Farmácia Popular” (FP) Program is one of the government’s key strategies to achieve this goal. Under FP, antihypertension (HTN) and antiglycemic (DM) medicines have been provided at subsidized prices in private retail settings since 2006, and free of charge since 2011. We aim to assess the impact of sequential changes in FP benefits on patient affordability and government expenditures for HTN and DM treatment under the FP, and examine their implications for public financing mechanisms and program sustainability.Increasing medicines availability and affordability is a key goal of Brazilian health policies. “Longitudinal, retrospective study using interrupted time series to analyze: HTN and DM treatment coverage; total and per capita expenditure; percentage paid by MoH; and patient cost sharing. Analyzes were conducted in the dispensing database of the FP program (from 2006 to 2012).reais, the Brazilian currency) to 18.74 for HTN and from R$33.07to R$15.05 for DM over the period analyzed, representing a reduction in per capita cost greater than 50%. The amount paid by patients for the medicines covered increased over time until 2011, but then declined to zero. We estimate that to treat all patients in need for HTN and DM in 2012 under FP, the Government would need to expend 97% of the total medicines budget.FP has increased its coverage over time; by December 2012 FP covered on average 13% of DM and 11.5% of HTN utilization, a growth of over 600 and 1500%, respectively. The overall cost per treatment to the MoH declined from R$36.43  and government (better negotiated prices). However, overall FP expenditures by MoH increased due to markedly increased utilization. The FP is sustainable as a complementary policy but cannot feasibly substitute for the distribution of medicines by the SUS. Treatment of hypertension (HTN) and diabetes (DM) is considered a health policy priority in Brazil, with particular attention paid to reducing preventable hospital admissions. Recent reductions have been associated with the expansion of the primary care, which facilitates early detection, and treatment of HTN and DM .Farmácia Popular” (FP), or by paying out-of-pocket at private retail pharmacies simultaneously.The main characteristics of the principal medicines provision mechanisms in place in Brazil are summarized in Table 4 in Farmácia Popular” (FP) is a medicines provision mechanism first implemented in 2004. In its first phase, medicines were provided in public health care facilities at a flat price, corresponding to the price obtained by the government in open bidding, plus administrative costs. This program was named “Farmacia Popular Rede Própria”. In 2006 the program, then named “Aqui tem Farmacia Popular - AFP” (“Farmácia Popular” is available here- AFP-I), was expanded to private pharmacies contracted with the Ministry of Health. Medicines were charged in a coinsurance model, with the government paying 90% of a reference price and patients paying 10% of the selling price (which might be higher than the reference price). To improve accountability of pharmacies, a new administrative system was implemented in 2009 (AFP-II) that provided information on each patient claim.The “n = 558) [n = 25,150, covering 63.4% of the 5570 municipalities [Saúde não tem preço - SNP” . Except for metformin 500 mg (extended release), all medicines covered in FP for these three conditions that are included in RENAME  were provided free-of-charge in SUS. The SNP makes treatment affordable for patients, but this benefit might be unsustainable in the long term, especially in a country that has other forms of provision.Subsequently, antihypertensive, antidiabetic (since 2011), and antiasthma medicines (since 2012) began to be dispensed with zero copayment from patients in both government-owned facilities (n = 558)  and contCurrently, the FP program continues to provide free medications for treatment of asthma, diabetes and hypertension, and co-finances drugs for treatment of dyslipidemia, osteoporosis, rhinitis, Parkinson’s and glaucoma, as well as contraceptives and geriatric diapers, totaling approximately R$2.9 billion in 2017 .FP is widely seen as a successful program that has expanded coverage in both the number of individuals treated for DM and HTN and in the number of dispensings per person –7. HowevFarmácia Popular interventions on patient affordability and government expenditures for HTN and DM treatment under the FP program, and consider their implications for public financing mechanisms and program sustainability.This paper aims to analyze the impact of the sequential This is a longitudinal, retrospective study using interrupted time series (ITS) to examine out-of-pocket payments and MoH expenditures for HTN and DM treatment. The main outcomes addressed are rates of HTN and DM treatment coverage, number of individuals in FP, total expenditures, percentages paid by MoH, treatment cost per capita and out of pocket payment.The Brazilian National Ethics Committee, by the National School of Public Health – Fiocruz – Brazil and the WHO ERC, approved the ISAUM-Br project, which is the basis for this paper.Saúde não tem preço” (SNP) program was implemented, under which all covered medicines for HTN and DM were dispensed free of charge to patients. FP private pharmacies were reimbursed according to a set of negotiated prices, while in government-owned pharmacies, medicines were fully subsidized. Only FP private pharmacies are addressed in this paper.The study interventions are two changes in patient cost sharing in AFP. The April 2009 AFP-II policy involved a reduction in reference prices for most FP medicines by an average of 24.5%, coupled with administrative changes aiming to improve accountability. In February 2011, the “The FP information system is the first widespread governmental administrative system on medicines dispensing in Brazil. The FP information system in contracted pharmacies is managed by the Unified Health System Informatics Department (DATASUS). Data include patient unique identification number (CPF), price paid, date of purchase, prescribed daily dose and amount procured. CPF allows linking to data on gender and date of birth. In the majority of cases, the buyer corresponds to the patient for patients over 18 years old. Other administrative systems cover a small set of medicines  and are not integrated at national level.FP program eligibility criteria have remained unchanged during the program: all medicines are sold only if a national ID and a valid prescription are presented. During the study period medicines were dispensed on a monthly basis, although prescriptions were valid for 120 days. Over time, the number of participating private sector pharmacies expanded substantially, especially in some regions .Data are derived from an electronic point-of-sales dispensing program implemented in 2006 in FP retail pharmacies and then integrated online by DATASUS. Available data include patient and pharmacy identifiers, patient age and gender, geographic location of the pharmacy, date of dispensing, name and quantity of medicine dispensed, daily prescribed dose, amount of MoH reimbursement, and patient copayment.We use data on dispensing of HTN and DM medicines from October 2006 to December 2012. All patients with at least one dispensing during the study period were included in this analysis. Dispensing data are of good quality and relatively complete, with duplicate cases accounting for less than 0.005% and individual-level missing data at less than 0.05%. We excluded encounters with missing data on any outcome variables from all analyses.Medicines covered by the program include four oral antidiabetic medications , insulin NPH and regular and six antihypertensive medications .program coverage, MoH expenditures, and affordability, as follows:We analyzed five study outcomes related to FP 1) Monthly number of individuals who received at least one dispensing at AFP pharmacies;reais (Brazilian currency), including total MoH expenditure and total patient payments;2) Total monthly program expenditure in 3) Monthly percentage of expenditure paid by the MoH;4) Monthly expenditure per treatment (per capita), which is the total monthly expenditure divided by the number of individuals in the program; and.5) Average monthly out-of-pocket payment, which is the average amount paid by patients per treatment.reais; the exchange ratio during the study period was roughly 2 Brazilian reais to 1 US dollar [Annual inflation was a relatively stable 3 to 7% during the study period. We performed a monthly inflation correction for all financial outcomes . We repoS dollar .program sustainability, we estimated the level of expenditure that would be needed to fully cover all individuals in Brazil with DM and HTN through the FP program, and calculated the percentage that would represent of total MoH expenditures on medicines, yearly from 2006 to 2012.As an indicator of potential It has been demonstrated that most people with HTN and DM diagnoses, respectively 95 and 85%, are under pharmacological treatment in Brazil . Thus, iproxy for the number of individuals who should be under treatment [To create this sustainability measure, we first developed two measures estimating annual FP program utilization: a) Number of unique individuals with at least one dispensing within a given year; b) Average number of individuals receiving at least one dispensing per month, averaged across 12 months in a given year . We used these to construct annual and monthly estimates of program coverage, where the denominator of each measure is an estimate of the annual prevalence of each disease in Brazil, as a reatment . [Pesquisa Nacional sobre acesso e utilização e promoção do Uso Racional de Medicamentos – PNAUM) [Vigilância de fatores de risco e proteção para doenças crônicas por inquérito telefônico – VIGITEL [In addition to deriving yearly coverage estimates using FP program data, we also used the FP coverage estimates reported in the following surveys: National Program for Improving Access and Quality of Primary Health Care PMAQ-AB) , Brazili– PNAUM) , Surveil VIGITEL  and Nati VIGITEL .We did not adjust the monetary values used in this analysis for inflation, since we are comparing the proportions of expenditures in each year, and not actual expenditures themselves.Farmácia Popular interventions on affordability and MoH expenditures, we used ITS segmented linear regression models to determine the effect of the FP policy changes on the study outcomes. In estimating effects, ITS models adjust for pre-existing trends in the period before the policy change [prais , corc” command in STATA v12 [To analyze the impact of y change . SegmentTATA v12 , we analTATA v12 –22.Our ITS models included three segments, baseline and one for each of the two program periods, with 29, 22, and 23 monthly observations, respectively. The segmented regression model was specified as follow , 20:\dot = outcome variable in month t; β0 = level at the start of the observation period (intercept); β1 = baseline trend; montht = number of months from start of observation; AFPIIt = whether month t is after AFPII; β2 = level change after the AFPII; β3 = trend change after the AFPII; SNPt = whether month t is after SNP; β4 = Level change after the SNP; β5 = trend change after the SNP; et = residual error.In this model, time (t) is a continuous variable indicating time in months from the start of the observation period; YThe baseline segment was fit with an intercept and a variable estimating trend. We estimate each policy effect by a variable representing the change in level of the outcome immediately after the policy and a second representing the change in trend of the post-policy segment. Patients would experience changes in copayment only when they presented to fill a prescription after the policy change. We thus defined a post-policy implementation period of 2 months for the program to take effect; these periods were excluded in the ITS models so that we could estimate stable post-intervention effects. Additionally, we performed a sensitivity analysis considering the possibility of autocorrelation, assessing the significance of the Durbin-Watson statistic. We found that all outcomes have some level of autocorrelation, we compare the use of “prais” alone, “prais, var rhotype (dw)”, and “prais var, corc” . We madep < 0.05. To create single number summaries of policy effects, we calculated estimates of the relative changes in outcomes compared to expected values based on prior trends in April 2010 and February 2012, about 1 year after the two copayment interventions.We retained all parameters in the models regardless of statistical significance. We highlight the results with A total of 6,059,643 and 14,447,006 patients received medicines for DM or HTN, respectively, from the FP program. The mean age was 55 years for diabetes and 56 for hypertension patients, with females comprising about 60% of patients for both diseases. The southeast region represented the majority of patients in the program Table .Table 1Annual coverage of unique patients varied from 3.2 to 16.7% for DM and 1.6 to 9.7% for HTN, from 2006 to 2008, while average monthly coverage varied from 0.9 to 3.7% for DM and 0.5 to 2.4% for HTN, respectively. In 2009 and 2010, just after the AFP-II, coverage of unique patients decreased from 17.4 to 12.9% for DM and 9.8 to 7.1% for HTN, while average monthly coverage decreased from 3.9 to 3.2% for DM and 2.4 to 1.9% for HTN, respectively. After SNP, there was an impressive increase in coverage of unique patients that reached 40.6% for DM and 32.6% for HTN by the end of follow-up, while average monthly coverage reached 13.0 and 11.5%, respectively , resulted in an increase in patient cost sharing and associated drop out of users. The SNP, on the other hand, resulted in the opposite effects on these measures.Affordability, as measured by level of patient out-of-pocket payment, decreased after AFP-II but improved substantially when medicines were dispensed for free following the SNP policy. Considering that, prior to the FP program, patients paid the full price of medicines out-of-pocket at retail pharmacies, the simple existence of a government subsidy mechanism increased overall affordability over time. This is important since it has been shown that there is a high burden of medicines expenditures at household level in Brazil , despiteThe effects of the reduction of reference prices following the AFP-II policy change have already been discussed in other publications , 24. MorOverall, the policy changes under AFP-II showed deleterious effects from both the government and patient perspectives. Despite reducing MoH overall expenditures by reducing their level of cost sharing, the savings were proportional to the number of individuals still using the program. There were no changes in efficiency, since there were no significant reductions in expenditure per treatment. After implementation of the free medicines SNP policy, overall MoH expenditures increased significantly because of increased coverage. However, the cost per treatment was about 40% more efficient, so the relative change in expenditures after 1 year of SNP was 700%, despite a 1600% increase in the number of individuals treated. This improvement in efficiency may be related to gains from economies of scale in drug purchasing and substitution of generic drugs . We prevWe found large discrepancies in estimates of the size of the DM and HTN populations and FP coverage in the literature , 28–30, Considering the estimated financial outlay that would be required for MoH to cover all HTN and DM patients in the country through FP, it is likely that the program is sustainable only as a complementary policy and not as a substitute to distribution of medicines by the SUS. This is in accordance with the program’s original purpose, which was to cover those low-income individuals with key chronic illnesses who used private health care services but could not afford medicines. However, due largely to deficiencies in the SUS, the FP came to be used as a substitute channel for provision of medicines . DespiteThe study limitations include having no data on medicines that are not part of the FP, making it impossible to evaluate the impact of FP policies on the utilization of other medicines used to treat diabetes and hypertension. Furthermore, since we use data from surveys to estimate program coverage, we need to also consider the limitations of these sources of secondary data.Individuals are allowed to be in multiple treatments. Per-capita cost was considered the total cost of all medicines covered in the month divided by the number of individuals in treatment under FP. As we used data at individual level, we think this is a reasonable approach, assuming that the distribution of medicines users would be similar in SUS and FP.Despite increased investment over time in SUS strengthening, there was not any specific new policy or intervention change that occurred at the same point in time of the studied interventions , 12. The, especially in the context of the Brazilian economic and political crises.The FP greatly increased its coverage during the period analyzed and substantially improved the affordability of chronic illness treatment when medicines for diabetes and hypertension became free-of-charge in 2011. There was also an improvement in government expenditure efficiency, since the treatment cost per capita declined after 2011 due to purchasing leverage. However, overall MoH expenditures in the FP program increased substantially because of the increased number of users. FP appears to be sustainable only as a complementary policy, not as replacement for the SUS provision of medicines at primary healthcare facilities. Considering the magnitude of the FP program in the Brazilian pharmaceutical market, future studies should address the optimum treatment cost per capitaAdditional file 1. Analysis Syntax - Diabetes and Hypertension interrupted time series models.Additional file 2. Diabetes and Hypertension interrupted time series models - original, analyzed data and graphs.


ARID1A has a high mutation rate in GI cancers and its deficiency is correlated with the microsatellite instability (MSI) genomic feature of cancer. We investigated the correlation between ARID1A mutations and tumor immunity using three GI cancer genomics datasets by the bioinformatic approach, and found that diverse antitumor immune signatures were more highly enriched in ARID1A-mutated GI cancers than in ARID1A-wildtype GI cancers. The elevated immune activity in ARID1A-mutated GI cancers was associated with the higher tumor mutation burden and lower tumor aneuploidy level, as well as a higher proportion of MSI cancers in this GI cancer subtype. Moreover, we found that ARID1A-mutated GI cancers more highly expressed PD-L1 than ARID1A-wildtype GI cancers. The elevated antitumor immune signatures and PD-L1 expression could contribute to the more active immunotherapeutic responsiveness and better survival prognosis in ARID1A-mutated GI cancers than in ARID1A-wildtype GI cancers in the immunotherapy setting, as evidenced in three cancer cohorts receiving immunotherapy. Thus, the ARID1A mutation could be a useful biomarker for identifying GI cancer patients responsive to immunotherapy.Because traditional treatment strategies for advanced gastrointestinal (GI) cancers often have a limited therapeutic effect, immunotherapy could be a viable approach for the therapy of advanced GI cancers, considering the recent success of immunotherapy in treating various refractory malignancies, including the DNA mismatch repair-deficient GI cancers. However, only a subset of cancer patients currently respond to immunotherapy. Thus, it is important to identify useful biomarkers for predicting cancer immunotherapy response. The tumor suppressor gene  TP53 and KRAS mutations with immunotherapy response in lung cancer [Gastrointestinal (GI) cancers are prevalent and account for a large number of cancer deaths globally . Traditig cancer . ARID1A is frequently mutated in a wide variety of cancers [ARID1A mutations were often mutually exclusive with TP53 mutations in cancer, indicating that ARID1A may also play an important role in tumor suppression [ARID1A deficiency with GI cancers [ARID1A loss was associated with poor prognosis in GI cancers [ARID1A (AT-rich interaction domain 1A) is a component of the ATP-dependent chromatin remodeling complex SNF/SWI which is involved in transcriptional activation of genes normally repressed by chromatin.  cancers  and its  cancers  and brea cancers . Previoupression . Many st cancers ,18,19,20 cancers ,21.ARID1A deficiency may promote antitumor immunity, as well as PD-L1 expression [ARID1A deficiency with tumor immunity has been attributed to the elevated tumor mutation load caused by the deficiency of DNA mismatch repair that is regulated by ARID1A [ARID1A mutations with tumor immunity in GI cancers by a bioinformatic approach. We confirmed that ARID1A mutations are associated with elevated immune activity in GI cancers and demonstrated that the increased tumor mutation load and reduced tumor aneuploidy in ARID1A-mutated GI cancers may explain the more active immune signatures in ARID1A-mutated GI cancers versus ARID1A-wildtype GI cancers. Furthermore, we found that ARID1A-mutated cancers likely exhibited a better survival prognosis than ARID1A-wildtype cancers in the immunotherapy setting. The reason behind this observation could be that the elevated immune activity and PD-L1 expression promote the immunotherapy response in ARID1A-mutated cancers. This study is a bioinformatic analysis based on publicly available datasets, which furnishes new insights into the association between ARID1A deficiency and antitumor immunity in GI cancers.A number of studies have demonstrated that pression ,22,23. Ty ARID1A . In thisy ARID1A ,25,26, wARID1A-mutated GI cancers than in ARID1A-wildtype GI cancers    genes were upregulated in ARID1A-mutated GI cancers relative to ARID1A-wildtype GI cancers   consistently showed significantly higher enrichment levels in  < 0.05) A. Moreov < 0.05) B. These  < 0.05) C, suggesARID1A-mutated GI cancers versus ARID1A-wildtype GI cancers by GSEA [ARID1A mutations are associated with increased immune signatures in GI cancers.Furthermore, we found that numerous immune-related KEGG  pathways by GSEA . These pARID1A-mutated GI cancers showed significantly higher TMB  than ARID1A-wildtype GI cancers    A. In con < 0.05) A. The siARID1A mutation, TMB, and aneuploidy level with immune activity in GI cancers, we built a logistic regression model with the three predictors  for predicting immune cytolytic activity in GI cancers. As expected, both ARID1A mutation and TMB were positive predictors and the aneuploidy level was a negative predictor for immune cytolytic activity . These data again suggest that ARID1A mutations are associated with increased immune signatures in GI cancers.To correlate activity B. In allARID1A mutation and the MSI genomic feature in all three GI cancer cohorts   (ARID1A deficiency is associated with the MSI genomic feature and impaired MMR in cancer [P < 0.001) (P < 0.001) (P < 0.01) (ARID1A mutations lead to MMR deficiency or MSI thereby contributing to the elevated tumor immunity in GI cancers.A previous study has shown that ARID1A can promote mismatch repair (MMR) by interacting with MMR protein MSH2 . As a re< 0.001) A. Moreov< 0.001) B. These n cancer . As expe< 0.001) C. In con< 0.001) C. Furthe < 0.01) D. This iARID1A-mutated and ARID1A-wildtype GI cancers within the MSI-L/MSS subtype. We found that the correlations between ARID1A mutations and tumor immune signatures were significantly weaker in the MSI-L/MSS subtype than in all GI cancers in STAD-2 and COAD  . The ratios of pro-/anti-inflammatory cytokines and M1/M2 macrophages were significantly higher in ARID1A-mutated MSI-L/MSS STAD-2 than in ARID1A-wildtype MSI-L/MSS STAD-2   . The imm < 0.05) . These rARID1A-mutated GI cancers had significantly higher PD-L1 expression levels than ARID1A-wildtype GI cancers  (PD-L1 expression in GI cancers [ARID1A mutations and immune checkpoint blockade therapy response using three cancer immunotherapy (anti-PD-1/PD-L1/CTLA-4) response-associated cohorts (Allen cohort [ARID1A-mutated and ARID1A-wildtype cancers (melanoma) in the Allen cohort and Hugo cohort and found that ARID1A-mutated cancers had higher response rates than ARID1A-wildtype cancers  (ARID1A-mutated and ARID1A-wildtype cancers in the three cohorts. In the Samstein cohort (pan-cancer), ARID1A-mutated cancers had a significantly better OS than ARID1A-wildtype cancers     . Nevertheless, the correlation between ARID1A mutations and OS was unlikely to be significant in the three GI cancer cohorts  not receiving immunotherapy (ARID1A-mutated cancer patients respond favorably to immune checkpoint blockade therapy versus ARID1A-wildtype cancer patients and therefore are likely to have a better OS prognosis. The more active immunotherapeutic responsiveness in ARID1A-mutated cancer patients could be attributed to the elevated tumor immunity and PD-L1 expression in this cancer subtype.We found that  < 0.01) A. This i cancers . Furthern cohort , Hugo con cohort , and Samn cohort ). We com cohort) B. Moreov= 0.005) C. Moreov= 0.031) C. In thectively) C. Furthe = 0.01) . Supplemotherapy D. These ARID1A mutations had a significant positive association with tumor immunity in GI cancers. This significant association was attributed to the dysfunction of ARID1A in regulating MMR proteins that results in deficient MMR and MSI genomic features in GI cancers. Besides the elevated antitumor immune signatures, ARID1A-mutated GI cancers exhibited significantly higher PD-L1 expression levels than ARID1A-wildtype GI cancers that would enhance the immunotherapy response and accordingly result in a better survival prognosis in this cancer subtype. Thus, the ARID1A mutation could be a predictive biomarker for the response to anti-PD-1/PD-L1 immunotherapy, as evidenced by the present and other studies [Our bioinformatic analysis revealed that  studies ,23,33. ARID1A-mutated GI cancers versus ARID1A-wildtype GI cancers, identified by GSEA [Besides a number of immune-related pathways, many cancer-associated pathways were upregulated in  by GSEA . These c by GSEA ,35,36,37 by GSEA ,39,40,41ARID1A-mutated GI cancers. However, we found that immune signature scores were still significantly higher in ARID1A-mutated than in ARID1A-wildtype GI cancers within the MSI-L/MSS subtype . In addition, we obtained three cancer genomics datasets  ), STAD-2n cohort , Hugo con cohort , and Samn cohort ) containThe enrichment level of an immune signature (represented by a set of marker genes) in a sample was quantified by the single-sample gene-set enrichment analysis (ssGSEA) score . A totalARID1A-mutated GI cancers relative to ARID1A-wildtype GI cancers using a threshold of FDR < 0.05. The pathways common in all three pathway sets were defined as the significantly upregulated pathways in ARID1A-mutated GI cancers versus ARID1A-wildtype GI cancers.Based on the three GI cancer gene expression profiling datasets , we used GSEA  to identFor each tumor sample, we used the ABSOLUTE algorithm  to calcuARID1A mutation, TMB, and tumor aneuploidy levels in predicting tumor immune activity. The three predictors ARID1A mutation (mutated or wildtype), TMB , and tumor aneuploidy level (defined as the tumor ploidy score generated by ABSOLUTE [We used logistic regression to assess the contributions of ABSOLUTE ) were biARID1A-mutated and ARID1A-wildtype cancers in the three GI cancer datasets and the three cancer immunotherapy response-associated datasets. Kaplan-Meier survival curves were used to show the survival differences and the log-rank test was used to evaluate the significance of survival time differences. We performed the survival analyses using R programming function “survfit” in “survival” package.We compared OS between


The data presented here are related to the research article “Energy-environmental efficiency and optimal restructuring of the global economy”   Specifications TableValue of the data•A combination of a theoretical framework and a computer program forms a tool for the analysis and development of environmentally friendly policies.•The computer program estimates the energy-environmental efficiency of the global economy and determines the ways of its optimal increase.•The computer program allows for analysis of the group efficiency of different economic systems.1This article presents an R program that utilizes and further develops a novel stochastic data envelopment analysis with a perfect object method (SDEA PO) introduced in Vaninsky 2This program advances a novel SDEA PO method introduced in


Industrial relevance: This investigation showed the potential production of a pasteurized RTF formula, a niche product, with a reduced amount of allergenic β-Lg.Infant milk formula (IMF) is designed to mimic the composition of human milk (9–11 g protein/L); however, the standard protein content of IMF (15 g/L) is still a matter of controversy. In contrast to breastfed infants, excessive protein in IMF is associated with overweight and symptoms of metabolic syndrome in formula-fed infants. Moreover, the beta-lactoglobulin (β-Lg) content in cow milk is 3–4 g/L, whereas it is not present in human milk. It is considered to be a major reason for cow milk allergy in infants. In this respect, to modify protein composition, increasing the ratio of alpha-lactalbumin (α-Lac) to β-Lg would be a pragmatic approach to develop a hypoallergenic IMF with low protein content. Such a formula would ensure the necessary balance of essential amino acids, as 123 and 162 amino acid residues are available in α-Lac and β-Lg, respectively. Hence, in this study, a pasteurized form of hypoallergenic and low-protein ready-to-feed (RTF) formula, a new product, is developed to retain heat-sensitive bioactives and other components. Therefore, the effects of high pressure processing (HPP) under 300–600 MPa at approximately 20–40 °C and HTST pasteurization (72 °C for 15 and 30 s) were investigated and compared. The highest ratio of α-Lac to β-Lg was achieved after HPP (600 MPa for 5 min applied at 40.4 °C), which potentially explains the synergistic effect of HPP and heat on substantial denaturation of β-Lg, with significant retention of α-Lac in reconstituted IMF.  Infant milk formula (IMF) is intended to serve as a functional substitute for infants under 12 months of age. The World Health Organization (WHO) recommends absolute breastfeeding of infants for the first 6 months of age , which iMoreover, beta-lactoglobulin (β-Lg) represents about 50% of total whey as a major whey protein in cow milk. It is also considered a major allergen to infants despite having numerous functional and nutritional roles in adult human health. However, interestingly, this β-Lg is absent in HM. To address the cow’s milk allergy (CMA), researchers and manufacturers introduced partially hydrolyzed formula (pHF) and extensively hydrolyzed formula (eHF), which are also recommended by paediatricians to reduce early allergy manifestation . In pHF On the other hand, alpha lactalbumin (α-Lac) is a bioactive protein present in all mammal milk, which is regarded as a component of lactose synthesis with antimicrobial, prebiotic, and Ca-binding capacity . The α-lThe standard protein content of infant formula is still a matter of controversy since formula production aims to mimic HM . HM contPreviously, Huppertz et al.  describeTherefore, the aim of this work was to achieve a higher ratio of α-Lac to β-Lg through investigating their retention after HPP at different pressure–temperature–time combinations in reconstituted IMF, fortified with α-Lac. For comparison, we also performed high-temperature short-time (HTST) pasteurization at 72 °C for 15 and 30 s, and thereafter measured the concentration of α-Lac and β-Lg. The combined effect of pressure and temperature on the kinetics of denaturation of both proteins was also analyzed.Bovine α-Lac powder (native form) was generously donated by Davisco Foods International, Le Sueur, MN, USA. The protein content and α-Lac level in the powder were more than 95% and 90%, respectively. Spray-dried IMF powder  was reconstituted into cooled, boiled milli-Q water, as per the instructions of the manufacturer. Finally, 100 mg of α-Lac was added into 100 mL of reconstituted IMF, and a magnetic stirrer was used for gentle mixing.HTST treatments at 72 °C for 15 and 30 s were performed in duplicate, using a sample (15 mL) in a copper tube, then immersing it in a water bath (72.2 °C). A thermocouple (K-type) was inserted at the geometric centre of the copper tube  to record the temperature–time profile using a data logger. The sample temperature increased sharply within 30 s to achieve the steady-state temperature. The sample tube was immediately transferred to an ice bath after treatment. It is to be noted that the determination of α-Lac and β-Lg content following ELISA  requiresA QFP 2L-700 HPP unit  was used to perform the combined pressure–heat treatments. The equipment has a 2 L cylindrical stainless steel pressure treatment chamber with two internal thermocouples for monitoring the temperature. The system also includes a heating system, water circulation, and a pumping system, along with a computer-operated control system. Distilled water was used as a pressure transmission fluid, and isostatic pressure transmission resulted in a uniform pressure in all directions .In HPP treatments, vacuum-packaged 15 mL samples were placed into the treatment chamber and immediately cooled in an ice water bath after treatment. HPP of 300 MPa (10 and 20 min), 400 MPa (10 and 20 min), 500 MPa (5 and 10 min), and 600 MPa (1 and 5 min) were applied at different temperatures, ranging from ambient temperature to ~40 °C. The treatment time refers to the duration of steady-state pressure conditions, as programmed in the operating system. The temperature increase was about 2.3 °C/100 MPa, due to the adiabatic heating during pressurization, and the decompression time was <20 s in all treatments.However, similar to Evelyn and Silva , the temTeff)”, to represent the entire treatment period (Equation (1)).T (K) is the absolute temperature recorded at a given time interval (t), E is the activation energy (kJ/mol) of the specific biological or chemical reaction, R is the gas constant (8.314 J/mol/K), and n is the number of temperature recording points measured at equal time intervals.Transient temperature change significantly impacts all chemical and biological reactions, due to its exponential effect on them. The average temperature during the steady-state pressure phase does not represent the accurate representation of the treatment temperature. For instance, as shown in Teff.From now on, the temperature in HPP treatments referred to in this paper will be the Defatted whey supernatant was prepared from both HTST- and HPP-treated samples by centrifuging them at 9000 g for 15 min at 4 °C. Then, the whey portion was obtained by adjusting the pH to 4.6 using 8 M acetic acid, followed by centrifugation at the same conditions. The pH of the supernatant was readjusted to 6.8 using 3 M NaOH.ct and co represent the concentrations of α-Lac or β-Lg after and before treatment, respectively.An enzyme-linked immunosorbent assay (ELISA) method was followed to measure the concentration of α-Lac and β-Lg. The sandwich ELISA was performed using bovine α-Lac and β-Lg Quantitation Kits  and an ELISA Starter Accessory Kit , purchased from Bethyl Laboratories, TX, USA. The ELISA plates were coated, and supplied bovine α-Lac and β-Lg were diluted to obtain standard curves, following the manufacturer’s protocols. A multimode plate reader with associated software  was used to read the absorbance of ELISA plates at 450 nm. The unknown concentration of α-Lac and β-Lg of treated samples was obtained in duplicate from the standard curves. Retention of α-Lac and β-Lg was calculated as a percentage of α-Lac and β-Lg in untreated samples, respectively, as given in Equation (2).Ea) for α-Lac and β-Lg denaturation was calculated using the results obtained from HPP-treated samples. The denaturation of α-Lac and β-Lg with time after HPP treatment can be described by the general rate equationk is the rate constant, c is the concentration of α-Lac or β-Lg, and n is the order of reaction.Activation energy (n = 1), the integration of Equation (3) givesIt has been reported that HPP denaturation of α-Lac follows first-order kinetics in whole milk . For firr2), and the value of the ordinate intercept b  appears close to zero. The slopes of the lines obtained correspond to the rate constant (k).The semi-logarithmic plot of Equation (4) gives a straight line with high coefficients of correlation (n ≠ 1), the integration of Equation (3) givesFurthermore, the reaction kinetics for β-Lg in HPP was also reported as a second-order reaction by Anema et al.  in skim t = 0) should be 1 if the treatment follows the estimated reaction order. The rate constant (k) is obtained from the slope of the lines. The Arrhenius equation relates the treatment temperature and the rate constant of a denaturation process as given in Equation (6):A is the pre-exponential factor, Ea is the activation energy, R is the universal gas constant, and T is the absolute temperature.The graphical representation of Equation (5) yields straight lines, and the ordinate intercept b  gives a linearized form asT) determines the effect of temperature on the rate of constant (k). The gradient of Equation (7) is equal to –Ea/R, and thus the activation energy (Ea) is calculated.The graphical representation of Equation (7) . The level of significance was set at Teff for HPP treatments on α-Lac and β-Lg was calculated using Equation (1) and tabulated in Teff was significantly lower than the maximum temperature in HPP (Tmax), which further establishes the application of the concept of Teff in denaturation studies.In this study, Ea) for α-Lac and β-Lg denaturation was calculated using the results obtained from HPP treated samples following k) at different temperatures and pressures, and to calculate the Ea. Experimental data obtained in this work and their graphical representation in n) as 1 and 2 for α-Lac and β-Lg, respectively. These orders consistently produced reasonably straight lines with good correlation of coefficients (r2 > 0.98) and agree well with previous studies [Activation energy  for the denaturation of α-Lac and β-Lg, respectively. Ea indicates the energy barrier that a protein is required to overcome to take part in a reaction. Values of Ea in β-Lg denaturation compared to α-Lac. These results correspond well with the previous studies investigated by Huppertz et al. [Furthermore, z et al.  for wholz et al.  for skimEa values obtained in this work for β-Lg denaturation differ to some extent from those reported earlier by Anema et al. [Ea as 103.8 and 114.35 kJ/mol for 500 and 600 MPa, respectively; twice higher than the reported values of this work.However, a et al.  from HPPEa could be attributed to the dissimilarity in treatment media, treatment duration, and the estimation of treatment temperature, since this study considered the temperature attained during the pressure come-up time.IMF is a complex food containing a variety of ingredients. Therefore, this difference in In this study, the retention of α-Lac and β-Lg after HTST and HPP was measured by ELISA and compared thereafter . Before The results found in this work show the potential route to develop an HPP-treated pasteurized RTF hypoallergenic formula because of having a higher ratio of α-Lac to β-Lg. In addition to this, such a formula would ensure the required amino acid balance in the treated product due to the α-Lac supplementation, which may also compensate the lower contribution of heavily denatured β-Lg in the amino acid profile. Thus, this work streamlines the possibility of manufacturing a hypoallergenic and low-protein pasteurized RTF formula. However, further investigation in post-treatment analysis  of HPP-treated formula is highly recommended to commercialize this research. Besides, the shorter shelf life at refrigerated conditions of this pasteurized product than that of the sterilized RTF formula would result in slower progress in gaining market. Moreover, HPP is still limited by its batch operation although the recent patent-pending concept of Hiperbaric, the HPP equipment manufacturer, to process liquid foods up to 10,000 L/h before bottling (aseptic packaging) is being considered as a promising innovation to address HPP’s batch operation .Our results demonstrated the synergistic effect of HPP and heat on the substantial reduction of β-Lg in reconstituted IMF added with α-Lac. Compared to HTST, the HPP treatment at 600 MPa for 5 min applied at 40.4 °C achieved the higher ratio of α-Lac to β-Lg. Overall, the pronounced reduction of β-Lg due to the combined effect of HPP and heat confirms the possibility to manufacture a hypoallergenic and low protein RTF formula, a niche product. Further investigation is necessary to explore the post-treatment effects on physicochemical and rheological properties, along with microbial studies.


Trypanosoma cruzi, which causes Chagas disease, is a significant health threat in many countries and affects millions of people. Given the magnitude of this disease, a broader understanding of trypanocidal mechanisms is needed to prevent and treat infection. Natural endoperoxides, such as ergosterol peroxide, have been shown to be toxic to parasites without causing harm to human cells or tissues. Although prior studies have demonstrated the trypanocidal activity of ergosterol peroxide, the cellular and molecular mechanisms remain unknown. The results of this study indicate that a free-radical reaction occurs in T. cruzi following ergosterol peroxide exposure, leading to cell death. Using a combination of biochemical, microscopic and in silico experimental approaches, we have identified, for the first time, the cellular and molecular cytotoxic mechanism of an ergosterol peroxide obtained from Pleurotus ostreatus (Jacq) P. Kumm. f. sp. Florida. Trypanosoma cruzi (Tc) is a hemoflagellate parasite that causes American trypanosomiasis, also known as Chagas disease [ disease . Althoug disease , it rema disease . Another disease ,5. Tc is disease . Every y disease ,8.Triatoma bite a human. After ingesting the human’s blood, the insect defecates on the skin, and the protozoa passes from the insect’s infected feces to the bite site. Infection can also occur through protozoa contact with mucus membranes or by the ingestion of contaminated food. In addition, Tc may be spread during blood transfusions or organ transplants [The clinical manifestations of Chagas disease include chronic cardiomyopathy and digestive or neurological alterations that require specialized medical treatment. The infection often occurs when one of several species from the genus nsplants . In a smnsplants .To address the health threat posed by this parasitic disease, two well-known trypanocidal drugs are currently used in clinical practice: nifurtimox and benznidazole ,11. ThesPleurotus ostreatus, through a mechanism that involves the target CYP51 protein necessary for triggering a free radical reaction that perturb cell organelles.Several studies have demonstrated the antiparasitic efficacy of compounds that have been isolated from plants and mushrooms. Endoperoxides, such as artemisinin and ergosterol peroxide, have anti-oncogenic and anti-proliferative properties . ErgostepropKa algorithm at pH 7 [The crystallographic structures for computational docking in this work are listed in  at pH 7 . All cryGasteiger charges were added and non-polar hydrogens merged. In addition, 27 crystalline protein structures and 3 homologous models from Tc were selected for docking experiments using Autodock Vina within Autodock 4.2 program software. All docking experiments were carried out in triplicate and Ki was estimated.Inverse docking assays were performed using Autodock v.4.2  and AutoKd values were calculated from sensorgrams sets using software BIAevaluation v.4.1 . Refractive indexes of compound samples and solvent contained run buffer were measured using a precision refractometer RX-5000 . The following reagents were obtained from manufacturer: HBS-N buffer , 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC), N-hydroxysuccinimide (NHS), 10 mM acetate buffer (pH 4.5), Ni-NTA-agarose , Bacto-Tryptone, Bacto-Peptone and Bacto-Yeast extract , and Bio-Gel HTP . Other analytical grade reagents were obtained from local suppliers.cDNA for protein expression was isolated using a “DNA-VK” kit of reagents for DNA isolation developed at the Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus. The surface plasmon resonance (SPR) study of intermolecular interactions was performed using the optical biosensor Biacore 3000 , operating on a surface plasmon resonance effect. The SPR signal was recorded independently in each channel of the biosensor in resonance units  as sensorgrams showing its changing in time. All experiments were performed at 25 °C using standard optical chip CM5 coated with the layer of carboxymethylated dextran. Escherichia coli strain HMS174(DE3) carrying the purified and isolated fragment in 12% PAAG gel, Tc cDNA fragments. Previously, the cDNA fragment was produced using GGGGTTGCCTATGCTGCC and CCCCAACGGATACGACGG, as the former forward and latter reverse PCR primers. The PCR amplification was done by using the following conditions: for 5′ at 94 °C, annealing for 1′ at 60 °C, followed extension for 1.5′ at 72 °C, for 30 cycles, and as extension for 10′ at 72 °C as previously described [C. albicans, cDNA samples were isolated from a patient exhibiting resistance to antifungal drugs, and were used for obtaining CYP51. Four amino acid substitutions  were found in the case of an C. albicans amino acid sequence [C. albicans and T. cruzi, the immobilization of CYP51 on the surface of the optical chip CM5. HBS-N buffer was used as a running buffer. Carboxyl groups of dextran were activated by passing the mixture of equal volumes of 0.2 M EDC and 0.05 M NHS for 7 min at a flow rate 5 µL/min followed by washing with a running buffer at the same speed for 1 min. Immobilization of CYP51 was performed by passing the protein solution (20 µg/mL) in 10 mM of acetate buffer (pH 4.5) for 10 min at a flow rate of 5 µL/min. After that, the chip surface was washed with running buffer for 1 h at a flow rate 5 µL/min. The effectiveness of protein immobilization was on average 10,000 RU.For the CYP51 Tc expression vector inserted into a pCWori vector  between escribed . CYP51 psequence . In bothPleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida was employed. Fruiting bodies were cultivated in barley straw as described in a previous report [A strain CP-26 of s report .Pleurotus ostreatus were taxonomically identified and dehydrated in an oven at 60 °C with airflow for 30 h. Distilled solvents were used. Chromatographic columns were dry packed with silica gel , and glass plates of silica gel (Merck 60 GF254 of 0.2 mm thickness) were used for thin-layer chromatography. Iodine vapors were used to crate spots. The melting point was determined by a Fisher–Johns apparatus and was not corrected. +H experiments were performed using a Varian Gemini 2000 apparatus Palo Alto, California  were obtained and identified as ergosterol peroxide based on its NMR spectroscopic data and compared to an authentic sample same as previously described [Fractions were eluted with hexane: EtOAc (7:3) of the chromatographic purification of the ethyl acetate extract from the 3.367 kg of dry fruiting body of Triatoma infestans. A liver infusion tryptone broth-LIT supplemented with 10% fetal bovine serum was used as a growing media [The strain used for the present study was the epimastigotes form of Tc Y in all experiments isolated from vector ng media .6 on a 50 mL volume until reaching the log phase epimastigotes measured by direct counting with a haemocytometer by mL. The cells were washed with sterile Phosphate-buffered saline (PBS) twice, each time centrifuging at 2000× g over 10′. The pellet was dissolved in an LIT complete media until reaching a density of 4 × 105 parasites per ml. An amount of 500 µL of cellular suspension was added to a 96-well plate and incubated until analysis. The parasites were incubated for 24 h at 28 °C and also treated with ergosterol peroxide (40 µM) that was previously dissolved in less than 1% dimethyl sulphoxide (DMSO). A negative control was added with H2O2, and another with no treatment other than ultrapure MQ H2O and a third one with a mix of ultrapure MQ H2O and Dodecyl Bisulfate DMSO in a concentration of less than 1%. All conditions were performed in triplicates and repeated three times independently and then to processed by a statistical analysis. For the carbonylation analyses, Trypanosomes were lysed and the quantification of carbonyl groups were determined. After treatment, a single ml mixture of reaction blended with 1 ml of 20 mM of 2,4-dinitrophenylhydrazine in 2 M HCl, which forms a stable dinitrophenyl product that is then read spectrophotometrically. The solution was incubated for at 25 °C and subsequently a 15% trichloroacetic acid was added to the tube. Plates were kept on an ice bucket for 10 min to right after spin down of the cells at 12,000× g for 20 min. The remaining protein pellet was kept and washed twice with an ethanol/ethyl acetate (1:1 v/v) solution and dissolved in 1.5 mL of 8 M guanidine-HCL (pH 7.4) and finally vortexed for suspension. The solubilized hydrazones were measured at 370 nm. The concentration of carbonyl content was counted by using the molar absorption coefficient 22,000 M−1 cm−1 D. Hartley, [The Tc culture strain Y started with a cell density of 1 × 10Hartley, . The conTc samples were treated with 10 µM ergosterol peroxide for 12 h were fixed to be analyzed using a JEOL JSM-7600F scanning electron microscope  at a working distance of 15 to 21 mm and a voltage of 1.2 to 5.0 kV after a preliminary wash in distilled water, followed by dehydration in a series of ethanol solutions of increasing concentration , air drying on 0.5″ aluminum mounts and sputter coating with gold-palladium using a Polaron SC7640 High Resolution Sputter Coater ; some micrographs were obtained using a Philips XL-30 environmental SEM (Pl. 7 and 8). Line drawings were made from digital images.Tc samples were treated with 20 µM ergosterol peroxide for 24 h were fixed to be analyzed. Images of confocal microscopy were obtained by using a Leica SP8 laser scanning confocal microscope , equipped with a HC PL APO CS2 63x/1.40 oil immersion lens , at 50% argon laser. A 545 nm Helium–Neon laser line was used for excitation of propidium iodide with a 645 nm emission filter.Tc samples were dosed with 20 µM ergosterol peroxide and after 24 h were fixed to be analyzed. Pellets were processed for standard electron microscopy . BrieflyC. albicans CAF2-1 [C. albicans SC5314 (ATCC MYA2876), C. glabrata (ATCC 2001) and S. cerevisiae BY4741. Yeast cells were cultivated overnight in 250 mL flasks in 50 mL of yeast extract–peptone–dextrose, YPD medium at 30 °C. A preculture was prepared by diluting the overnight culture to an optical density OD620 of 0.2 in 25 mL of YPD medium, and the yeast cells were allowed to grow for 3 h so that they reached the exponential growth phase. The OD was determined in a 180-ll sample volume with the microtiter plate reader lQuant . The working culture was prepared by diluting the preculture to an OD620 of 0.2. After cultivation for another 3 h, the OD620 was recorded, the whole suspension was taken into a Falcon tube, and cells were harvested by centrifugation (Eppendorf centrifuge 5804 R) at room temperature at 5000 rpm for 5 min and washed carefully three times with phosphate-buffered saline (PBS). The washed cells were resuspended in 1 mL of PBS. To test the electrochemical activity of nonliving organisms (dead cells), the working culture was autoclaved at 121 °C for 20 min. The cells were harvested into lysogeny broth, LB broth media and resuspended in a 96-well plate in a concentration of with 105 CFU/mL to be treated with the ergosterol peroxide using the final concentrations of 1, 5, 10, 20, 40, 70 µM. The C. albicans yeast cells were grown at 30 °C for 3, 18, 24 and 48 h and were harvested, resuspended to be counted in a Neubauer camera MilliporeSigma  for final estimation of concentration and statistical analysis.The following microbial strains were used: s CAF2-1 , which wKd), sets of sensorgrams were obtained by injection of compound solutions for 6 min at a flow rate of 30 µL/min in the range of 10–75 µM concentrations. Dissociation of the complexes was recorded for at least 30 min at a flow rate 30 µL/min. After that, the sensor chip surface was regenerated by injections of the regenerating solution  for 17 s at a flow rate 35 µL/min.Stock solutions of the compound were prepared by dissolving it in DMSO at a concentration of 5 mM. These stock solutions were further diluted by HBS-N buffer up to a concentration of 100 µM. To obtain solutions with lower concentrations of compounds, HBS-N buffer with the addition of DMSO in the same concentration as that of 100 µM solution was used. To prevent bulk-effects during SPR analysis DMSO contained, HBS-N buffer was used as a run buffer. The alignment of DMSO concentration in HBS-N buffer and compound samples was performed by value of refractive index. To determine the equilibrium dissociation constant of compound complex with immobilized CYP51A1  homologous of this protein. As shown below, there is a concordance at the effect in association and disassociation and ergosterol peroxide concentration and resonance with the Tc version of CYP51; there was no correlation effect with the C. albicans counterpart  and nterpart . Interes74 µg/mL .To examine the ergosterol peroxide activity from a biochemical perspective, we tested its cytotoxicity on the Tc hemoflagellate. These results suggest that the compound’s trypanocidal activity is due to the breakage of its oxygen-oxygen bond, unleashing oxidation onto lipids, proteins and nucleic acids. To determine if this free radical damage was caused by the rupture of the peroxide bridge, we measured the post-treatment carboxyl output using a carbonylation assay . ConfocaSignificant concentrations of carbonylated compounds were observed in the Tc hemoflagellate samples that were treated with ergosterol peroxide compared with those that were treated with ergosterol or the vehicle (dimethyl sulfoxide) and compared to the samples that received no treatments. The detection of carbonylated proteins after ergosterol peroxide treatment suggests that the trypanocidal activity is related to the peroxide bridge, whose homolytic cleavage triggers oxidation.To examine the evidence of cellular membrane disruptions, we treated Tc hemoflagellate specimens with a cytotoxic concentration of ergosterol peroxide (10 µM) and incubated the specimens for 12 h. Enlarged extracellular vesicles were observed in all the analyzed specimens that were treated with ergosterol peroxide compared to the non-treated specimens A. The siSimilar experimental conditions were used with a different microscopy approach to evaluate cell viability and to identify the active site of the ergosterol peroxide within the Tc cells. The ergosterol peroxide was applied for a longer period, and confocal electron microscopy was used to observe the Tc cells. The Tc samples were treated with a 20 µM cytotoxic concentration of ergosterol peroxide, stained with propidium iodine, incubated for 24 h and then analyzed with confocal electron microscopy. The fluorescence was detected by probing the in situ interactions between the ergosterol peroxide and the Tc nucleic acids, the result of nuclear and cytoplasmic membrane disruptions caused by the ergosterol peroxide.These interactions were made evident by the positive signal produced by the binding of the propidium iodine and the Tc nucleic acids A,B.After a longer ergosterol peroxide exposure, we observed changes in the Tc nuclear and cytoplasmic membranes. In addition, the Golgi apparatus disappeared .The biological effects of ergosterol peroxide  range from immunosuppressive to cytotoxic to anti-microbial and include anti-plasmodial, anti-leishmanial, anti-amoebic and trypanocidal activities ,36,49,50Trypanosoma brucei [The key identified proteins included enzymes that are involved in sugar and lipid metabolism, but the highest docking predictions were for enzymes that are indispensable in sterol metabolism, rather than glucose metabolism. The top match that was predicted in silico to have molecular interactions with ergosterol peroxide was CYP51, a sterol demethylase and lipid transporter. This protein has been recently proved to be essential for a brucei . CYP51 cC. albicans version of CYP51. These results are consistent with our previous studies that have shown ergosterol peroxide cytotoxicity in Tc but not in C. albicans. The Candida genus is capable of synthesizing sterols, so these organisms do not depend on sterol intake from the environment. Tc may also synthesize its own sterols, as it is able to grow in potato-dextrose agar broth, which lacks ergosterol. The resistance to EP by Candida albicans might be due to differences in CYP51 aminoacid sequence.The molecular docking results for CYP51 were confirmed with SPR, which indicated a significant interaction with the Tc version of CYP51 and no evidence of affinity with the However, specific sterols are required to maintain Tc viability and proliferative capacity during the parasite’s infection and life cycle. Sterols are also indispensable for several membrane-related cellular functions, including the integration of hydrophobic domains, membrane fluidity and permeability, and the formation of ion channels, enzymes and cellular compartments ,54. CYP5The structural similarity of ergosterol peroxide and ergosterol explains both our initial in silico results as well as the cytotoxic effect of ergosterol peroxide on Tc. The structural resemblance of cholesterol, ergosterol and ergosterol peroxide further explains the ability of ergosterol peroxide to incorporate into the parasite’s plasma membranes.The reverse docking results indicated that ergosterol peroxide had a high affinity with transport enzymes and sterol synthesis proteins (CYP51). This might explain how ergosterol peroxide is dispersed across the cytoplasm. Enzymes involved in transport and sterol synthesis were identified as having a high probability of interacting with ergosterol peroxide. Consequently, once ergosterol peroxide enters a cell, it may disrupt metabolic output and lipid-mediated transport by interacting with CYP51, an important sterol synthesis enzyme. Notably, CYP51 is a primary drug target for several microbial infections in both animals and plants; its potential pharmacological role should not be overlooked.The transmission electron microscope images revealed that the predominant changes following ergosterol peroxide treatment occurred at the nuclear, kynetoplasm and cytoplasmic membranes. The cytoplasmic membrane, in particular, exhibited lineal and equidistant pores. After ergosterol peroxide treatment, the nucleic acid content was disseminated throughout the cytoplasm, as shown by the propidium iodide staining.These results were further confirmed by the carbonylation test, which demonstrated an increase in carbonyl groups after ergosterol peroxide treatment. These carbonyl groups are generated by free radical reactions, which can be produced through the homolytic cleavage of peroxide bonds and which may be enhanced by the transition metals present in Tc. The ergosterol peroxide mechanism of action may involve a reactive oxygen species that causes the membrane alterations we observed.Biological systems are constantly exposed to reactive oxidants that are either the result of endogenous production or are externally delivered . These reactive oxidants cause molecular transformations and oxidative damage. Inside cells, proteins are the primary targets for oxidative damage due to their abundance and naturally-engineered reaction rates, but the high reactivity of oxidants means their detrimental effects can be traced to all molecular components, including proteins, lipids, DNA, RNA, carbohydrates and even antioxidants . OxidatiPrevious studies have reported that iron catalyzes the formation of alkoxy radicals from hydroperoxides of cholesterol through the Fenton reaction. Based on the results of the present study and the close similarity between ergosterol, cholesterol and ergosterol peroxide, we propose that a Fenton mechanism is likely involved in the homolytic cleavage of the peroxide bridge. Since the active site of CYP51 contains a heme iron, a radical burst could be triggered by its interaction with ergosterol peroxide, which would act as an enzymatic inhibitor generating long-lived radicals or carbonyl moieties to name a few examples . The funOur results indicate that a specific lipid/protein oxidation is involved in ergosterol peroxide’s interaction with CYP51. Ergosterol peroxide’s high affinity for interacting proteins allows it to deliver a deathly cargo. This mechanism links alkoxy radical formation to the production of lipid aldehydes and protein carbonylation and explains the rapid (within 24 h) cytotoxic effect of ergosterol peroxide on Tc. Ergosterol peroxide promotes proteomic and lipid carbonylation, providing evidence of the development of free radicals and a specific directed mechanism. In the Tc cell membrane, when ergosterol is replaced by ergosterol peroxide, the peroxide moiety is broken, triggering a series of free radical reactions . The alk


Many health programs can assess coverage using standardized cluster survey methods, but estimating the coverage of nutrition programs presents a special challenge due to low disease prevalence. Used since 2012, the Semi-Quantitative Evaluation of Access and Coverage (SQUEAC) employs both qualitative and quantitative methods to identify key barriers to access and estimate coverage of therapeutic feeding programs. While the tool has been increasingly used in programs, the validity of certain methodological elements has been the subject of debate.We conducted a study comparing a SQUEAC conjugate Bayesian analysis to a two-stage cluster survey estimating the coverage of a therapeutic feeding program in Niger in 2016.We found that the coverage estimate from the conjugate Bayesian analysis was sensitive to the prior estimation. With the exception of prior estimates produced by an external support team, all prior estimates resulted in a conflict with the likelihood result, excluding interpretation of the final coverage estimate. Allowing for increased uncertainty around the prior estimate did not materially affect conclusions.SQUEAC is a demanding analytical method requiring both qualitative and quantitative data collection and synthesis to identify program barriers and estimate coverage. If the necessary technical capacity is not available to objectively specify an accurate prior for a conjugate Bayesian analysis, alternatives, such as a two-stage cluster survey or a larger likelihood survey, may be considered to ensure valid coverage estimation.NCT03280082. Retrospectively registered on September 12, 2017.The online version of this article (10.1186/s12963-018-0167-3) contains supplementary material, which is available to authorized users. Valid estimates of nutrition program coverage in low- and middle-income countries are needed to assess program performance and inform allocation of resources and policy.The Semi-Quantitative Evaluation of Access and Coverage (SQUEAC) methodology represents a step forward in coverage assessment of therapeutic feeding programs by providing a routine means by which to simultaneously identify barriers to accessing care and estimate program coverage.The SQUEAC conjugate Bayesian analysis proposed for coverage estimation can be a technically demanding method that depends on accuracy of the prior estimate. Methods to develop a final prior estimate have been suggested, but there is little evidence regarding the validity of their application in capacity-limited settings. Subjective and overly certain prior estimates produced from these methods may render the final coverage estimate invalid. The appropriate technical capacity is necessary during prior estimation to ensure informative results. More research to review and validate methods to produce accurate prior estimates may be needed.Program managers should decide on the appropriate method to be used based on information needed and availability of appropriate capacity to implement the SQUEAC method.Seventeen million children are estimated to suffer from severe acute malnutrition (SAM) each year  and expeCoverage of many health programs can be assessed using cluster survey methods, but coverage estimates for nutrition programs present special challenges due to the low prevalence of disease and often limited resources for data collection. Since 2012, the Semi-Quantitative Evaluation of Access and Coverage (SQUEAC) methodology has been widely used to assess the coverage of SAM treatment programs . In contWhile SQUEAC was developed as a practical, low-cost tool for programs to assess the quality of therapeutic feeding programs, questions have been raised over the optimal balance between validity and operational feasibility of the conjugate Bayesian analysis . In partThis study was conducted in the Madaoua health district in the Tahaoua region of southern Niger. Madaoua is situated in the Sahel region and is characterized as largely agricultural with a high seasonal burden of acute malnutrition. In support of the Ministry of Health, Médecins Sans Frontières (MSF) has been operational in the region since 2006, supporting one inpatient and six outpatient centers for the management of SAM with community surveillance and outreach teams operational in 80 villages.The SQUEAC methodology was designed to be used on a regular basis to monitor program performance, identify barriers to service access and uptake, and estimate coverage . It usesPrior estimates of coverage were estimated using five recommended methods  and threScenario 1 represented a “broad program staff implementation” that included all methods for prior estimation but did not include estimates from the external support team. Scenario 2 represented a more “basic program staff implementation” that did not include weighted scores, which may be considered to be more time- and resource-intensive methods that are less likely to be utilized in constrained settings, nor estimates from the external support team. Scenarios 1 and Scenario 2 are likely to represent common field conditions, where recently trained program staff  weight boosters and barriers to develop a prior without input from experts. Scenario 3 represents the case of “external implementation” when expert opinion is available to weight boosters and barriers and included only prior estimates developed by the external support team.It is recommended to triangulate credible prior information to develop a single, final prior estimate for use in the conjugate Bayesian analysis . We therCurrent guidance suggests greater uncertainty be assigned to the prior estimate when the position of the prior mode is not certain, or the priors from various methods and sources show high variability. We therefore allowed standard (± 25%) and greater uncertainty (± 35%) for each prior estimate.Village selection was made using spatial stratified systematic sampling. A complete list of villages was sorted by geographically delimited health area, and villages were selected systematically to yield a reasonably even spatial sample across health areas. Within the selected villages all SAM cases were found using exhaustive active and adaptive case-finding, a census sampling method  in a two-stage cluster survey was estimated using Eq. The sample size required for the SQUEAC likelihood survey (Stage 3) depended on the prior distribution (described by the mode and two shape parameters α and β) and the precision  desired for the posterior estimate Eq. . DependiSingle coverage was estimated using the observed number of uncovered SAM cases, observed number of covered recovering cases, and an estimate of uncovered recovering cases  resulted in a conflict with the likelihood result, invalidating any interpretation of the resulting posterior estimate of coverage  was 42% and was the only prior estimate that did not conflict with the likelihood data. The final coverage estimate from the external support team was 30.3% .Table The SQUEAC methodology was developed to allow frequent evaluation of nutrition programs by program staff; the method produces qualitative insights into barriers to care and quantitatively estimates program coverage. In this analysis, we compare coverage estimates obtained through a two-stage cluster survey and the SQUEAC methodology. We found SQUEAC to be a technically demanding method for estimation which would appear to require a high level of objectivity possible with support from an experienced external or program team.A limitation common to Bayesian methods, final coverage estimates can be biased if the prior estimate is inaccurate  and strong , and if the sample size used for the likelihood survey is small . In these cases, the prior may dominate the analysis and result in a conflict that invalidates the final coverage estimate. Methods to develop a final prior estimate are presented in the technical guidance . These mUnfortunately, there is no way to a priori assess the accuracy of the prior, and this becomes evident only during formal testing for conflicts at the time of final analysis. In the case of conflict, current guidance instructs that the prior should be redefined in Stage 1 and Stage 2, and the Stage 3 likelihood survey conducted again. However, resource constraints rarely allow for this repetition. A practical alternative may be for the Stage 3 likelihood survey data alone to inform a final coverage estimate. The sample size for a Stage 3 likelihood survey, however, is small, and any resulting estimate would be less precise than desired. Therefore, when there is uneasiness around the prior development process, a larger likelihood sample size may be considered. The larger likelihood sample size would provide more precision to final estimates in the event that the final Bayesian estimate was not valid. While it is also recommended to increase the uncertainty around the final prior estimate  in such cases, doing so in this analysis did not prevent the resulting conflict.To avoid the potential for biased estimation, implementers must be realistic about the position of the prior and the level of certainty. In our analysis, individual prior estimates were consistently higher than the population survey estimates of coverage, and estimates varied substantially by source. This is consistent with the potential for those intimately involved with the program  to positively assess their own program, with recent evidence noting weighting and program assessment can be sensitive to the participant group . MultiplOur findings suggest several strategies that could ensure appropriate and quality implementation of the SQUEAC methodology. To begin, the selection of the appropriate method to describe program performance should depend on program needs and priorities. If qualitative information on barriers to access and a coverage estimate are required (both important for program assessment and improvement), the complete SQUEAC methodology may be implemented. The process of prior development may begin with review of relevant coverage estimates from similar programs or contexts. This may provide some external perspective and help orient initial estimates as the team gathers and triangulates additional secondary program data and qualitative information. If there is uncertainty in the prior, a larger likelihood survey may be allowed for to increase the precision of the likelihood estimate. This will increase costs but provide more protection against the possibility of not having a final coverage estimate to report if a conflict arises that invalidates the posterior coverage estimate in the conjugate Bayesian analysis. Implementation of the complete SQUEAC methodology with a larger likelihood survey may be particularly of interest in the first assessment of a new program, as the SQUEAC methodology ensures programs engage with communities and allows for a comprehensive understanding of community structures, communication, and health-seeking behavior that is critical for building an effective community mobilization strategy. If there is good certainty of the prior estimate, for example one which is informed by a quality SQUEAC assessment recently conducted, then the complete SQUEAC methodology with standard sample size for the likelihood survey may be appropriate. If only a quantitative estimate of coverage is required, or if there is doubt regarding the prior estimate produced during the SQUEAC Stages 1 and 2, it may be preferable to minimize influence of the qualitative and routine quantitative data review and shift limited resources to a two-stage cluster survey that uses a larger sample size to produce a robust coverage estimate. Lastly, if a coverage estimate is not a priority but qualitative information on barriers and boosters is more important for program monitoring and improvement, then a standalone qualitative assessment  can be conducted.While the choice in assessment design should be guided by program needs and priorities, it should also be noted that the two-stage cluster survey and SQUEAC require different resources, in terms of time, costs, and technical capacity. As coverage estimation in the SQUEAC requires development of a final prior estimate, a process that should be objective and iterative, correct implementation could require additional time and the engagement of higher-level program staff or external support. In settings where routine monitoring is well integrated into routine activities, and/or barriers and boosters of access are already well understood, the time needed to develop a final prior may be shorter. Qualitative data collected during Stage 1, however, may be useful for program improvement and warrant the additional commitment of resources and capacity in some settings.Our study has some important limitations. First, we used a simple mean of individual prior estimates to calculate a single, final prior estimate. While this approach ensures that no individual prior estimate is used alone , final prior estimation would ideally be a more iterative process that allows for reflection and revision of the final prior estimate with the critical evaluation of new information. The choice to calculate an unweighted mean of multiple individual priors resulted in a more linear approach but reflects common field practice. A weighted approach could also be adopted to assign relative importance to individual estimates for which there is more confidence; however, specific guidance on such methods is not yet available. Second, we present individual prior estimates developed by a range of participant sources, including local program staff and caregivers who may lack the necessary expertise, training, or objectivity to develop an accurate prior. The scenarios presented here, therefore, are not ideal but likely representative of pragmatic field conditions.Valid estimates of program coverage are needed to assess programs, inform allocation of resources, and improve evidence-based policy. Several methodologies are available to monitor program coverage, but methods differ in structure, priority, and cost. The overall SQUEAC methodology represents a step forward in the coverage assessment of nutrition programs by providing a means to both identify barriers to accessing care and estimate program coverage. We show that implementation of SQUEAC, however, can be technically demanding and requires the appropriate capacity during prior estimation to be informative. Program priorities and reporting requirements should inform the choice of study design, balancing feasibility and validity that are likely given available capacity.Additional file 1:Supplementary Appendix. (DOCX 39 kb)