Effect of exercise detraining in cognitive functions of older adults: A systematic review.

OBJECTIVE: Provide a synthesis of the current literature about the effects of detraining on cognitive functions in older adults. METHODS: The PICOS acronym strategy was performed in PubMed/MEDLINE, Web of Science, Cochrane Library and PsycINFO database. The Preferred Reporting Items for Systematic Review and Meta-Analyses statement had been followed in the present study, in which the search was conducted on October 2023. The study selection consisted in original articles including older adults, detraining after training exercise period, use of tests or scales to measure cognitive function. The Downs and Black checklist had been used to assess the studies quality. Sample characteristics, type of previous training, detraining period, cognitive functions measurements and main results were extracted by 2 investigators. RESULTS: From 1927 studies, 12 studies were included, being 11 studies identified via systematic research, and 1 study by citation search. Older adults, ranged from 60 to 87 years old, were assessed after detraining. The cognitive functions most evaluated were global cognition and executive functions. One study evaluated both cognitive outcome and cerebral blood flow. Most of the studies demonstrated a decline in the cognitive function after detraining. CONCLUSION: Exercise detraining period, ranging from 10 days to 16 weeks, can effect negatively the cognitive function in older adults.

Impaired signaling pathways on Berardinelli-Seip congenital lipodystrophy macrophages during Leishmania infantum infection.

Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.

Erosion of the Capital City Advantage in Child Survival and Reproductive, Maternal, Newborn, and Child Health Intervention Coverage in Sub-Saharan Africa.

The place of residence is a major determinant of RMNCH outcomes, with rural areas often lagging in sub-Saharan Africa. This long-held pattern may be changing given differential progress across areas and increasing urbanization. We assessed inequalities in child mortality and RMNCH coverage across capital cities and other urban and rural areas. We analyzed mortality data from 163 DHS and MICS in 39 countries with the most recent survey conducted between 1990 and 2020 and RMNCH coverage data from 39 countries. We assessed inequality trends in neonatal and under-five mortality and in RMNCH coverage using multilevel linear regression models. Under-five mortality rates and RMNCH service coverage inequalities by place of residence have reduced substantially in sub-Saharan Africa, with rural areas experiencing faster progress than other areas. The absolute gap in child mortality between rural areas and capital cities and that between rural and other urban areas reduced respectively from 41 and 26 deaths per 1000 live births in 2000 to 23 and 15 by 2015. Capital cities are losing their primacy in child survival and RMNCH coverage over other urban areas and rural areas, especially in Eastern Africa where under-five mortality gap between capital cities and rural areas closed almost completely by 2015. While child mortality and RMNCH coverage inequalities are closing rapidly by place of residence, slower trends in capital cities and urban areas suggest gradual erosion of capital city and urban health advantage. Monitoring child mortality and RMNCH coverage trends in urban areas, especially among the urban poor, and addressing factors of within urban inequalities are urgently needed.

Evaluation and discovery of novel benzothiazole derivatives as promising hits against Leishmania infantum.

An early exploration of the benzothiazole class against two kinetoplastid parasites, Leishmania infantum and Trypanosoma cruzi, has been performed after the identification of a benzothiazole derivative as a suitable antileishmanial initial hit. The first series of derivatives focused on the acyl fragment of its class, evaluating diverse linear and cyclic, alkyl and aromatic substituents, and identified two other potent compounds, the phenyl and cyclohexyl derivatives. Subsequently, new compounds were designed to assess the impact of the presence of diverse substituents on the benzothiazole ring or the replacement of the endocyclic sulfur by other heteroatoms. All compounds showed relatively low cytotoxicity, resulting in decent selectivity indexes for the most active compounds. Ultimately, the in vitro ADME properties of these compounds were assessed, revealing a satisfying water solubility, gastrointestinal permeability, despite their low metabolic stability and high lipophilicity. Consequently, compounds 5 and 6 were identified as promising hits for further hit-to-lead exploration within this benzothiazole class against L. infantum, thus providing promising starting points for the development of antileishmanial candidates.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

Child immunization status according to number of siblings and birth order in 85 low- and middle-income countries: a cross-sectional study.

Background: Identification of unvaccinated children is important for preventing deaths due to infections. Number of siblings and birth order have been postulated as risk factors for zero-dose prevalence. Methods: We analysed nationally representative cross-sectional surveys from 85 low and middle-income countries (2010-2020) with information on immunisation status of children aged 12-35 months. Zero-dose prevalence was defined as the failure to receive any doses of DPT (diphtheria-pertussis-tetanus) vaccine. We examined associations with birth order and the number of siblings, adjusting for child's sex, maternal age and education, household wealth quintiles and place of residence. Poisson regression was used to calculate zero-dose prevalence ratios. Findings: We studied 375,548 children, of whom 13.7% (n = 51,450) were classified as zero-dose. Prevalence increased monotonically with birth order and with the number of siblings, with prevalence increasing from 11.0% for firstborn children to 17.1% for birth order 5 or higher, and from 10.5% for children with no siblings to 17.2% for those with four or more siblings. Adjustment for confounders attenuated but did not eliminate these associations. The number of siblings remained as a strong risk factor when adjusted for confounders and birth order, but the reverse was not observed. Among children with the same number of siblings, there was no clear pattern in zero-dose prevalence by birth order; for instance, among children with two siblings, the prevalence was 13.0%, 14.7%, and 13.3% for firstborn, second, and third-born, respectively. Similar results were observed for girls and boys. 9513 families had two children aged 12-35 months. When the younger sibling was unvaccinated, 61.9% of the older siblings were also unvaccinated. On the other hand, when the younger sibling was vaccinated, only 5.9% of the older siblings were unvaccinated. Interpretation: The number of siblings is a better predictor than birth order in identifying children to be targeted by immunization campaigns. Zero-dose children tend to be clustered within families. Funding: Gavi, the Vaccine Alliance.

Progesterone-mediated remodeling of the maternal-fetal interface by a PGRMC1-dependent mechanism.

Implantation and maintenance of pregnancy involve intricate immunological processes that enable the developing fetus to coexist with the maternal immune system. Progesterone, a critical hormone during pregnancy, is known to promote immune tolerance and prevent preterm labor. However, the mechanism by which progesterone mediates these effects remains unclear. In this study, we investigated the role of the non-classical progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling at the maternal-fetal interface. Using JEG3 cells, a trophoblast model cell line, we observed that progesterone stimulation increased the expression of human leukocyte antigen-C (HLA-C) and HLA-G, key molecules involved in immune tolerance. We also found that progesterone upregulated the expression of the transcription factor ELF3, which is known to regulate trophoblast-specific HLA-C expression. Interestingly, JEG3 cells lacked expression of classical progesterone receptors (PRs) but exhibited high expression of PGRMC1, a finding we confirmed in primary trophoblasts by mining sc-RNA seq data from human placenta. To investigate the role of PGRMC1 in progesterone signaling, we used CRISPR/Cas9 technology to knockout PGRMC1 in JEG3 cells. PGRMC1-deficient cells showed a diminished response to progesterone stimulation. Furthermore, we found that the progesterone antagonist RU486 inhibited ELF3 expression in a PGRMC1-dependent manner, suggesting that RU486 acts as a progesterone antagonist by competing for receptor binding. Additionally, we found that RU486 inhibited cell invasion, an important process for successful pregnancy, and this inhibitory effect was dependent on PGRMC1. Our findings highlight the crucial role of PGRMC1 in mediating the immunoregulatory effects of progesterone at the maternal-fetal interface.

Biofilm viability and microbial community of non-inoculated moving bed biofilm reactor in Nile tilapia Oreochromis niloticus cultivation.

The aim of this study was to evaluate two moving bed biofilm reactors (MBBR) without nitrifying bacteria inoculation. Biofilms and viable bacterial colonies were evaluated after 124 days. MBBR bioreactors received water from Oreochromis niloticus fish farming and water quality parameters were monitored daily. Four distinct phases with different fish stocking density were established.: phase 1 (2.40 kg m-3), phase 2 (4.95 kg m-3), phase 3 (8.71 kg m-3) and phase 4 (12.23 kg m-3). The successful maturation of the bioreactors occurred around on the 100th experimental day when the nitration rate increased to 57 % in MBBR1 and 38 % in MBBR2. 105 species were identified in the biofilms, which were grouped into 65 genera, three of which were essential: Pseudomonas (21.7 %), Nitrospira (15.1 %) and Gemmobacter (11.2 %). MBBR start-up without bacterial inoculation is time-consuming, however, strengthened by important nitrifying groups.

Genetic structure and diversity of the rfb locus of pathogenic species of the genus Leptospira.

Leptospirosis is caused by pathogenic strains of the genus Leptospira and is considered the most widespread zoonotic bacterial disease. The genus is characterized by the large number of serology variants, which challenges developing effective serotyping methods and vaccines with a broad spectrum. Because knowledge on the genetic basis of the serological diversity among leptospires is still limited, we aimed to explore the genetic structure and patterns of the rfb locus, which is involved in the biosynthesis of lipopolysaccharides, the major surface antigen that defines the serovar in leptospires. Here, we used genomic data of 722 pathogenic samples and compared the gene composition of their rfb locus by hierarchical clustering. Clustering analysis showed that the rfb locus gene composition is species-independent and strongly associated with the serological classification. The samples were grouped into four well-defined classes, which cluster together samples either belonging to the same serogroup or from different serogroups but sharing serological affinity. Our findings can assist in the development of new strategies based on molecular methods, which can lead to better tools for serological identification in this zoonosis.

Chitosan surface modification modulates the mucoadhesive, permeation and anti-angiogenic properties of gellan gum/bevacizumab nanoparticles.

Bevacizumab (BVZ) was the first monoclonal antibody approved by the FDA and has shown an essential advance in the antitumor therapy of colorectal cancer (CRC), however, the systemic action of BVZ administered intravenously can trigger several adverse effects. The working hypothesis of the study was to promote the modulation of the mucoadhesion properties and permeability of the BVZ through the formation of nanoparticles (NPs) with gellan gum (GG) with subsequent surface modification with chitosan (CS). NPs comprising BVZ and GG were synthesized through polyelectrolyte complexation, yielding spherical nanosized particles with an average diameter of 264.0 ± 2.75 nm and 314.0 ± 0.01 nm, polydispersity index of 0.182 ± 0.01 e 0.288 ± 0.01, and encapsulation efficiency of 29.36 ± 0.67 e 60.35 ± 0.27 mV, for NPs without (NP_BVZ) and with surface modification (NP_BVZ + CS). The results showed a good ability of nanoparticles with surface modification to modulate the NPs biological properties.

A composite index; socioeconomic deprivation and coverage of reproductive and maternal health interventions.

Objective: To examine inequalities in the coverage of reproductive and maternal health interventions in low- and middle-income countries and territories using a composite index of socioeconomic deprivation status. Methods: We obtained data on education and living standards from national household surveys conducted between 2015 and 2019 to calculate socioeconomic deprivation status. We assessed the coverage of reproductive and maternal health interventions, using three indicators: (i) demand for family planning satisfied with modern methods; (ii) women who received antenatal care in at least four visits; and (iii) the presence of a skilled attendant at delivery. Absolute and relative inequalities were evaluated both directly and using the slope index of inequality and the concentration index. Findings: In the 73 countries and territories with available data, the median proportions of deprivation were 41% in the low-income category, 11% in the lower-middle-income category and less than 1% in the upper-middle-income category. The coverage analysis, conducted for 48 countries with sufficient data, showed consistently lower median coverage among deprived households across all health indicators. The coverage of skilled attendant at delivery showed the largest inequalities, where coverage among the socioeconomically deprived was substantially lower in almost all countries. Antenatal care visits and demand for family planning satisfied with modern methods also showed significant disparities, favouring the less deprived population. Conclusion: The findings highlight persistent disparities in the coverage of reproductive and maternal health interventions, requiring efforts to reduce those disparities and improve coverage, particularly for skilled attendant at delivery.

Identifying and Characterizing the Poorest Urban Population Using National Household Surveys in 38 Cities in Sub-Saharan Africa.

Identifying and classifying poor and rich groups in cities depends on several factors. Using data from available nationally representative surveys from 38 sub-Saharan African countries, we aimed to identify, through different poverty classifications, the best classification in urban and large city contexts. Additionally, we characterized the poor and rich groups in terms of living standards and schooling. We relied on absolute and relative measures in the identification process. For absolute ones, we selected people living below the poverty line, socioeconomic deprivation status and the UN-Habitat slum definition. We used different cut-off points for relative measures based on wealth distribution: 30%, 40%, 50%, and 60%. We analyzed all these measures according to the absence of electricity, improved drinking water and sanitation facilities, the proportion of children out-of-school, and any household member aged 10 or more with less than 6 years of education. We used the sample size, the gap between the poorest and richest groups, and the observed agreement between absolute and relative measures to identify the best measure. The best classification was based on 40% of the wealth since it has good discriminatory power between groups and median observed agreement higher than 60% in all selected cities. Using this measure, the median prevalence of absence of improved sanitation facilities was 82% among the poorer, and this indicator presented the highest inequalities. Educational indicators presented the lower prevalence and inequalities. Luanda, Ouagadougou, and N'Djaména were considered the worst performers, while Lagos, Douala, and Nairobi were the best performers. The higher the human development index, the lower the observed inequalities. When analyzing cities using nationally representative surveys, we recommend using the relative measure of 40% of wealth to characterize the poorest group. This classification presented large gaps in the selected outcomes and good agreement with absolute measures.

Structure-activity relationships of novel N-imidazoylpiperazines with potent anti-Trypanosoma cruzi activity.

Background: Chagas disease is caused by the parasite Trypanosoma cruzi, and the lack of effective and safe treatments makes identifying new classes of compounds with anti-T. cruzi activity of paramount importance. Methods: Hit-to-lead exploration of a metabolically stable N-imidazoylpiperazine was performed. Results: Compound 2, a piperazine derivative active against T. cruzi, was selected to perform the hit-to-lead exploration, which involved the design, synthesis and biological evaluation of 39 new derivatives. Conclusion: Compounds 6e and 10a were identified as optimized compounds with low micromolar in vitro activity, low cytotoxicity and suitable preliminary absorption, distribution, metabolism and excretion and physicochemical properties. Both compounds reduced parasitemia in mouse models of Chagas disease, providing a promising opportunity for further exploration of new antichagasic compounds.

Human vs machine towards neonatal pain assessment: A comprehensive analysis of the facial features extracted by health professionals, parents, and convolutional neural networks.

Neonates are not able to verbally communicate pain, hindering the correct identification of this phenomenon. Several clinical scales have been proposed to assess pain, mainly using the facial features of the neonate, but a better comprehension of these features is yet required, since several related works have shown the subjectivity of these scales. Meanwhile, computational methods have been implemented to automate neonatal pain assessment and, although performing accurately, these methods still lack the interpretability of the corresponding decision-making processes. To address this issue, we propose in this work a facial feature extraction framework to gather information and investigate the human and machine neonatal pain assessments, comparing the visual attention of the facial features perceived by health-professionals and parents of neonates with the most relevant ones extracted by eXplainable Artificial Intelligence (XAI) methods, considering the VGG-Face and N-CNN deep learning architectures. Our experimental results show that the information extracted by the computational methods are clinically relevant to neonatal pain assessment, but yet do not agree with the facial visual attention of health-professionals and parents, suggesting that humans and machines can learn from each other to improve their decision-making processes. We believe that these findings might advance our understanding of how humans and machines code and decode neonatal facial responses to pain, enabling further improvements in clinical scales widely used in practical situations and in face-based automatic pain assessment tools as well.

How to Test Human CAR T Cells in Solid Tumors, the Next Frontier of CAR T Cell Therapy.

Chimeric antigen receptor (CAR) T cell therapy has proven to be a successful treatment option for leukemias and lymphomas. These encouraging outcomes underscore the potential of adoptive cell therapy for other oncology applications, namely, solid tumors. However, CAR T cells are yet to succeed in treating solid tumors. Unlike liquid tumors, solid tumors create a hostile tumor microenvironment (TME). CAR T cells must traffic to the TME, survive, and retain their function to eradicate the tumor. Nevertheless, there is no universal preclinical model to systematically test candidate CARs and CAR targets for their capacity to infiltrate and eliminate human solid tumors in vivo. Here, we provide a detailed protocol to evaluate human CAR CD4+ helper T cells and CD8+ cytotoxic T cells in immunodeficient (NSG) mice bearing antigen-expressing human solid tumors.

Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors.

The adaptive immune system exhibits exquisite specificity and memory and is involved in virtually every process in the human body. Redirecting adaptive immune cells, in particular T cells, to desired targets has the potential to lead to the creation of powerful cell-based therapies for a wide range of maladies. While conventional effector T cells (Teff) would be targeted towards cells to be eliminated, such as cancer cells, immunosuppressive regulatory T cells (Treg) would be directed towards tissues to be protected, such as transplanted organs. Chimeric antigen receptors (CARs) are designer molecules comprising an extracellular recognition domain and an intracellular signaling domain that drives full T cell activation directly downstream of target binding. Here, we describe procedures to generate and evaluate human CAR CD4+ helper T cells, CD8+ cytotoxic T cells, and CD4+FOXP3+ regulatory T cells.

Coverage, Trends, and Inequalities of Maternal, Newborn, and Child Health Indicators among the Poor and Non-Poor in the Most Populous Cities from 38 Sub-Saharan African Countries.

Rapid urbanization is likely to be associated with suboptimal access to essential health services. This is especially true in cities from sub-Saharan Africa (SSA), where urbanization is outpacing improvements in infrastructure. We assessed the current situation in regard to several markers of maternal, newborn, and child health, including indicators of coverage of health interventions (demand for family planning satisfied with modern methods, at least four antenatal care visits (ANC4+), institutional birth, and three doses of DPT vaccine[diphtheria, pertussis and tetanus]) and health status (stunting in children under 5 years, neonatal and under-5 mortality rates) among the poor and non-poor in the most populous cities from 38 SSA countries. We analyzed 136 population-based surveys (year range 2000-2019), contrasting the poorest 40% of households (referred to as poor) with the richest 60% (non-poor). Coverage in the most recent survey was higher for the city non-poor compared to the poor for all interventions in virtually all cities, with the largest median gap observed for ANC4+ (13.5 percentage points higher for the non-poor). Stunting, neonatal, and under-5 mortality rates were higher among the poor (7.6 percentage points, 21.2 and 10.3 deaths per 1000 live births, respectively). The gaps in coverage between the two groups were reducing, except for ANC4, with similar median average annual rate of change in both groups. Similar rates of change were also observed for stunting and the mortality indicators. Continuation of these positive trends is needed to eliminate inequalities in essential health services and child survival in SSA cities.

A theoretical protocol for the rational design of the bioinspired multifunctional hybrid material MIP@cercosporin.

CONTEXT: Rational design of polymeric materials prepared with the molecular imprinting technology is gaining even more space, as it can provide the optimal conditions to direct the laboratory molecularly imprinting polymer (MIP) preparation, maximizing their efficiency while reducing costs and preparation time, when compared to try-and-error approaches. We perform a rational design of an MIP with specific cavities for cercosporin accommodation by means of computational tools. The main steps of an MIP preparation were simulated and it was found that the most appropriated functional monomer to be used in the MIP preparation for cercosporin is the acrylamide, while the most suitable crosslinking agent is found to be p-divinylbenzene. Also, the most suitable solvents to remove cercosporin from the cavity are those with low dielectric constant, such as chloroform. This kind of solvent can then be used in washing step, in the case of use the MIP for sensing destinations. On the other hand, solvents like water, which has high dielectric constants, can efficiently improve the interactions between cercosporin and the functional monomer acrylamide, being indicated when the objective is to attract or maintain the cercosporin inside the MIP cavity. Thus, a MIP@cercosporin hybrid material can be used in aqueous solutions more reliably, or even the cercosporin detection in this media can be favoured. In the selectivity analysis of the material prepared in this specific condition, the results point that this MIP can also detect elsinochrome A with high efficiency, and could be more selective for hypericin, altertoxin, hypocrelin A, and phleichrome mycotoxins. METHOD: The main steps of a MIP synthesis were theoretically simulated trough density functional theory (DFT) calculations aiming to direct and optimize the synthesis and applications of the material before the bench tests. Initially, in order to choose the most suitable functional to be employed for cercosporin calculations, eight of the DFT functionals that had been previously used for cercosporin calculations in literature were tested, which were the LCWPBE, B3LYP, CAM-B3LYP, M062-X, mPW1PW91, PBE0, TPSSh, and ωb97Xd. The theoretical 1H NMR chemical shifts for cercosporin molecule were calculated and compared with experimental results to analyze the performance of the functionals. Of all these, the best results were obtained with the TPSSh functional, employing the 6-31G(d,p) basis set, and this level of theory was then used for all the following steps. All the simulations were performed by means of geometry optimizations and frequency calculations. Additionally, AIM calculations were employed for further analysis of the interactions between the chosen functional monomer and cercosporin template in step 1, which was functional monomer selection. In washing step, the calculations were done using implicit solvation model, and finally, in selectivity tests, the putative "solid" MIP was simulated by freezing the positions of the monomers after the template remotion, and then other structurally similar toxins were placed in its cavity for the geometry optimizations and frequency calculations.