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BACKGROUND: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking. RESULTS: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports. CONCLUSIONS: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.

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Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.

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Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is still neither vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation. In the present study, we describe an experimental screening of 2,320 compounds from the chemical library of the Muséum National d'Histoire Naturelle of Paris on ZIKV NS2B-NS3 protease. A total of 96 hits were identified with 90% or more of inhibitory activity at 10 µM. Amongst the most active compounds, five were analyzed for their inhibitory mechanisms by kinetics assays and computational approaches such as molecular docking. 2-(3-methoxyphenoxy) benzoic acid (compound 945) show characteristics of a competitive inhibition (Ki = 0.49 µM) that was corroborated by its molecular docking at the active site of the NS2B-NS3 protease. Taxifolin (compound 2292) behaves as an allosteric inhibitor whereas 3,8,9-trihydroxy-2-methyl-1H-phenalen-1-one (compound 128), harmol (compound 368) and anthrapurpurin (compound 1499) show uncompetitive inhibitions. These new NS2B-NS3 protease inhibitors are valuable hits to further hit-to-lead optimization.

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The Arbovirus (Arthropod-borne virus) is a group which comprises viruses whose transmission is carried out by arthropod vectors infecting vertebrates. Some arboviruses related to human diseases have been given considerable relevance as Chikungunya and Mayaro of the family Togaviridae, genus Alphavirus. The lack of proper specific treatment has prompted the requirement for deeper structural studies that could unveil leads to new drugs. Among possible targets, viral proteases are recognized as proteins with big potential. These proteins, termed nsP2 in Alphavirus, have the function of cleaving certain regions of the viral polyprotein, being vital to the viral cycle. In this research, we used docking and molecular dynamics to analyze the contact between the protease nsP2 of Alphavirus Chikungunya and Mayaro and substrates formed by peptides with ten amino acid residues. A model of the Mayaro nsP2 was constructed based on homologous proteases. Our study suggests that the glycine specificity motif, a region where a highly conserved glycine residue in position P2 of the protease substrate is positioned, facilitates the nucleophilic attack by assisting in placing the P1 carbonyl group carbon. Stabilization of different substrate regions maybe explained by relevant contacts with the enzyme. Besides that, the phi and psi angles in the outlier region of the Ramachandran plot found for the P2 glycine of the Chikungunya substrate seems to indicate the necessity of this residue that can accommodate angles not allowed to other residues.Communicated by Ramaswamy H. Sarma.

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Humicola grisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichia pastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40-50 °C and pH 5.0-9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

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OBJECTIVES: Investigate polymorphisms and expressions of human leukocyte antigen-G (HLA-G), galectin-1 (Gal-1), and interleukin-10 (IL-10) in people living with HIV (PLHIV) with and without comorbidities to help understanding the mechanisms involved in triggering these disorders in PLHIV and in their prognosis. DESIGN: Here we evaluated the potential correlation between the genetic polymorphism and/or protein levels of HLA-G, Gal-1, and IL-10 with and without comorbidities of PLHIV. METHODS: Two hundred HIV patients under antiretroviral treatment (83 with comorbidities and 117 without comorbidities) and 200 healthy individuals (controls) were genotyped, using PCR, for HLA-G 14-base pair polymorphism located at the 3' untranslated region in exon 8 insertion/insertion (Ins/Ins: low HLA-G expression) or deletion/deletion (Del/Del: high HLA-G expression). Soluble levels of HLA-G (sHLA-G), Gal-1, and IL-10 were quantified by enzyme-linked immunosorbet assay. RESULTS: HIV patients without comorbidities exhibited higher frequency of 14-base pair Del/Del genotype than HIV patients with comorbidities. As expected, HIV patients Ins/Ins with and without comorbidities produced less sHLA-G than controls. However, HIV patients Del/Del with comorbidities expressed sHLA-G more than controls and HIV patients Del/Del without comorbidities. Interestingly, patients that showed low levels sHLA-G, and presence of comorbidities, exhibited high Gal-1 serum levels. However, an increase in soluble levels of IL-10 in PLHIV was observed when compared to controls, especially in the PLHIV group without comorbidities suggesting, a protective role of IL-10 in the development of comorbidities. CONCLUSIONS: These data suggested that the high expression of sHLA-G and IL-10 or Gal-1 could be associated and could be associated with the development or not of comorbidities in PLHIV.

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Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

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Abstract Background The early detection of vascular damage in subclinical stages of hypertensive disease may be the key point in the prevention of cardiovascular outcomes. Objectives to correlate parameters of structural vascular damage (measurement of the carotid intima-media thickness) with parameters of functional vascular damage (central hemodynamic measurements) in pre-hypertensive and hypertensive patients taking up to two classes of anti-hypertensive drugs. Methods This was a cross-sectional descriptive study conducted with a convenience sample of patients attending the Liga de Hipertensão Arterial , a multidisciplinary program for the diagnosis and treatment of systemic hypertension, of the Federal university of Goias. Patients with arrythmia, diabetes, previous cardiovascular or cerebrovascular diseases, and end-stage diseases were excluded. Carotid Doppler test, measurements of peripheral and central blood pressure by applanation tonometry (Sphygmocor®) and oscillometry (Mobil-O-Graph®) were performed. The t-test was used for comparisons and the Pearson correlation test for correlations, considering a p<0.05 statistically significant. Results twenty patients (12 women) were evaluated, mean age 53.8 ± 14.3 years. Higher values of central pulse pressure (42.9±13.9 vs. 34.7±9.6, p=0.01) and pulse wave velocity (PWV) (9.0±1.9 vs. 7.9±1.5, p=0.01) were obtained by applanation tonometry compared with oscillometry. No difference between the methods was observed for the other measures. A significant correlation was found between carotid artery intima-media thickness (CA-IMT) and PWV (r=0.659; p=0.002) by the oscillometric test, but not with applanation tonometry. No correlation was found between central hemodynamic variables and the presence of carotid artery plaques. Conclusion PWV, estimated by oscillometry, was the only central hemodynamic parameter that correlated significantly with CA-IMT in pre-hypertensive and hypertensive patients at low cardiovascular risk. International Journal of Cardiovascular Sciences. 2020; [online].ahead print, PP.0-0

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The investigation of the views of the stakeholders involved in the municipal solid waste separate collection programme (CP) performed in the East Zone of the city of São Paulo is presented in this paper. Aiming to obtain the necessary information to be analysed, interviews with the manager of the Municipal Urban Cleaning Authority (MUCA) of the city, with leaders of recycling worker cooperatives (WCs), and citizens were performed using semi-structured questionnaires. The analysis was performed using the software "IRAMUTEQ". The results show that, in the view of the manager of the MUCA of the city and the WCs' leaders, the low adhesion of the population to the separate CP is the greatest obstacle to a further expansion of the existing programme. At the same time, the citizens have blamed the low diffusion made by the municipality about the separate CP in the East Zone of São Paulo. Thus, it can be concluded that the separate CP available in the East Zone of São Paulo is still flawed; since neither the public power, the WCs, nor the population are satisfied and point out several flaws. This situation highlights the need to establish better venues for discussion between the population, WCs, and the municipality to jointly design a separate collection system with a more participatory approach.

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RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (K) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.

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Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from Tityus obscurus scorpion venom was characterized and named ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray structure of the ToPI1:trypsin complex, in association with the mass spectrometry data, indicate a sequential set of events: the complex formation with the inhibitor Lys32 in the trypsin S1 pocket, the inhibitor C-terminal residue Ser33 cleavage, and the cyclization of ToPI1 via a peptide bond between residues Ile1 and Lys32. Kinetic and thermodynamic characterization of the complex was obtained. ToPI1 shares no sequence similarity with other PIs characterized to date and is the first PI with CS-α/ß motif described from animal venoms. In its cyclic form, it shares structural similarities with plant cyclotides that also inhibit trypsin. These results bring new insights for studies with venom compounds, PIs, and drug design.

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This study aimed to evaluate the effect of sodium caseinate added into freezing extender on the sperm parameters of cryopreserved bull semen and in vitro and in vivo fertility. One ejaculate of 30 bulls was used and processed using Botu-Bov (Botupharma, Botucatu, Brazil) with the addition of 20% egg yolk (EY) or 15% egg yolk with 2% sodium caseinate (EY + SC), subsequently submitted to freezing. Semen from both groups were evaluated immediately after thawing (T0) and after thermic stress at 37 °C for 90 min (T90), for sperm kinetics, by CASA method, and plasma membrane integrity (PMI), superoxide (O2-) concentration and high mitochondrial potential (HMP) by flow cytometry. In vitro fertilization (IVF) was performed to assess embryo cleavage rate on day 3, and blastocyst rate on day 8. The in vivo fertility test was performed using fixed-time artificial insemination (FTAI). In sperm evaluation, trajectory velocity, linear velocity, curvilinear velocity, and lateral head movement were higher (P < 0.05) in EY + SC at T0. At T90, while rectilinearity and linearity did not differ between EY and EY + SC (P > 0.05), the other parameters evaluated were higher in EY + SC. Similarly, the integrity of the plasma and acrosomal membranes (iPAM) was higher (P < 0.05) at T90 in EY + SC, but did not differ (P > 0.05) between the groups at T0. For O2- and HMP, the values were lower (P < 0.05) in EY + SC group in both moments; furthermore, EY + SC showed higher cleavage and blastocyst rates in IVF. Likewise, pregnancy rates by FTAI were higher (P < 0.05) in the EY + SC group. In conclusion, the addition of sodium caseinate into freezing extender improves sperm parameters of frozen-thawed bull semen and fertility rates on during in vitro and in vivo tests.

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Construction and demolition wastes have been studied by technical means aiming at the development of management tools to reduce their environmental impacts. Among these, recycling can be highlighted. This paper aims at the technical assessment of basket gabions filled with construction and demolition solid waste. Gabions are usually used for retaining walls construction, and these are commonly filled with rocks. Retaining walls are essential for earthfill slopes stabilization, and they must have proper characteristics of strength. However, depending on the slope height or the technical responsibility of the retaining wall, alternative materials with proper characteristics could be used to fill the gabions. The use of recycled material for gabion filling could reduce environmental impacts and costs caused by retaining wall construction. So, basket gabion cells were filled with construction solid waste and basaltic rocks, both crushed into coarse granulometry aiming to compare technical characteristics among them. The performed laboratory tests showed that the horizontal and vertical displacements determined for gabion filled with wastes were near to those obtained for gabions filled with basaltic rocks for a retaining wall of 5 m and up. In conclusion, it can be drawn that basket gabions filled with construction and demolition waste may be a technical alternative for civil construction, reducing environmental impacts and raw material consumption for retaining wall execution.

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Abstract Background Changes in arterial compliance are among the first changes detectable in hypertensive syndrome. Methods with good reproducibility as compared with the gold standard for identifying such changes are desirable in clinical practice. Objectives To compare central pressure measurements and arterial stiffness obtained by two non-invasive methods (tonometry and oscillometry). Methods This was a descriptive, cross-sectional study with a convenience sample of patients with borderline hypertension or stage 1 hypertension. Peripheral and central blood pressure measurements were obtained by tonometry (SphygmoCor®), considered the gold standard, and oscillometry (Mobil O´graph®). Comparisons of results were made by unpaired t-test, and p values < 0.05 were considered statistically significant. Results No difference was found in central pressure measurements obtained by SphygmoCor® (117 x 80.1 mmHg) compared with Mobil O'graph (112 x 81.4 mmHg). Mean augmentation index (AIx) was 26.1% and 21.3%, and mean pulse pressure (PP) amplification 10.7 mmHg and 10.0 mmHg by Sphygmocor® and Mobil O´graph®, respectively (p > 0.05). Mean pressure wave velocity (PWV), 8.4 m/s vs. 7.4 m/s (p = 0.013) and mean central pulse pressure, 37.7 mmmHg and 30.9 mmHg (p = 0.013) were significantly higher by SphygmoCor® than Mobil O´graph®. Conclusion Values of central systolic blood pressure, AIx and pulse pressure amplification obtained by oscillometry were not statistically different compared with tonometry; values of PWV and cPP, however, were underestimated by oscillometry. (Int J Cardiovasc Sci. 2020; 33(2):145-150)

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An elevated prevalence of cryptococcal infection is a tendency in low-income countries and constitutes a global public health problem due to factors such as the limited efficacy of antifungal therapy and the AIDS/transplant immunocompromised patients. The fungus Cryptococcus neoformans, implicated in this burden, has had several genes validated as drug targets. Among them, the thioredoxin system is one of the major regulators of redox homeostasis and antioxidant defense acting on protein disulfide bonds. Thioredoxin 1 from C. neoformans (CnTrx1) was cloned and expressed in E. coli and the recombinant protein was purified and crystallized. Functional assay shows that CnTrx1 catalyzes the reduction of insulin disulfide bonds using dithiothreitol, while acting as a monomer in solution. The crystal structure of oxidized CnTrx1 at 1.80 Å resolution presents a dimer in the asymmetric unit with typical Trx-fold. Differences between the monomers in the asymmetric unit are found specially in the loop leading to the Cys-Gly-Pro-Cys active-site motif, being even larger when compared to those found between reduced and oxidized states of other thioredoxins. Although the thioredoxins have been isolated and characterized from many organisms, this new structural report provides important clues for understanding the binding and specificity of CnTrx1 to its targets.

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Bovine papillomavirus proteins were extensively studied as a prototype for the human papillomavirus. Here, the crystal structure of the extended E2 DNA-binding domain of the dominant transcription regulator from the bovine papillomavirus strain 1 is described in the space group P31 21. We found two protein functional dimers packed in the asymmetric unit. This new protein arrangement inside the crystal led to the reduction of the mobility of a previously unobserved loop directly involved in the protein-DNA interaction, which was then modeled for the first time.

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Resumo Cerca de 40% da população mundial está acima do peso, sendo a obesidade mais frequente nos estratos com menores rendimentos e tempo de estudo. A relação entre ganho de peso e fatores sociodemográficos é bem documentada, mas poucos pesquisadores buscam associar obesidade com raça/cor. Desta forma, este artigo visa mapear, na literatura científica, a extensão, o alcance e a natureza da relação entre obesidade e raça, por meio de revisão de escopo. As fontes informacionais foram os bancos de dados Medical Literature Analysis and Retrieval System Online (Medline), Excerpta Medica Database (Embase), Web of Science, Health InterNetwork Access to Research Initiative (Hinari) e Scopus, além da literatura cinza. Foram encontrados 2.526 artigos, permanecendo 10 documentos após eliminadas as duplicatas e aplicados os critérios de inclusão e exclusão. Existe uma relação complexa entre raça, obesidade, nível socioeconômico e gênero, cuja especificidade se dá em função do contexto sócio-histórico. As possíveis explicações para as disparidades raciais na obesidade residem nos efeitos fisiológicos, psicológicos e culturais do estresse devido à discriminação racial. Novos estudos devem ser realizados considerando as diferenças regionais, pois a desigualdade racial, embora aconteça em todos os lugares, assume diferentes formatos.

Abstract About 40% of the world's population is overweight. Obesity is most prevalent among social strata with lower income and education. Although the association between sociodemographic factors and weight gain is well documented, few researchers associated obesity with race/color. This article aims to map the extent, scope, and nature of the association between obesity and race in the scientific literature by conducting a scoping review. Data sources were the Literature Analysis and Retrieval System Online (Medline), Excerpta Medica Database (Embase), Web of Science, Health InterNetwork Access to Research Initiative (Hinari), and Scopus databases, as well as the gray literature. In total, 2,526 articles were found. After duplicates were excluded and inclusion and exclusion criteria applied, 10 articles remained. Race, obesity, socioeconomic status, and gender are tied into a complex relationship whose specificity lies on the socio-historical context. Racial disparities in obesity may be explained by physiological, psychological, and cultural effects of stress due to racial discrimination. Although racial inequality happens everywhere, it assumes different forms. Considering that, further studies should approach regional differences.

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Ecological monitoring research relies heavily on signals to detect ecosystem changes, making the selection of indicators a crucial methodological requirement. Over the years, individual species and species assemblages have been widely used, thereby, giving rise to reference methods that support the detection of ecological indicators. One such method, the Indicator Value Analysis (IndVal), has been adapted to identify not only species but also combinations of species, assuming collective responses to environmental factors. However, the IndVal method requires a pre-selection of species before performing the analysis, especially in the case of large datasets (e.g. high species richness), when it becomes ineffective. Species pre-selection might introduce subjectivity and a bias into the database, which can cause possible impacts on the final set of indicators. To address these issues, the authors propose the use of Market Basket Analysis (MBA) – a data mining method – which is mathematically similar to IndVal but designed to handle large amounts of data. Both methods were applied to select indicators from gradually larger datasets of Soil Surface Dwelling Arthropods from the Brazilian Amazon, using threshold-dependent indices to assess concordance between results. In general, the results obtained by applying both methods were found to be similar, with an average Jaccard's distance of 0.432 (±0.346) and an average True Skill Statistic of 0.991 (±0.012). As expected, MBA was able to select ecological indicators without species pre-selection as well as from datasets where IndVal had been unsuccessful. In such cases, and by means of objective association rules, the authors demonstrate that MBA could be used to pre-select ecological indicators, which can then be further processed and summarized with the IndVal method. In this study, the authors briefly outline the potential of MBA to complement IndVal and discuss advantages and disadvantages of using MBA for ecological indicators (pre-) selection.