Machine Learning-Enhanced Quantum Chemistry-Assisted Refinement of the Active Site Structure of Metalloproteins.

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Elucidating the concentration-dependent effects of thiocyanate binding to carbonic anhydrase.

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP's therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG's anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1ß, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol.

Teleost fish skin-scales are essential for protection and homeostasis and the largest tissue in direct contact with the environment, but their potential as early indicators of pollutant exposure are hampered by limited knowledge about this model. This study evaluated multi-level impacts of in vivo exposure of European sea bass to fluoxetine (FLX, a selective serotonin-reuptake inhibitor and an emerging pollutant) and 17ß-estradiol (E2, a natural hormone and representative of diverse estrogenic endocrine-disrupting pollutants). Exposed fish had significantly increased circulating levels of FLX and its active metabolite nor-FLX that, in contrast to E2, did not have estrogenic effects on most fish plasma and scale indicators. Quantitative proteomics using SWATH-MS identified 985 proteins in the scale total proteome. 213 proteins were significantly modified 5 days after exposure to E2 or FLX and 31 were common to both treatments and responded in the same way. Common biological processes significantly affected by both treatments were protein turnover and cytoskeleton reorganization. E2 specifically up-regulated proteins related to protein production and degradation and down-regulated the cytoskeleton/extracellular matrix and innate immune proteins. FLX caused both up- and down-regulation of protein synthesis and energy metabolism. Multiple estrogen and serotonin receptor and transporter transcripts were altered in sea bass scales after E2 and/or FLX exposure, revealing complex disruptive effects in estrogen/serotonin responsiveness, which may account for the partially overlapping effects of E2 and FLX on the proteome. A large number (103) of FLX-specifically regulated proteins indicated numerous actions independent of estrogen signalling. This study provides the first quantitative proteome of the fish skin-scale barrier, elucidates routes of action and biochemical and molecular signatures of E2 or FLX-exposure and identifies potential physiological consequences and candidate biomarkers of pollutant exposure, for monitoring and risk assessment.

Transcriptional profiling and physiological responses reveal new insights into drought tolerance in a semiarid adapted species, Anacardium occidentale.

Water stress affects plant performance at various organisational levels, from morphological to molecular, with a drastic drop in crop yield. Integrative studies involving transcriptomics and physiological data in recognized tolerant species are appropriate strategies to identify and understand molecular and functional processes related to water deficit tolerance. The cashew tree (Anacardium occidentale) is a species naturally adapted to environments with low water availability associated with adverse conditions such as heat, high radiation and salinity. We used an integrative strategy, combining classical physiological measurements with high throughput RNA-seq to understand the main adaptive mechanisms of cashew to water deficit followed by recovery. Physiological analyses indicate that young cashew plants display typical isohydric behaviour. They first exhibit rapid stomatal closure, followed by CO2 assimilation, thus preserving the relative water content, membrane integrity and photosystem II activity. Differential expression was observed in 1733 genes from plant leaves exposed to water deficit stress for 26 days. Among them, 705 were upregulated and 1028 were downregulated. After rewatering, 1330 (76.7%) genes returned to their basal expression level. Transcriptional, combined with physiological data, reveal that cashew plants display high phenotypic plasticity and resilience to acute water deficit, and do not activate senescence pathways. A series of genes/pathways and processes involved with drought tolerance in cashew are evidenced, particularly in carbon metabolism, photosynthesis and chloroplast homeostasis.

Environmental and anthropic variabilities at Guanabara Bay (Brazil): A comparative perspective of metal depositions in different time scales during the last 5,500 yrs.

The determination of age-dated metal sediment accumulation rates in a representative South American bay is able to portray the dimension of human impacts during the colonial occupation and industrial periods. Many studies have assessed metal distribution and chronology at Guanabara Bay, in Brazil. However, understanding natural variabilities associated to paleoclimatic changes and comparing these natural variabilities to anthropogenic processes are not well established to date. Accurate geochronological control integrating ages determined by 210Pb and 14C chronologies through an exponential spline fit model allowed for a precise definition of changes associated to the holocene marine transgression, as well as the colonial period, leading to intense land use changes, and the industrial period. The reference values of the system were defined based on their concentrations and the accumulation rates of ecotoxicologically important metals. Al, Ba, Fe, Cd, Cu, Cr, Li, Ni, Mn, Pb, Si, Ti, V, and Zn distributions were determined in a Guanabara Bay core (BG-28) by the EPA 3051 method. Elemental distribution profile assessment allowed for the identification of variabilities associated to weathering processes, predominantly of lithogenic origin, mainly for Al, Ba, Fe, Li, Si, and V. Weathering processes occurred simultaneously to land use changes in the drainage basin since the colonial period, at 400 cal yr BP, and during the industrial period, mainly after the 1960s, denoted by Cd, Cr, Cu, Mn, Pb and Zn increases. The highest average metal enrichment values metals associated to industrial processes reached 5.95, with 119.1-fold higher accumulation rates than the background accumulation values observed between 4200 and 500 cal yr BP.

The Occurrence of Apparent Competition and Apparent Mutualism in a Modeled Greenhouse System with Two Non-competing Pests and a Shared Biocontrol Agent.

This work puts forward a dynamical population model to qualitatively reproduce the phenomena of apparent competition and apparent mutualism found in an experiment with two arthropods being attacked by a predator in a context of pest biological control in greenhouse crops. The two agricultural pests consist of one species of thrips (Frankliniella occidentalis (Pergande 1895)) and one species of whiteflies (Trialeurodes vaporariorum Westwood, 1956), and the shared predator is a predatory mite (Amblyseius swirskii Athias-Herriot, 1962). The predatory mite is the biocontrol agent employed in order to achieve the biological control. The proposed model successfully reproduces this density mediated indirect interactions between pests when their carrying capacities are increased. Moreover, the pests' final population levels may depend on their initial densities and those of their predator. With these results, the proposed model may have the potential to assess whether these indirect pest interactions disrupt or enhance biological control. Additionally, it can also be used as an ancillary tool to theoretically assess the effects of pest biocontrol strategies in the referred experimental setup.

Changes in land use affect anuran helminths in the South Brazilian grasslands.

Degradation and habitat loss of natural grasslands in Southern Brazil has a negative impact on native organisms, potentially including the composition of anuran helminth communities. Here, we characterized the richness, abundance, taxonomic composition, prevalence and intensity of helminth infection in four anuran species. Host anurans were collected in 34 ponds (19 in native grasslands with livestock and 15 in agricultural cultivation) from the highland grasslands in the Brazilian states of Santa Catarina and Paraná. Our results showed a significant difference between native grasslands with livestock and agricultural cultivation regarding the structure of helminth communities for the hosts Aplastodiscus perviridis and Pseudis cardosoi. We also found a greater prevalence and intensity of infection in anurans in areas of agricultural cultivation than in native grasslands with livestock. We found that the environmental descriptors (local and landscape) seem to explain most of the differences in anuran parasitism recorded between native grasslands with livestock and agricultural areas. Thus, we emphasized that the loss of grassy habitat due to conversion to agricultural cultivation can alter helminth communities in anurans, with further work needed to understand the mechanisms involved.

Can differences between continental and insular habitats influence the parasites communities associated with the endemic frog Haddadus binotatus?

Habitats characterized by geographic isolation such as islands have been studied using different organisms as models for understanding the dynamic and insular patterns of biodiversity. Determinants of parasite richness in insular host populations have been conducted mainly with mammals and birds, showing that parasite richness decreases in insular areas. In the present study, we predicted that the type of environment (insular or continental) can influence the richness, diversity and abundance of parasites associated with the endemic frog Haddadus binotatus (Spix, 1824). We sampled frogs in two insular and two mainland fragments to survey their helminth parasites. The total richness was composed of 15 taxa of Nematoda and two of Acanthocephala, and the community composition of the two islands had more similarities between them than the two mainland localities. The insular effect was positive for richness and abundance of helminths, and no significant effect was observed on helminth diversity - even the mean diversity presented high numbers for the islands. We presumed that insular hosts could have lost some parasites in the colonization process when these continental islands were separated from the mainland, approximately 11,000 years ago. However, the high richness and abundance on islands can be explained by an epidemiological argument, which considers high population density due to insularity and other features of the host as factors that increase parasite transmission success among individuals.

How epidemiological patterns shift across populations in an exotic lizard.

Several factors influence the dynamics and structure of parasite communities. Our goal was to investigate how the community composition, prevalence and abundance of parasites change across seven populations of the exotic lizard Hemidactylus mabouia in Northeast Brazil, and to describe ontogenetic and sex variations. We found differences in the composition of component communities and patterns of infection according to the host body size across the lizard populations. We did not find any variation between the sexes regarding epidemiological patterns, which can probably be explained by the similar diet and habitat use of male and female H. mabouia. An unusually high abundance and prevalence of trematodes infecting this host lizard was apparent when we compared other native lizard hosts, and we suggest that local environmental conditions might be advantageous to the development and life cycle of these parasites due to the abundance of all the intermediate and definitive hosts.

Metacercariae of Heterodiplostomum lanceolatum (Trematoda: Proterodiplostomidae) found in Leptodactylus podicipinus (Anura: Leptodactylidae) from Brazil: a morphological, molecular and ecological study.

The trematodes from South American reptiles are poorly known, with only one life cycle completely characterized. We used molecular and morphological methods to characterize diplostomoid metacercariae found in 29 of 86 pointedbelly frogs, Leptodactylus podicipinus (Cope, 1862) collected in a marsh pond in Selvíria, in the central-west region of Brazil. The metacercariae were identified as Heterodiplostomum lanceolatum Dubois, 1936 (Proterodiplostomidae), a rarely reported species that matures in snakes. In phylogenetic analysis of partial sequences from 28S rDNA, H. lanceolatum fell within a polytomy with the proterodiplostomid Crocodilicola pseudostoma (molecular divergence of 4.1%) and other members of the superfamily Diplostomoidea. Our collections provide insights into the ecology of this parasite, in that infected frogs were smaller than uninfected frogs, and metacercariae were more numerous in the abdominal cavity and hindlimb muscles than in abdominal muscles, which suggests directions for future research on the transmission and pathology of this proterodiplostomid.

How Do Nuclei Couple to the Magnetic Moment of a Paramagnetic Center? A New Theory at the Gauntlet of the Experiments.

The recent derivation, based on pure quantum chemistry (QC) first-principles, of the pseudocontact shifts (PCSs) caused by a paramagnetic metal center on far away nuclei has cast doubts on the validity of the semiempirical (SE) theory, predicting PCSs to arise from the metal magnetic susceptibility anisotropy. The SE theory has been used and applied countless times, especially in the last 2 decades, to obtain structural information on proteins containing paramagnetic metal ions. We show here that the QC and SE predictions can be directly tested against experiments, provided a suitable macromolecular system is used. The SE approach yields a good prediction of the experimental PCSs while the QC one does not. It appears that the classic theory is able to grasp satisfactorily the underlying physics.

Non-crystallographic symmetry in proteins: Jahn-Teller-like and Butterfly-like effects?

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.

Metal centers in biomolecular solid-state NMR.

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.

Vertebrate SLRP family evolution and the subfunctionalization of osteoglycin gene duplicates in teleost fish.

BACKGROUND: Osteoglycin (OGN, a.k.a. mimecan) belongs to cluster III of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). In vertebrates OGN is a characteristic ECM protein of bone. In the present study we explore the evolution of SLRP III and OGN in teleosts that have a skeleton adapted to an aquatic environment. RESULTS: The SLRP gene family has been conserved since the separation of chondrichthyes and osteichthyes. Few gene duplicates of the SLRP III family exist even in the teleosts that experienced a specific whole genome duplication. One exception is ogn for which duplicate copies were identified in fish genomes. The ogn promoter sequence and in vitro mesenchymal stem cell (MSC) cultures suggest the duplicate ogn genes acquired divergent functions. In gilthead sea bream (Sparus aurata) ogn1 was up-regulated during osteoblast and myocyte differentiation in vitro, while ogn2 was severely down-regulated during bone-derived MSCs differentiation into adipocytes in vitro. CONCLUSIONS: Overall, the phylogenetic analysis indicates that the SLRP III family in vertebrates has been under conservative evolutionary pressure. The retention of the ogn gene duplicates in teleosts was linked with the acquisition of different functions. The acquisition by OGN of functions other than that of a bone ECM protein occurred early in the vertebrate lineage.

Basal metabolic rate in pregnant adolescents.

BACKGROUND & AIMS: In most clinical settings basal metabolic rate (BMR) is estimated by predictive equations (PE) but there is no specific algorithm for pregnant women. To this end, this study aimed to measure BMR (BMRm) in single-fetus primigravida 63 adolescent pregnant women recruited from primary health care units from a tropical municipality in Brazil. Additionally, BMRm was compared with estimated BMR from internationally recommended PE for nonpregnant adolescents. METHODS: BMR was measured by indirect calorimetry early in the morning in the fasting pregnant adolescents in a quiet, noise and luminosity controlled room. Anthropometry was measured by conventional research protocols and pre-pregnancy anthropometry was obtained from a questionnaire. RESULTS: Mean BMRm was 5447.3 (SD, 917.3) kJ/day. The PE provided biased (overestimation of 747.2 ± 774.1 kJ/day representing 15.7 ± 17.6%) and inaccurate (approximately 40% fell within ±10% of BMRm). CONCLUSIONS: It is necessary to develop accurate estimates of BMR and energy requirements during pregnancy especially in adolescents who are still growing but already nurture a new human being.

Toward multifaceted roles of sucrose in the regulation of stomatal movement.

Plant atmospheric CO2 fixation depends on the aperture of stomatal pores at the leaf epidermis. Stomatal aperture or closure is regulated by changes in the metabolism of the two surrounding guard cells, which respond directly to environmental and internal cues such as mesophyll-derived metabolites. Sucrose has been shown to play a dual role during stomatal movements. The sucrose produced in the mesophyll cells can be transported to the vicinity of the guard cells via the transpiration stream, inducing closure in periods of high photosynthetic rate. By contrast, sucrose breakdown within guard cells sustains glycolysis and glutamine biosynthesis during light-induced stomatal opening. Here, we provide an update regarding the role of sucrose in the regulation of stomatal movement highlighting recent findings from metabolic and systems biology studies. We further explore how sucrose-mediated mechanisms of stomatal movement regulation could be useful to understand evolution of stomatal physiology among different plant groups.

Chronic Kidney Disease Circulating Calciprotein Particles and Extracellular Vesicles Promote Vascular Calcification: A Role for GRP (Gla-Rich Protein).

OBJECTIVE: Inhibition of mineral crystal formation is a crucial step in ectopic calcification. Serum calciprotein particles (CPPs) have been linked to chronic kidney disease (CKD) calcification propensity, but additional knowledge is required to understand their function, assemblage, and composition. The role of other circulating nanostructures, such as extracellular vesicles (EVs) in vascular calcification is currently unknown. Here, we investigated the association of GRP (Gla-rich protein) with circulating CPP and EVs and the role of CKD CPPs and EVs in vascular calcification. APPROACH AND RESULTS: Biological CPPs and EVs were isolated from healthy and CKD patients and comparatively characterized using ultrastructural, analytic, molecular, and immuno-based techniques. Our results show that GRP is a constitutive component of circulating CPPs and EVs. CKD stage 5 serum CPPs and EVs are characterized by lower levels of fetuin-A and GRP, and CPPs CKD stage 5 have increased mineral maturation, resembling secondary CPP particles. Vascular smooth muscle cell calcification assays reveal that CPPs CKD stage 5 and EVs CKD stage 5 are taken up by vascular smooth muscle cells and induce vascular calcification by promoting cell osteochondrogenic differentiation and inflammation. These effects were rescued by incubation of CPPs CKD stage 5 with γ-carboxylated GRP. In vitro, formation and maturation of basic calcium phosphate crystals was highly reduced in the presence of γ-carboxylated GRP, fetuin-A, and MGP (matrix gla protein), and a similar antimineralization system was identified in vivo. CONCLUSIONS: Uremic CPPs and EVs are important players in the mechanisms of widespread calcification in CKD. We propose a major role for cGRP as inhibitory factor to prevent calcification at systemic and tissue levels.

Helminth community structure of 13 species of anurans from Atlantic rainforest remnants, Brazil.

This study aimed to evaluate the community composition and structure of the helminths found in 13 anuran species, and to evaluate whether this parasite community is determined by anuran characteristics. We found that the helminth fauna of the amphibians from five anuran families consisted of 13 taxa and that Cosmocercidae gen. sp. was the most prevalent taxon, followed by Oswaldocruzia subauricularis. Host body size was a determining factor of the composition and structure of the parasitic fauna. Helminth abundance and richness were positively correlated with host body size. The host Leptodactylus latrans had the highest helminth richness (n = 8). The frog Hypsiboas faber had the greatest helminth diversity (H' = 0.711). The mean helminth species richness and diversity differed significantly between host species (P < 0.05). Taken together, our data indicate that, in sympatric species of amphibians, the morphological and behavioural characteristics of the hosts are important for structuring the helminth parasite communities.

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: Implications for calcification-related chronic inflammatory diseases.

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and γ-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein γ-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNFα, IL-1ß and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.