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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Nematode infection patterns in a Neotropical lizard species from an insular mountain habitat in Brazil.

Neotropical lizards are known to harbour rich nematode parasite faunas; however, knowledge of the diversity and patterns of infection are still lacking for many species. This is true for the genus Tropidurus, in which data on patterns of parasitism are known for only approximately 11 of its 30 species. We show that the nematode fauna associated with a population of Tropidurus montanus is composed of three species of host-generalist parasites with high overall prevalence. Male and female lizards did not differ in infection pattern and there was no relationship between host body size and intensity of infection for the most prevalent parasite species. Nevertheless, overall prevalence changed seasonally, with a higher proportion of parasitized individuals being found in the dry period than in the rainy period. We discuss our findings in the context of diet patterns of T. montanus, which we suggest may explain the similarities in prevalence and intensity of infection between the sexes. In addition, seasonal changes in diet are considered to be related to the observed differences in prevalence between dry and rainy periods.

Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system.

OBJECTIVE: Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K-dependent Gla-rich protein (GRP) in vascular and valvular calcification processes. APPROACH AND RESULTS: Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both γ-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP-fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion. CONCLUSIONS: GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A-MGP calcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.

Immunohistochemical assessment of PTEN in vulvar cancer: best practices for tissue staining, evaluation, and clinical association.

BACKGROUND: Pten encodes a well-characterized protein that is important in several cancers due to its tumor suppressor function. Yet, the detection and evaluation of PTEN by immunohistochemistry (IHC) for clinical practice have not been standardized. Thus, in this study, we performed a literature review of protocols for PTEN assessment by IHC and the possible differences in evaluation, based on our experience with vulvar carcinomas. Also, we report some of our most recent findings regarding the clinical impact of PTEN in this type of tumor. METHODS: In total, 150 FFPE vulvar carcinoma samples in a tissue microarray were examined by IHC with regard to PTEN, PI3K, AKT, and mTOR. All evaluations were performed by slide digitalization and quantification using APERIO ImageScope software. All measurements were converted into HScore values for the statistical analysis. RESULTS: Sharp and specific PTEN expression was observed in the nuclei and cytoplasmic compartments. Its HScore values ranged from 3.5 to 226, with a median of 92.5. mTOR expression was robust in all cases (mean HScore=248.1). AKT and PI3K had median HScore values of 200.5 and 156.5, respectively. In addition, PTEN expression was associated with higher rates of patient survival. CONCLUSION: The preanalytical step is the first issue in the immunohistochemical evaluation of PTEN. With regard to the analytical procedure, the antigen retrieval step yielded better stains for protocols with high-pH buffers, and antibody clone 6H2.1 effected the most reliable results. PTEN is a good prognostic marker for vulvar cancer, correlating with higher rates of patient survival. Our data underscore the importance of technical standardization to ensure more reliable and reproducible evaluation of PTEN in clinical practice.

Microsatellite loci and genetic structure of artificial populations of Cotesia flavipes (Hymenoptera, Braconidae).

Cotesia flavipes (Cameron) is a parasitoid wasp used in the biological control of the sugarcane borer (Diatraea saccharalis) (Fabr., 1794). Studies on the genetic diversity of C. flavipes are hampered by the lack of highly polymorphic molecular markers. In this report, a set of 11 microsatellite loci were developed from an enriched library of C. flavipes. Four microsatellite loci were polymorphic and were screened in 212 C. flavipes individuals (183 females and 29 males) that were randomly sampled from seven rearing laboratory populations. The number of alleles ranged from two to three. The average inbreeding coefficient (FIS) among all laboratory populations was 0.120, indicating an excess of homozygotes. The average genetic diversity within the laboratory populations was 0.292, which is lower than the values reported for wild Cotesia spp populations. Genetic diversity was most pronounced within laboratory populations (70 to 90%). Most of the observed alleles were fixed or close to fixation. This low overall genetic diversity may have originated from a founder effect, i.e., the contribution of a small number of individuals (genes and alleles) to the formation of these populations. To our knowledge, this study is the first to provide microsatellite loci and an analysis of the genetic structure of C. flavipes. Our results suggest that new introductions of C. flavipes may increase genetic diversity and improve the efficiency of the biological control of D. saccharalis. In addition, population structure data could be used to estimate the minimum number of wasps to be imported.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

Helminth communities of Leptodactylus latrans (Anura: Leptodactylidae) from the Atlantic rainforest, south-eastern Brazil.

The helminth fauna associated with Leptodactylus latrans, a large frog living in a disturbed environment of Atlantic rainforest in south-eastern Brazil, was evaluated. We found eight helminth taxa, including five nematode species, Falcaustra mascula, Oswaldocruzia subauricularis, Physaloptera sp., Rhabdias sp. and an unidentified cosmocercid, two trematodes, Gorgoderina parvicava and Haematoloechus fuelleborni, and one larval cestode. The overall prevalence of infection was 63.2% with a mean intensity of 11.3 ± 3.8. The cosmocercid nematode and O. subauricularis showed the highest prevalences, although the trematode G. parvicava was the most abundant and dominant parasite species. Host size positively influenced both the intensity of infection and parasite species richness. Our data suggest that the juvenile individuals of L. latrans are more susceptible to parasitic infection than the adults. The comparison of the similarity of this community component with that found in other studies in South America shows that, as well as the characteristics of the host, the sampling area also influences the parasitic fauna. Therefore, the results of this study agree that the helminth communities of frogs have relatively low species richness and dominance of generalist species.

Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11).

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11-cellohexaose and CtCBM11-cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand-protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

Phylogeny and micro-habitats utilized by lizards determine the composition of their endoparasites in the semiarid Caatinga of Northeast Brazil.

Trophic networks can have architectonic configurations influenced by historical and ecological factors. The objective of this study was to analyze the architecture of networks between lizards, their endoparasites, diet, and micro-habitat, aiming to understand which factors exert an influence on the composition of the species of parasites. All networks showed a compartmentalized pattern. There was a positive relation between diet and the diversity of endoparasites. Our analyses also demonstrated that phylogeny and the use of micro-habitat influenced the composition of species of endoparasites and diet pattern of lizards. The principal factor that explained the modularity of the network was the foraging strategy, with segregation between the "active foragers" and "sit-and-wait" lizards. Our analyses also demonstrated that historical (phylogeny) and ecological factors (use of micro-habitat by the lizards) influenced the composition of parasite communities. These results corroborate other studies with ectoparasites, which indicate phylogeny and micro-habitat as determinants in the composition of parasitic fauna. The influence of phylogeny can be the result of coevolution between parasites and lizards in the Caatinga, and the influence of micro-habitat should be a result of adaptations of species of parasites to occupy the same categories of micro-habitats as hosts, thus favoring contagion.

Gastrointestinal nematodes of the lizard Tropidurus hispidus (Squamata: Tropiduridae) from a semi-arid region of north-eastern Brazil.

The tropidurid lizard Tropidurus hispidus has a wide distribution in South America. However, knowledge about its helminth fauna is patchy and has been reported for only a few localities along its range of distribution. This study presents data on helminth fauna composition and parameters of infection for a population of T. hispidus from an area within the Brazilian Caatinga biome (semi-arid physiognomy). We found five nematode species within the gastrointestinal tract of lizards: Parapharyngodon sceleratus (Pharyngodonidae); Physaloptera lutzi, Physaloptera retusa and Physalopteroides venancioi (Physalopteridae); and Strongyluris oscari (Heterakidae). The overall prevalence was 84.2% and the mean intensity of infection was 8.5 ± 1.1. The body size of adult male lizards influenced positively the intensity of infection. The infracommunities of nematodes presented an intermediate aggregated distribution (discrepancy index; D= 0.519) and a depauperate nematode fauna. The presence of generalist parasite species has contributed to an increase in the overall richness of the component community. This sampled host population presented the highest prevalence of parasites compared with other studies on T. hispidus, but their relatively low richness can be related to the disturbed environment of the study area.