modifications . Among the flavonoids tested, taxifolin showed the most effective inhibition result (88%) at 100 μM. Although chalcones and chalcone-based compounds have been described as inhibitors of other Leishmania enzymes, to the best of our knowledge, this is the first report on the inhibition of Leishmania arginase by this class of flavonoids.Chen et al.  showed tLeishmania trypanothione reductase -1-(2-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one] with a prenyloxy group in ring A and a nitro group in ring B demonstrated a potent inhibitory activity against To gain further insights into the mechanism of inhibition, the three most active chalcone derivatives, those with percentages of inhibition greater than that of quercetin  , were doL. mexicana arginase , Asn143(H), Asp194(H), Ala192(A), Thr257(A) residues   with milin silico results support the findings of de Mello et al. . LC32 exhibited the greatest effect against L. infantum promastigotes with an IC50 of 74.1 ± 10.9 μM. Then, chalcones LC14, LC41, and LC39 showed similar IC50 values of 283.4 ± 14.2, 319.1 ± 14.3, and 398.0 ± 44.2 μM, respectively. Lastly, LC34 showed an IC50 of 747.2 ± 22.3 μM, and LC37 was the only chalcone derivative that did not display in vitro activity against L. infantum promastigotes at the highest concentration tested.After this first screening, the chalcone derivatives exhibiting more than 50% LiARG inhibition   were sel50 of 4,531.0 ± 212.0 μM and thus a SI of 11.4, proving to be more than 10 times selective against L. infantum promastigotes. In contrast, LC14 displayed a CC50 of 75.1 ± 8.9 μM (SI < 1), being more toxic to the host cell than the parasite. The chalcone derivatives LC34, LC41, and LC32 showed CC50 values of 3,010.9 ± 88.0 (SI of 4.0), 1,146.8 ± 58.8 (SI of 2.6), and 479.1 ± 19.5 μM (SI of 6.5), respectively. Indeed, the replacement of a bromide at position 4 of ring A of LC39 by a methoxy group in LC41 may play an important role in the cytotoxicity for macrophages. In addition, LC37, which did not show anti-L. infantum promastigote activity, also did not inhibit the growth of RAW 264.7 macrophages at the highest concentration tested.Next, we evaluated the cytotoxicity of the selected chalcone derivatives against RAW 264.7 macrophages, enabling us to determine their selectivity against parasite cells. LC39 showed a CCL. infantum promastigote activity and selectivity against parasite cells (SI > 1.0), the chalcone derivatives LC32, LC34, LC39, and LC41 were selected for the analysis of their activity against L. infantum intracellular amastigotes. Fungizone® was again used as a reference drug control. L. infantum-infected peritoneal macrophages were treated with the selected chalcone derivatives and the viability of promastigotes recovered from infected macrophages was measured. All chalcone derivatives were able to reduce the parasite load when compared to untreated control cells, displaying IC50 values of 42.3 ± 17.1 (LC39), 43.7 ± 13.7 (LC41), 65.4 ± 10.9 (LC34), and 111.5 ± 19.8 μM (LC32)  . MaquiavM (LC32)  demonstr50 value against intracellular amastigotes, resulting in a SI of 107.1, even greater than that of the reference drug Fungizone® . It is noteworthy that a SI over 10 is considered ideal according to the hit and lead criteria in drug discovery for infectious diseases previously established by Katsuno et al. . Indeed, previous studies have already connected the anti-inflammatory effect of chalcones to the inhibition of NO production  revealed that arginase downregulates several virulence factors, including LPG, PPG, GP63, and amastin  increased NO production by  control . The res control , which sL. infantum arginase. Among the chalcone derivatives tested against LiARG, three  showed inhibitory potential greater than the reference inhibitor quercetin. In silico studies indicated the direct interaction of chalcone derivatives with LiARG's active site residues as well as their low toxicity and good oral bioavailability. In addition, the chalcone derivatives LC34, LC39, and LC41 were effective against the promastigote and intracellular amastigote forms of L. infantum. Remarkably, LC39 stood out for being highly selective to the parasite, even more so than the reference drug Fungizone®. Interestingly, our results point at the nitro group substituent in ring B as an important factor for the antileishmanial effect of chalcones, corroborating previous findings. Taken together, the results presented here bring new perspectives for Leishmania arginase inhibitors and the development of chalcone-based drug candidates against visceral leishmaniasis.We report for the first time on the ability of chalcones to inhibit The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in silico analysis. IL, RS, and LM synthesized the chalcone derivatives. AG performed the biological assays. All authors contributed to the article and approved the submitted version.AV, AP, and IR designed the work. AG, JJ, AP, and IR wrote the manuscript. AG and DO performed the enzymatic assays and data analysis. AMTS, and ARS performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As of 18 October 2020, over 39.5 million cases of coronavirus disease 2019 (COVID-19) and 1.1 million associated deaths have been reported worldwide. It is crucial to understand the effect of social determination of health on novel COVID-19 outcomes in order to establish health justice. There is an imperative need, for policy makers at all levels, to consider socioeconomic and racial and ethnic disparities in pandemic planning. Cross-sectional analysis from COVID Boston University’s Center for Antiracist Research COVID Racial Data Tracker was performed to evaluate the racial and ethnic distribution of COVID-19 outcomes relative to representation in the United States. Representation quotients (RQs) were calculated to assess for disparity using state-level data from the American Community Survey (ACS). We found that on a national level, Hispanic/Latinx, American Indian/Alaskan Native, Native Hawaiian/Pacific Islanders, and Black people had RQs > 1, indicating that these groups are over-represented in COVID-19 incidence. Dramatic racial and ethnic variances in state-level incidence and mortality RQs were also observed. This study investigates pandemic disparities and examines some factors which inform the social determination of health. These findings are key for developing effective public policy and allocating resources to effectively decrease health disparities. Protective standards, stay-at-home orders, and essential worker guidelines must be tailored to address the social determination of health in order to mitigate health injustices, as identified by COVID-19 incidence and mortality RQs. The high incidence of novel coronavirus disease 2019 (COVID-19) in the United States is concerning; there have been approximately 8.2 million confirmed cases and nearly 220,000 deaths . Th. Th13]. Data on White, Black, Hispanic/Latinx, Asian, AIAN, and NHPI people were evaluated. We excluded the multiple race/ethnicity category from our analysis, as there was insufficient data for analysis. COVID-19 incidence data classified by race and ethnicity were available in 46 of 50 US states. Racial and ethnic data from Louisiana, New York, North Dakota, and Rhode Island were not available and were excluded from our analysis. Cases and deaths where race or ethnicity were unknown were also excluded. The data were restricted to reports from 1 March to 14 June 2020, when public policy stay-at-home orders were in effect, regardless of their enforceability .A representation quotient (RQ) was utilized. RQs were defined as the proportion of a particular subgroup in the total number of COVID-19 cases or mortality relative to the corresponding estimated proportion of that subgroup in the United States population. For example, COVID-19 incidence RQ for White people would be given by stralia) .p < 0.05) differences between the average state RQs between all racial/ethnic groups.After calculation of incidence and mortality RQ by race and ethnicity, Tukey’s test for post-hoc analysis was completed with an ANOVA to evaluate statistically significant  published categorized the following industries in an advisory memorandum as critical infrastructure sectors, and therefore as essential work: medical and health care, telecommunications, information technology systems, defense, food and agriculture, transportation and logistics, energy, water and wastewater, law enforcement, and public works . This seAccording to the Center for Economic and Policy Research’s 2019 analysis of the essential workforce, over 31 million Americans are deemed essential. Approximately 50.9% of these workers are in the health care sector, 20.6% in food and agriculture, and 7.2% in transportation and delivery . These eIn the United States, more than 36% of essential workers are non-White . FurtherThere are distinct differences in the proportion of workers who are eligible to work from home by race and ethnicity. The US Bureau of Labor Statistics reported that in the period 2017–2018, 42 million wage and salary workers (29%) had the option to work from home. Of these, 36 million reported working from home at least occasionally . While 2Personal transportation methods vary widely across the US. In rural areas, Americans rely heavily on personal vehicles to travel to their place of employment . ConversEarly reports from Japan show COVID-19 incidence rates are heavily influenced by population density . DensityLimited access to potable water or indoor plumbing on Native American reservations makes it difficult to follow handwashing guidelines. The study by Rodriguez-Lonebear et al. found that among AIAN, increased likelihood of COVID-19 was associated with the lack of indoor plumbing . The assSocially assigned race has been shown to have greater impact on health than self-reported race . These sBeginning in May, Black Lives Matter and other social justice movement demonstrations were held nationwide. These public demonstrations were sparked by the murders of unarmed Black people by police, including Breonna Taylor and George Floyd, among others. Public health officials initially raised concerns that these protests could potentially increase the spread of COVID-19. However, it is unclear whether these large-scale protests have directly impacted COVID-19 outcomes. A notable increase in COVID-19 incidence occurred shortly after the protests began, but this also coincided with the “reopening” of many states and a marked increase in overall social activity. Interestingly, studies have shown that cities which had large protests actually had a net increase in social distancing behavior .Many argue that racial injustice, and the longstanding mistreatment of Black people by the criminal justice system, is a public health crisis and necessitates these protests. Indeed, the incarceration of Black people occurs at a rate 5 times higher than their White counterparts . Black aStructural racism contributes to disproportionate rates of incarceration; there are higher rates of incarceration among Black, Indigenous, and Hispanic/Latinx individuals, particularly those of low socioeconomic status and those with mental illness, than their White counterparts . MoreoveAlmost 30 million Americans do not have health insurance coverage . AlthougHealth insurance programs may be expensive, inaccessible for many individuals, and have complex policies for covered expenses. Even when enrolling in marketplace plans with tax credits, only 69% of individuals had premiums of <$100 per month . This baAccess to testing and treatment varies across the US. Of greatest concern is affordability of care. Several states, such as Michigan, Wisconsin, and New York, have reported that the zip codes most impacted by COVID-19 are also the poorest . In New In an effort to expand services to rural health clinics, the US Department of Health and Human Services allocated $225 million dollars to support expansion of COVID-19 testing, telemedicine services, and notification/information services . Using tIn 2017, the Federal Communications Commission identified approximately 25 million Americans that do not have internet access, of which, approximately 19 million live in rural areas . FurtherInsufficient access to nutritious foods, low health literacy, and lack of culturally appropriate health education compound risk of hypertension, diabetes, and obesity—all of which are risk factors for COVID-19 morbidity and mortality ,56,57. AVitamin D deficiency has also been found to be associated with increased risk for COVID-19, further worsening the outlook for the undernourished . StudiesWe suspect that COVID-19 incidence and mortality may be under-reported given the initial difficulty in testing. Studies indicate that, after controlling for COVID-19 deaths, the overall mortality rate has increased during the March–May 2020 period compared to average mortality rates for the past 10 years . This suAnother limitation to this study was the variability in state-level data. There is no standard method for data collection by race, sex, and ethnicity between states. For example, some states categorized more than 25% of reported cases as “other” race or ethnicity. This aggregation of data may obscure other disparities, yet to be defined, and may have led to underestimation of incidence and mortality RQs. Additionally, our study only evaluated state-level incidence and mortality, which may have masked greater disparities between counties and cities within states.During the study period, 8 states had fewer than 100 COVID-19 deaths, some with less than 40. This may overestimate racial and ethnic disparities due to a low sample size. Cases and mortality continue to fluctuate nationwide and as more data become available, repeat analysis may be warranted using an extended time frame.National data may mask racial and ethnic disparities in COVID-19. Microcosmic data, from city or state-level reporting, better illustrate the disparate health outcomes during the pandemic. The evidence suggests that there is significant disparity in COVID-related health outcomes by race and ethnicity regarding representation by state. We suspect that these disparities would be even more apparent at the county and city level and this warrants further investigation. There is a need for public reporting of disaggregated COVID-19 data by county and zip code. Transparency of local data would allow for greater precision in allocation of resources and establishment of effective policy changes to disrupt the social determination of poor health outcomes, moving the United States towards the goal of health justice.Infectious disease, including COVID-19, does not selectively affect individuals based on race. However, the social dynamics which perpetuate racial, economic, and environmental disparity create a system of injustice which sustains health inequality, thereby resulting in disparate susceptibility to infection, morbidity, and mortality among marginalized communities. It is an exercise in futility to search for any single element that predisposes people of color, in particular Black people, to adverse COVID-19 outcomes. Instead, we need to explore the intersectionality of inequity and the social determination of health. Therefore, we believe that racial and societal disparities are not only cultural and economic issues, but also an issue of public health and social justice.Many of these inequalities are longstanding and rooted in our society’s infrastructure. The dismantling of systems of oppression which drive the ongoing health injustice epidemic is requisite for a reduction in health disparities and relief of COVID-19 disease burden. Limiting and reducing mortality require identification of disparity dynamics in our communities and addressing the social determination of these vulnerabilities through specific, intentional interventions. Although systemic change may take time, we hope that health injustices, as exemplified by the COVID-19 pandemic, serve to spark action and create momentum towards achieving social justice and eliminating health disparities.


It has been a long-standing puzzle why electrons with repulsive interactions can form pairs in unconventional superconductors. Here we develop an analytic solution for renormalization group analysis in multiband superconductors, which agrees with the numerical results exceedingly well. The analytic solution allows us to construct soluble effective theory and answers the pairing puzzle: electrons form pairs resonating between different bands to compensate the energy penalty for bring them together, just like the resonating chemical bonds in benzene. The analytic solutions allow us to explain the peculiar features of critical temperatures, spin uctuations in unconventional superconductors and can be generalized to cuprates where the notion of multibands is replaced by multipatches in momentum space. The exotic phenomena of superconductors remained mysterious until Bardeen, Cooper and Schrieffer (BCS)2 came up with a complete theory in 1957. There are two key ingredients in BCS theory. Firstly, a negatively charged electron distorts the nearby lattice formed by positively charged ions and another electron thus feels an effective attraction toward the original electron. In professional jargons, one can say that the effective attraction is mediated by electron-phonon interactions. The second key ingredient is pair formation: an electron with specific momentum and spin pairs up with another electron with opposite momentum and spin via the effective attraction. These electron pairs, referred as Cooper pairs, form a Bose-Einstein condensate in the superconducting state4.It has been over one hundred years since Heike Kamerlingh Onnes discovered the resistance of mercury suddenly drops to zero7, heavy-fermion compounds10, organic superconductors11, iron-based superconductors13 and recently found superconducting graphene superlattices14, remaining queer16 and unexplained by the electron-phonon interactions alone. It is generally believed that the Coulomb interaction between electrons may be responsible for the emergent superconductivity4. However, there is a huge mismatch of the energy scales – the critical temperature is much smaller than the bare electronic interactions. Perhaps the most intriguing puzzle for these unconventional superconductors is the pairing mechanism: what is the glue to pair up electrons from mutual repulsive interactions? The experimental evidences seem to suggest that pairing in the unconventional superconductors is not due to electron-phonon interactions13. Due to strong magnetic correlations20 in these materials, it is proposed that spin fluctuations27 may play the role of glue to pair electrons up. The renormalization-group (RG) studies3,29 seems to indicate that the antiferromagnetic spin fluctuations may be the cause of unconventional superconductivity in iron-based materials.Despite the celebrating success of BCS theory, there are other superconductors including cuprates32, we integrate out quantum fluctuations at shorter length scales within the one-loop approximation and seek for the ground state in the low-energy limit. Because the RG equations are intrinsically non-linear, numerical analysis is already challenging, rendering simple understanding beneath the messy numerics inaccessible in most cases. We adapt the classification scheme of relevant couplings developed in previous works35 to overcome the challenge and analyze how superconductivity emerges in RG transformations.In this Article, we investigate the pairing mechanism in multiband iron-based superconductors by RG analysis. As in previous RG studiesg and g⊥, standing for intraband and interband pair hopping. The functional form of the gap function computed within the mean-field theory dictates the RG equations for g and g⊥. In fact, these RG equations derived from the mean-field theory are so simple so that analytic solutions can be found easily.In addition, we make use of the scaling relation to bridge the results obtained by RG analysis and those in the conventional mean-field theory. While it is well known that the mean-field theory provides a self-consistent description for various physical quantities, it is less familiar to the scientific community that the mean-field theory also lays out the RG equations for its parameters. For example, for the generalized multiband BCS Hamiltonian, the vanilla version of the mean-field theory is characterized by two important parameters: Here comes the surprise. For multiband iron-based superconductors, we find the RG flows are well captured by simple analytic solutions for intraband and the interband pairing hopping derived from the mean-field theory. The solutions are elegant and simple enough for us to reveal the pairing mechanism in multiband superconductors.g and g⊥ are positive before RG transformations. However, it turns out that there is no need to search for attractive glues to pair electrons up anymore. Through interband pair hopping g⊥, electron pairs hop between different bands to lower the energy, compensating the penalty for pair formation due to repulsion. Our findings are consistent with the previous studies37. The pairing mechanism in multiband superconductors is resonating pair hopping between different bands, just like the resonating chemical bonds in benzene. In fact, the effective theory we found is consistent with the previous mean-field theories for multiband superconductors in the interband-dominated regime44.Because the electronic interactions are repulsive in nature, both couplings g and g⊥ are large, the binding energy of Cooper pairs  is determined by their difference δ, which is one order smaller than the bare couplings as detailed later. (3) The interband pair hopping not only explains why strong magnetic fluctuations often show up in unconventional superconductors but also pins down at what momentum the spin fluctuations should appear.However, the RG analysis makes stronger predictions than the mean-field theories and sews several different phenomena into one coherent picture. Let us explain the major findings without digging into the technical details. The binding energy for Cooper pair formation is dictated by a small parameter δ = g, just the intraband pair hopping. Thus, for electrons to pair up, one needs some sort of attractive interactions (g < 0). Besides, the binding energy is of the same order of the effective attraction. But the interband pair hopping completely changes the story: attractive interaction is no longer a necessity. Despite of repulsive interactions, as long as the interaction profile produces a larger interband pair hopping, Cooper pairs form and superconductivity sets in. The interband pair hopping, driving unconventional superconductivity in the presence of repulsion, provides natural explanations, not only for the spin fluctuations at the momentum connecting the Fermi surfaces, but also for the mismatched energy scales of the critical temperature and the bare interaction strength.The difference between pairing mechanism for multiband superconductors and that for the conventional BCS theory lies in the presence of interband pair hopping. In its absence, the binding energy is solely determined by To put our feet on the firm ground, we choose iron-based superconductor as a demonstrating example. In the following, we elaborate the numerical and analytical details how the above conclusions are achieved. It is worth emphasizing that most conclusions are not limited to iron-based superconductors. Generalization of our theory will be discussed at later paragraphs.46. The mutual repulsion between electrons is model by the simplest on-site interactions, containing intra-orbital repulsion U1, inter-orbital repulsion U2 and the Hund’s coupling JH between different orbitals. Detail descriptions of the model can be found in Methods.The kinetic energy in iron-based superconductors is described by a five-orbital tight-binding model with appropriate hopping matrix elementss± pairing symmetry31. Due to the s-wave symmetry, it is enough to sample one point on each Fermi surfaces for qualitative understanding. It turns out that this simplification is equivalent to put the iron-based superconductor on the four-leg ladder with periodic boundary conditions along the x direction. It will become clear later that this geometry does not change the ground state but simplify the algebra significantly. Later, we will come back to two-dimensional calculations and reaffirm the validity of our major claims.The above Hamiltonian has been studied by functional renormalization group (fRG) method, which shows that the ground state is a superconductor with sign-reversed kx and the chirality R/L as shown in Fig. 49, the allowed interactions are Cooper scattering σ, ρ denote the spin and charge channels respectively. The RG equations for all couplings are given explicitly in Methods. Though the RG equations can be written down explicitly, the solutions are still too complicated. With l = In(Λ0/Λ) being the logarithm of the ratio between bare energy cutoff Λ0 and the running cutoff Λ, we integrate the coupled differential equations numerically, and elaborate the details in Methods. We find all couplings are captured by the scaling Ansatz35,gi are generally referred to the Cooper and forward scatterings mentioned above, Gi is an order one constant and 0 ≤ γi ≤ 1. The scaling exponent γi help us to build the hierarchy of all relevant couplings without ambiguity. For example, starting with U1 = 2.8 eV, U2 = 1.4 eV and JH = 0.7 eV, the RG exponents for all couplings are shown in Fig. x = 0 to x = 0.12.In weak coupling, the relevant degrees of freedom can be labeled by the quantized momentum mentclass2pt{minimγi = 1 are the most dominant, including intraband Cooper scattering c12 leads to the s± pairing symmetry. Note that our RG Ansatz predicts the dominant superconducting bands with the correct pairing symmetry as obtained by the fRG method. In fact, a detail analysis including all subdominant relevant couplings lead to correct signs for all gap functions in different bands as those obtained in the fRG method. Thus, the ground state in the ladder geometry is the same as that in the two-dimensional system.The couplings with g(l) and g⊥(l),l = ld, it fixes that c(0), c⊥(l) are given and ld is found in numerics already, the values of g(0) and g⊥(0) are pretty much determined.However, due to the explicit form of the RG equations for ladder geometry, the pairing mechanism in the multiband superconductor can be studied analytically. Construct the couplings, c(l) and c⊥(l) from integrating the whole set of nonlinear differential equations (about 50 equations for this case) can be captured by the analytic solutions given in Eq. (c(l), c⊥(l) and the analytic ones g(l), g⊥(l) are shown in Fig. The above simplification is inspiring: it implies that the RG flows for the intraband and the interband pair hopping n in Eq. . Comparig < 0 arisen from effective attraction and Λ is the Debye energy. But, it is not widely known that the RG equation for g can be derived from the dependence of the gap function. In the RG method, the gap function is rescaled with rescaling energy of systems in RG steps. By integrating high-energy modes, the rescaling of the gap function is described by the RG equation dΔ/dl = Δ. It is said that gap functions at different energy scales can be related by 50. Taking the derivative, d/dl, on the both sides, the RG equation reads, dg/d = −g2l. Therefore, if the RG flow for some system is described by the derived RG equation, its effective theory is just the single-band BCS Hamiltonian.To dig out the secret message behind the analytic solutions, let us switch gear to review the well-known single-band BCS Hamiltonian first. The gap function takes the form g and g⊥ stand for intraband and interband pair hopping. The above non-linear coupled equations can be solved exactly, giving the analytic solutions we discussed previously. Therefore, the effective theory for iron-based superconductors is the multiband BCS Hamiltonian, proven by matching the RG flows together.We can generalize the scaling argument to multiband BCS Hamiltonian as elaborated in Methods. In particular, when two bands reign interband pair hopping, the corresponding RG equations areg = g⊥ < 0), the ground state flows toward the Fermi-liquid fixed point and no superconductivity occurs. However, if the interband pair hopping is larger (g = g⊥ < 0), the ground state flows toward the superconducting phase with s± pairing symmetry. It is worth mentioning that, if the initial couplings are close to the symmetric ray g = g⊥, it will flow toward the Fermi-liquid fixed point first and then turns around to the unconventional superconducting state. This means that, upon cooling down the system toward the critical temperature, it exhibits non-trivial crossover properties over a wide range of temperatures. This crossover is shown in the orange-shaded regime in Fig. g and g⊥ are small.RG flows for repulsive interactions are shown in Fig. g⊥ = 0). As is clear in Fig. g(0), i.e. attractive interaction, is required to trigger the superconducting instability. For repulsive interaction g(0) > 0, the RG flow always bring the system to the trivial Fermi-liquid fixed point. The RG flows in the special case g⊥ = 0 give rise to the commonly accepted criterion that an effective attraction is necessary for Cooper pair formation. However, the criterion is obviously wrong as the RG flows in the upper plane is quite different from those in the horizontal axis.It is interesting to compare with the pairing instability in the absence of interband pair hopping  instability if the momentum Q = K1 − K2, connecting the two Fermi surfaces, is close to half of the reciprocal lattice vectors, i.e. Q = G/2. In the case of iron-based superconductor in ladder geometry, Q =  satisfies the above condition. Therefore, it is expected that the antiferromagnetic spin fluctuations at the momentum Q are enhanced along with unconventional superconductivity. Note that, in conventional superconductors, Q is automatically zero and no antiferromagnetic spin fluctuations are expected to grow hand-in-hand with the superconductivity.Finally, the interband pair hopping 50 on RG in two dimensions. In stead of following Shankar’s original approach, we follow closely the methodology developed in functional RG. Consider two circular Fermi surfaces as shown in Fig. θi represents the angle of the corresponding momentum.Now that we realize the importance of interband pair hopping, we are ready to generalize Shankar’s seminal workDerivations of the RG equation in two dimensions are provided in Mathods. Here we concentrate on the final results and their implications. The RG equations in two dimensions are,In the absence of interband pair hopping, we have checked that the above RG equations reduce to those derived by Shankar with just one circular Fermi surface. The general RG equations involving integration over internal angles can be simplified to the previous result. Assuming the density of states and the bare couplings are rotationally invariant, i.e. s in Eq.  again. TThe two-pocket model presented here provides another perspective on the relation between superconductivity and antiferromagnetic spin fluctuations. In iron-based superconductors, the momentum connecting two Fermi surfaces is Our previous calculations concentrate on the dominant bands in the iron-based superconductor to reveal the pairing mechanism in multiband superconductors on firm ground. But, it is reasonable to include all active bands in the generalized BCS Hamiltonian when quantitative accuracy is required. In addition, it is intriguing to check whether the fRG flows can be captured by the analytic solutions in Eq.  as well.d-wave symmetry. It implies that pairing mechanism in cuprates arises from resonating pair hopping between antinodal regimes51. The interpatch pair hopping between antinodal regimes also explains the enhance spin fluctuations at . The pairing instability in the nodal regimes is only triggered when approaching close to the critical temperature. How to rigorously generalize the analysis presented here from multiband superconductors to multipatch single-band system remains an open question at this point.For single-band materials but with significant variations in density of states at different momenta, one should divide the Fermi surface into appropriate patches and applies the method developed here. We have carried out primitive RG analysis on cuprates by cutting the single-band Fermi surface into 16 patches . The dominant patches locate at  and  with sign-reversed gap functions, agreeing with the g⊥ and suppresses the intraband one g. It is not yet clear whether the optimal electronic interaction for different materials share a generic profile, or the answer may vary from one material to the other.We would like to emphasize that the weak-coupling RG analysis demonstrated here may work well in strong coupling if no quantum phase transition is encountered. That is to say, the generalized BCS Hamiltonian with multiple bands (or different momentum patches) may be the mother Hamiltonian for most unconventional superconductors in the low-energy limit. Even the pairing mechanism is revealed, non-trivial crossovers in the unconventional superconductors are expected at wide temperature range and a complete phenomenological description is still lacking. Finally, the pursue for higher critical temperatures should not be on seeking for stronger glues. In stead, it is time to search for optimal interaction profile which gives the largest interband pair hopping Kvw in the momentum space has been constructed in previous studies45. The Fermi surface with electron doping π, π) in the Brillouin zone. In addition, there are two electron pockets located at  and  points46.The band structure of the iron-based superconductor can be described by the five-orbital tight-binding model,mentclass2pt{minim30 satisfying the SU(2) symmetry  arei runs over all lattice sites. Note that, due to the multiple orbitals, even the simplest on-site interactions contain three terms: intra-orbital repulsion U1, inter-orbital repulsion U2 and Hund’s coupling JH between different orbitals.To investigate the correlation effects, the most general on-site interactionsx direction, we quantize the momentum kx and reduce the five active bands to five pairs of Fermi points, as show as Fig. 49, and can be written down explicitly,By putting the iron-based superconductor on the four-leg ladder with periodic boundary conditions along the 0 and the running cutoff Λ. The coefficient tensor vi for each band. We intentionally separate the intraband and interband RG equations for clarity. Various symmetries ensure that cij = cji and fij = fji. To avoid double counting, we choose fii = 0 here.The notation 49, i.e.To compute the initial conditions of the RG transformation, we rewrite the field operators in the five-orbital tight-binding Hamiltonian by the chiral field decompositionuR/Lia, a = 1, 2, …, 5, are the five orbital components of the Bloch wave functions associated with the R/L (or +y/−y direction) members of the i-th Fermi pair, and cσ and cσ terms can be represented asHere With the similar computation, we also obtain the initial conditions of the forward couplings,g and g⊥ of our effective theory in the main text are We notice that the initial conditions of U1 = 2.8, U2 = 1.4 and JH = 0.7, we numerically solve the RG equations to obtain the RG flows with different filling factors. We find that the coupling constants diverge at the same length scale ld. By showing the RG flows versus 1/(ld − l) in the log-log plot, as illustrated in Fig. 34. The RG exponents for different filling factors are showed in Fig. Within the initial conditions for 54, the four-fermion vertex function Γ(4) is expressed asα, β are spin indices, ω and K =  are labelled as the frequency and the two-dimensional momentum respectively.Following standard derivations developed in functional RG approachN patches for each band in the vicinity of the two Fermi surfaces, determined by the energy cut-off Λ0, as shown in Fig. kFP and vP respectively being the Fermi momentum and Fermi velocity of the band P. We also approximate that the dispersion of the two Fermi pockets are independent of the angular direction. As a consequence, the tree-level RG analysis, demonstrated by Shankar50, shows that the only marginal couplings are the forward and Cooper scattering in this convention i-th patch. With the convention of the band indices V = C, F.The derivation is aided by dividing the Fermi surfaces into two bands and U(1) and SU(2) symmetries, the generic form of functional RG equations for Vs and V1 in one-loop approximation are represented asLpp and Lph functions, depicting particle-particle and particle-hole bubble diagrams respectively, are denoted asUnder the P-th Fermi pocket as 50.We elaborate the computation of the first and second contributions of ) in Eq. , as an en of Eq.  is repreWith the same spirit, we compute the full set of RG equations. Furthermore, to simplify the coefficients in the RG equations, we rescale the couplings asThus, the full set of non-trivial RG equations is represented as,N → ∞ and the two dimensional RG emerges. Terms with 1/N factor but without summation vanish in the two dimensional limit. This simplification is the same as the elegant “phase-space argument” invented by Shankar50 before. Terms with summations smoothly evolve back into angular integral,Take 50.The identity 56.i-th band. To simplify the calculations, we drop the momentum dependence of the interaction 55 with different instabilities, depending on two parameters D is the density of states at the Fermi energy.We consider the standard multiband BCS Hamiltonians± pairing symmetry, i.e. the gap functions are related s-wave pairing symmetry, i.e. the gap functions are related For s± pairing symmetry for instance, by integrating high-energy modes, the rescaling of the gap function is described by the RG equation 50. It is said that gap functions at different energy scales can be related by l in the both side, we obtain the equation,,We connect the gap function to the RG approach, where the gap function is rescaled with rescaling energy of systems in RG steps. Taking the gap function with We combine with the gap function from generalized two-band BCS Hamiltonian Eq. , and ends-wave pairing symmetry leading to the RG equation,In the same manner, the generalized two-band BCS Hamiltonian gives the Combining the above RG equations together, we obtain Eq. .


Identification of molecular determinants of receptor-ligand binding could significantly increase the quality of structure-based virtual screening protocols. In turn, drug design process, especially the fragment-based approaches, could benefit from the knowledge. Retrospective virtual screening campaigns by employing AutoDock Vina followed by protein-ligand interaction fingerprinting (PLIF) identification by using recently published PyPLIF HIPPOS were the main techniques used here. The ligands and decoys datasets from the enhanced version of the database of useful decoys (DUDE) targeting human G protein-coupled receptors (GPCRs) were employed in this research since the mutation data are available and could be used to retrospectively verify the prediction. The results show that the method presented in this article could pinpoint some retrospectively verified molecular determinants. The method is therefore suggested to be employed as a routine in drug design and discovery. The results were confirmed by site-directed mutagenesis (SDM) studies and identified Asn147, Glu182, Thr323, and Gln347 as the molecular determinants [Development of computational methods to accurately identify molecular determinants of the receptor-ligand binding is of considerable interest. Istyastono et al.  combinedrminants . On the rminants  employedrminants ,16, and rminants  in a retrminants . Unforturminants  offer po2a receptor (AA2AR), β2 adrenergic receptor (ADRB2), C-X-C chemokine receptor type 4 (CXCR4), and Dopamine D3 receptor (DRD3). These receptors were selected because they are members of GPCRs, their ligands and decoys datasets are available in the enhanced version of the database of useful decoys (DUDE) [The upgraded version of PyPLIF called PyPLIF HIPPOS was recently made publicly available . The sofs (DUDE) , and theThe proposed method identified 23 probable molecular determinants of the ligand binding to the studied receptors . ThirteeIn In Similar to Two branches are leading to ligand identification in https://gpcrdb.org/) (accessed on 20 March 2021) [The proposed computational methods presented in this article  predicted in total 23 molecular determinants of the ligand binding to some GPCRs, i.e., AA2AR, ADRB2, CXCR4, and DRD3. Thirteen out of these 23 molecular determinants (circa 56.52%) were retrospectively verified by observing the mutant data compiled in GPCRdb ch 2021) ,9. There21 ,9. ThThe computational techniques employing the combination of retrospective SBVS campaign and RPART analysis were originally used to increase the prediction quality of the developed SBVS to identify ligand for ERα . SubsequThe most recent GPCRdb updates have just been published , which a4 receptor (HRH4) as the molecular determinant of the ligand binding could be used further to fine-tune the HRH4 affinity and the selectivity toward the histamine H3 receptor (HRH3) [The information of the molecular determinants could be used further in structure-based drug design and discovery, especially in fragment-based approaches to perform optimization rationally in order to fine-tune the affinity and the selectivity for a particular receptor target . For exar (HRH3) . In the r (HRH3) . The infr (HRH3) ,27. In or (HRH3) ,35 and dr (HRH3) ,37.All the identified molecular determinants in this receptor were verified in the GPCRdb . Only onThree out of nine identified molecular determinants in this receptor were verified in the GPCRdb . There aThree out of four identified molecular determinants in this receptor were verified in the GPCRdb . SimilarThree out of six identified molecular determinants in this receptor were verified in the GPCRdb . There a® Xeon® CPU E5-2620 v4 @ 2.10GHz as the processor and 64GB of random-access memory (RAM). There were in total 16 central processing units (CPUs) from 8 cores @ 2 threads. The following were the software used in the research presented in this article: AutoDock Vina version 1.1.2 [https://github.com/radifar/PyPLIF-HIPPOS/releases/tag/0.1.2, accessed on 20 March 2021); PLANTS docking software 1.2 [https://www.rcsb.org/) (accessed on 20 March 2021) [https://gpcrdb.org/) (accessed on 20 March 2021) [The computational simulations were performed in a 64-bit Linux (CentOS Linux release 7.4.1708) machine with Intelon 1.1.2 ; PyPLIF on 1.1.2  version ware 1.2 ,30; SPORware 1.2 ; Open Baware 1.2 ; ADFRsuiware 1.2 ; and RPAware 1.2 . Compounch 2021)  with thech 2021)  were useThe generic procedure consisted of 3 steps: (i) retrospective SBVS campaigns using AutoDock Vina; (ii) PLIF identification using PyPLIF HIPPOS followed by ensPLIF calculation; and (iii) RPART analysis using R.The retrospective SBVS using molecular docking simulations started with the preparation of the compounds obtained as mol2 files from DUDE, followed by preparation of the virtual target obtained from RCSB PDB, and preparation of the configuration file to perform docking. Then, the docking simulations were performed for all prepared compounds. Module “separate” from Open Babel was used to split the obtained files from DUDE into a single file for a single compound. The mol2 files were then subjected to the module “prepare_ligand” from ADFRsuite to be converted into the AutoDock Vina readily format pdbqt. The module “splitpdb” from SPORES was used to split the protein part from others into the pdb files obtained from RCSB PDB. The “protein.mol2” resulted from the module “splitpdb” of SPORES was then subjected to the module “prepare_receptor” from ADFRsuite to be converted into the AutoDock Vina readily format pdbqt. The generic configuration for the docking simulations were set as follows: num_modes = 10; energy_range = 5; cpu = 8; log = out.log. The XYZ coordinate position and the size of the docking box were specific for each virtual target. The center of the co-crystal ligand was set as the XYZ coordinate position, and the distance of 5 Å from the surface of the co-crystal was used to calculate the docking box size. The module “bind” from PLANTS was used to obtain the values of the XYZ coordinate positions and the size of the docking boxes. Each prepared compound was then docked using AutoDock Vina. The module “bind” from PLANTS also provided a list of residues in the docking box. The residues were used to create configuration files for PLIF identification using PyPLIF HIPPOS. By employing the configuration files, the PLIF identifications were performed for all docking poses resulted from the retrospective SBVS . The opthttps://www.rcsb.org/structure/3eml (accessed on 20 March 2021) [http://dude.docking.org/targets/aa2ar (accessed on 20 March 2021) [The human AA2AR with the PDB ID of 3EML was downloaded from ch 2021) , while thttps://www.rcsb.org/structure/3ny8 (accessed on 20 March 2021) [http://dude.docking.org/targets/adrb2 (accessed on 20 March 2021) [The human ADRB2 with the PDB ID of 3NY8 was downloaded from ch 2021) , while thttps://www.rcsb.org/structure/3odu (accessed on 20 March 2021) [http://dude.docking.org/targets/cxcr4 (accessed on 20 March 2021) [The human CXCR4 with the PDB ID of 3ODU was downloaded from ch 2021) , while thttps://www.rcsb.org/structure/3pbl (accessed on 20 March 2021) [http://dude.docking.org/targets/cxcr4 [The human DRD3 with the PDB ID of 3PBL was downloaded from ch 2021) , while tThe combination of retrospective SBVS campaigns, PLIF-derived ensPLIF descriptors using PyPLIF HIPPOS, and RPART analyses provide a full in silico complementary method to SDM studies for the molecular determinants of the ligand binding to the corresponding GPCRs. Notably, the method shows better prediction quality indicators of the SBVS protocols compared to the original protocols. Moreover, for a particular receptor target, there are options to optimize the prediction quality, e.g., fine-tuning the configuration of the docking simulations or filtering poses prior to ensPLIF calculation based on the docking score.