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The management of schizophrenia necessitates a comprehensive treatment paradigm that considers individual patient nuances and the efficacy of lurasidone in addressing schizophrenia symptoms, particularly at elevated dosages. Numerous randomized trials have affirmed the efficacy of lurasidone across various dimensions of schizophrenia, demonstrating marked enhancements in positive, negative and cognitive symptoms compared to a placebo. In addition, lurasidone exhibits potential in ameliorating agitation amongst acutely ill patients, showcasing greater efficacy at higher doses. However, despite the favourable outcomes observed with higher lurasidone doses, routine clinical practice often opts for lower doses, potentially limiting its maximal therapeutic impact. Furthermore, lurasidone also shows efficacy in reducing post-psychotic depression in dual psychosis. Moreover, practical insights into lurasidone usage encompass swift dose escalation within a 1-5-day span and recommended combination strategies with other medications such as benzodiazepines for insomnia or agitation, beta-blockers for akathisia, and antihistamines or antimuscarinic drugs for patients transitioning rapidly from antipsychotics with substantial antihistamine and/or anticholinergic effects. Finally, a series of clinical cases is presented, highlighting benefits of lurasidone in terms of cognitive function, functional recovery and other therapeutic aspects for the management of schizophrenia., Competing Interests: Disclosure and potential conflicts of interest: JLPO has received honoraria as a consultant and/or advisor to Angelini, Janssen/J&J and Lundbeck. AC is an employee of Angelini Pharma España SLU. All other authors declare that they have no conflicts of interest relevant to this manuscript. The International Committee of Medical Journal Editors (ICMJE) Potential Conflicts of Interests form for the authors is available for download at: https://www.drugsincontext.com/wp-content/uploads/2024/07/dic.2024-4-4-COI.pdf, (Copyright © 2024 Guarro Carreras MT, Jiménez Suárez L, Lago García L, Montes Reula L, Neyra del Rosario A, Rodríguez Batista FA, Velasco Santos M, Prados-Ojeda JL, Diaz-Marsà M, Martín-Carrasco M, Cardenas A.)

Snake robots, also known as apodal robots, are among the most common and versatile modular robots. Primarily due to their ability to move in different patterns, they can evolve in scenarios with several constraints, some of them hardly accessible to other robot configurations. This paper deals with a specific environment constraint where the robot needs to climb a prismatic obstacle, similar to a step. The objective is to carry out simulations of this function, before implementing it in the physical model. To this end, we propose two different algorithms, parameterized by the obstacle dimensions determined by image processing, and both are evaluated in simulated experiments. The results show that both algorithms are viable for testing in real robots, although more complex scenarios still need to be further studied.

Alpha-terpineol is a monoterpene alcohol found in essential oils from medicinal plants with some well-known pharmacological activities and widely used in cosmetics. However, the toxicological effects and additional pharmacological activities need to be clarified. Thus, the study evaluated the toxic, cytotoxic, genotoxic, hemolytic, and oxidative potential of alpha-terpineol in non-clinical bioassays. Different concentrations of alpha-terpineol were used in bioassays, including MTT (50, 100, 200, and 400 μg/mL), Artemia salina (6.25-400 μg/mL), Allium cepa (10, 50, and 100 μg/mL), comet assay (100, 200, and 500 μg/mL), cytokinesis-block micronucleus (100, 250, and 500 μg/mL), confocal microscopy for apoptosis quantification (100 and 500 μg/mL), hemolysis and Saccharomyces cerevisiae central disk test (10, 35, and 75 μg/mL). For the MTT test, alpha-terpineol was more cytotoxic on melanoma murine B16-F10 cells rather than macrophages. For A. salina test, alpha-terpineol showed LC 50 of 68.29 and 76.36 μg/mL for 24 h and 48 h of exposure time, respectively. Meanwhile, alpha-terpineol was also cytotoxic to meristematic cells, which revealed inhibition of cellular division and mutagenic action by formation of bridges and delayed anaphases. The compound increased damage index and frequency of damage corroborated by the presence of micronuclei, bridges and nuclear buds at 500 μg/mL, but it caused neither hemolysis, oxidative damage on the S. cerevisiae nor cell death in normal fibroblasts. The findings indicate alpha-terpineol has cytotoxic potential by cytogenetic and molecular mechanisms associated with apoptosis and probable target effects against melanoma cells.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH 2 ), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC 50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 μM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC 50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC 50 value of 23 μM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Halicephalobus gingivalis is a free-living nematode that occasionally causes infections in horses. We report a rare case of limb fracture of horse caused by infection with H. gingivalis. An 8-year-old mare was referred to the Veterinary Hospital of the Federal University of Lavras with claudication grade 5 of the right hind limb, that had been started 3 months ago. The patient had aseptic arthritis in the tarsal joint and edema that extended to the quartile. The radiographic examination showed punctate osteolysis with exacerbation of bone trabeculation along the calcaneus, talus, proximal epiphysis of the third metatarsal and distal epiphysis of the tibia. Treatment for arthritis was initiated, and the animal showed a slight improvement in limb function. However, 21 days after hospitalization, due to a comminuted fracture of the tibia, it was euthanized. At necropsy, yellowish masses were found from the metatarsal to the tibia, and around the tarsal bones and joint. Similar masses were also found in the left kidney. Numerous nematodes compatible with H. gingivalis were identified. This is the first description of a pathological fracture caused by H. gingivalis infection in an equine limb.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme-inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1 Cat ) without a pro-region. High-resolution 3D structures of PvS1 Cat , alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1' and P2' positions of the inhibitor, although P' residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.

Transthyretin was discovered in the 1940s, named after its ability to bind thyroid hormones and retinol. In the genomic era, transthyretins were found to be part of a larger family with homologs of no obvious function, then called transthyretin-related proteins. Thus, it was proposed that the transthyretin gene could be the result of gene duplication of an ancestral of this newly identified homolog, later found out to be an enzyme involved in uric acid degradation, then named HIUase (5-hydroxy-isourate hydrolase). Here, we sought to re-enact the evolutionary history of this protein family by reconstructing, from a phylogeny inferred from 123 vertebrate sequences, three ancestors corresponding to key moments in their evolution-before duplication; the common transthyretin ancestor after gene duplication and the common ancestor of Eutheria transthyretins. Experimental and computational characterization showed the reconstructed ancestor before duplication was unable to bind thyroxine and likely presented the modern HIUase reaction mechanism, while the substitutions after duplication prevented that activity and were enough to provide stable thyroxine binding, as confirmed by calorimetry and x-ray diffraction. The Eutheria transthyretin ancestor was less prone to characterization, but limited data suggested thyroxine binding as expected. Sequence/structure analysis suggests an early ability to bind the Retinol Binding Protein. We solved the X-ray structures from the two first ancestors, the first at 1.46 resolution, the second at 1.55 resolution with well-defined electron density for thyroxine, providing a useful tool for the understanding of structural adaptation from enzyme to hormone distributor.

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cyto F730A ) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys 824 at the C-terminal of ADAM17cyto with the Cys 73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases., (Copyright © 2020. Published by Elsevier B.V.)

Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant of Plasmodium falciparum ATC ( Pf ATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to Pf ATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting Pf ATC as a target of this drug and a promising target for the development of novel antimalarials.

The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction : Malaria is one of the most prevalent human infections worldwide with over 40% of the world's population living in malaria-endemic areas. In the absence of an effective vaccine, emergence of drug-resistant strains requires urgent drug development. Current methods applied to drug target validation, a crucial step in drug discovery, possess limitations in malaria. These constraints require the development of techniques capable of simplifying the validation of Plasmodial targets. Areas covered : The authors review the current state of the art in techniques used to validate drug targets in malaria, including our contribution - the protein interference assay (PIA) - as an additional tool in rapid in vivo target validation. Expert opinion : Each technique in this review has advantages and disadvantages, implying that future validation efforts should not focus on a single approach, but integrate multiple approaches. PIA is a significant addition to the current toolset of antimalarial validation. Validation of aspartate metabolism as a druggable pathway provided proof of concept of how oligomeric interfaces can be exploited to control specific activity in vivo . PIA has the potential to be applied not only to other enzymes/pathways of the malaria parasite but could, in principle, be extrapolated to other infectious diseases.

Chitosan has become promising as a biomaterial because of its biocompatible, biodegradable nature and non-toxic. The biochemical and antioxidant properties of chitosan modified with acetylacetone and ethylenediamine (Cacen) or diethylenetriamine (Cacdien) associated with ceftazidime (F) were investigated. Chitosan was characterized using Elemental Analysis (CHN), Thermal Analysis (TG/DTG/DSC), X-Ray Diffractometry (XRD), and was investigated an in vitro hemolytic cytotoxicity test, Artemia saline larvae for toxicity and in vitro antioxidant activity by DPPH (2,2-diphenyl-1-picrylhydrazyl). The chemical modification was confirmed by CHN, XRD and TG. The Cacen, Cacdien, CacenF and CacdienF derivatives presented high percentages of nitrogen (7.6%, 7.9%, 14.1% and 19.2%, respectively), confirming the modification and drug adsorption. The average molecular weight of chitosan and its derivatives were 132 kDa, with very few variations after modification. This incorporation decreased in the crystallinity index of Cacen (7.1%) and Cacdien (4.3%), as well as the incorporation of the drug (CacenF 0.3% and CacdienF 3.4%). FTIR spectra showed bands at 1580-1654 cm -1 referring to carbonyl and imine group and bands at 3500-3300 and 1462-1264 cm -1 related to vibration and deformation the amine group. 13 C NMR spectroscopy was observed new peaks in carbonyl and imine regions (170-200 ppm). The thermal stability of the derivatives without the drug improved according to TG/DTG/DSC analysis, however there were decreased in the derivatives with the drug. The biocompatibility of the derivatives was confirmed by the low hemolytic rate (<5%), non-toxic on Artemia salina (LD 50% >3000 ppm), and significant index (1-34%) of antioxidant activity. The results demonstrated that chitosan and its derivatives are promising biomaterials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.

Objective: The transcriptional repressor DREAM is involved in thyroid-specific gene expression, thyroid enlargement and nodular development, but its clinical utility is still uncertain. In this study we aimed to investigate whether DREAM mRNA levels differ in different thyroid tumors and how this possible difference would allow the use of DREAM gene expression as molecular marker for diagnostic and/or prognosis purpose., Materials and Methods: We quantified DREAM gene mRNA levels and investigated its mutational status, relating its expression and genetic changes to diagnostic and prognostic features of 200 thyroid tumors, being 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesions [49 goiter and 50 follicular adenomas (FA)]., Results: Levels of mRNA of DREAM gene were higher in benign (0.7909 ± 0.6274 AU) than in malignant (0.3373 ± 0.6274 AU) thyroid lesions (p < 0.0001). DREAM gene expression was able to identify malignancy with 66.7% sensitivity, 85.4% specificity, 84.2% positive predictive value (PPV), 68.7% negative predictive value (NPV), and 75.3% accuracy. DREAM mRNA levels were also useful distinguishing the follicular lesions FA and FVPTC with 70.2% sensitivity, 73.5% specificity, 78.5% PPV, 64.1% NPV, and 71.6% accuracy. However, DREAM gene expression was neither associated with clinical features of tumor aggressiveness, nor with recurrence or survival. Six different genetic changes in non-coding regions of DREAM gene were also found, not related to DREAM gene expression or tumor features., Conclusion: We suggest that DREAM gene expression may help diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions; however, it is not useful as a prognostic marker.

Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence-labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell-based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP-binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Investigate the clinical utility of serum interleukin dosages of IL-2, IL-2R, IL-4, IL-6, IL-6R, IL-8, IL-10 and IL-12 in the diagnosis and characterization of patients with DTC. In particular, verify ILs utility in the identification of individuals who are evolving disease-free or with the active disease., Methods: We evaluated 200 patients with malignant nodules (100 patients disease-free and 100 patients with recurrence/active disease); 60 benign nodules and 100 healthy controls, serum levels were assessed by ELISA., Results: All ILs, but not IL-4, differentiated these three groups. We observed that IL-2, 2R and 10 serum concentrations were associated with thyroglobulin levels. Serum IL-2 was able to differentiate patients with active disease from the disease-free with a sensitivity of 98%, specificity of 58%, positive predictive value (PPV) of 70% and negative predictive value (NPV) of 97% (p=0.0007). IL-6R levels differentiated patients with active disease from the disease-free patients with 56% sensitivity, 63% specificity, PPV of 60% and NPV of 59% (p<0.0001). IL-8 values also distinguished patients with active disease from the disease-free ones with sensitivity of 50%, specificity of 76%, PPV of 68% and NPV of 60% (p=0.0025); using IL-12, we obtained a sensitivity value of 73%, specificity of 66%, PPV of 68% and NPV of 71% (p<0.0001). Furthermore, interleukin levels showed association with some tumor characteristics of aggressiveness., Conclusion: We suggest that the serum concentration of ILs may assist in the diagnosis and characterization of tumor malignancy helping identify patients with active disease who deserve closer medical attention., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro , and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro . Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879's mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.

Introduction: Tumors of the proximal tibia, in children, can affect the growth plate and pose a challenge to further reconstruction of the bone defects resulting from tumor resection. Reconstruction methods do not always compensate the potential for bone growth in this segment. We present a new surgical technique of bone reconstruction, based on the transposition of the ipsilateral fibula with its growth plate and the use of an internal sliding fixation device, without need for microsurgical technique., Case Description: We report two patients with osteosarcoma of the proximal tibia affecting the growth cartilage who were treated with the new technique., Discussion and Evaluation: In both cases, bone healing, hypertrophy and longitudinal growth of the transposed fibula were documented., Conclusions: This new technique preserves the blood supply of the auto-transplanted bone segment, maintaining physeal growth potential, with no need for microsurgery. The implant allows longitudinal bone growth, which was radiographically confirmed., Level of Evidence: Case report, Level IV.

Purpose: The role of thyroid-specific transcription factors in thyroid malignancy is still poorly understood, so we investigate thyroid-specific transcription factors gene expression both in benign and in malignant thyroid nodules, aiming to study a possible clinical utility of these molecules., Methods: We quantified TTF-1, FOXE1 and PAX8 mRNA levels, relating their expression to diagnostic and prognostic features of thyroid tumors. RNA was extracted from 4 normal thyroid tissues, 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesion tissues [49 goiter and 50 follicular adenomas (FA)]., Results: Levels of mRNA of both FOXE1 (P < 0.0001) and PAX8 (P < 0.0001) genes, but not TTF-1 (P = 0.7056), were higher in benign than in malignant thyroid lesions. FOXE1 was able to identify malignant nodules with 75.8 % sensitivity, 76.1 % specificity, 75.8 % positive predictive value, 76.1 % negative predictive value and 75.9 % accuracy. PAX8 was able to identify malignancy with 60.6 % sensitivity, 81.1 % specificity, 76.9 % positive predictive value, 66.4 % negative predictive value and 70.6 % accuracy. Both FOXE1 and PAX8 gene expression patterns were also able to differentiate FA from the follicular variant of PTC-FVPTC. However, the investigated gene expression was neither associated with any clinical feature of tumor aggressiveness nor associated with recurrence or survival., Conclusions: We suggest that FOXE1 and PAX8 gene expression patterns may help to diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions, but are not determinants of thyroid tumor progression.

Objective: To conduct a retrospective analysis on cases undergoing inspection of orthopedic damage, at an orthopedic emergency service in a teaching hospital, with the aim of evaluating patients with postoperative infection after conversion to internal osteosynthesis., Methods: This was a retrospective analysis covering the period from June 2012 to June 2013, on patients who underwent inspection of orthopedic damage due to external fixation and subsequently were converted to definitive osteosynthesis using a nail or plate., Results: We found an infection rate of 13.3% in our sample and, furthermore, found that there had been technical errors in setting up the fixator in 60.4% of the cases., Conclusion: We found an infection rate that we considered high, along with inadequacies in constructing the external fixator. We emphasize that this procedure is not risk-free and that training for physicians who perform this procedure should be mandatory.

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90., (© 2014 FEBS.)

Proteins participate in almost every cell physiological function, and to do so, they need to reach a state that allows its function by folding and/or exposing surfaces of interactions. Spontaneous folding in the cell is in general hindered by its crowded and viscous environment, which favors misfolding and nonspecific and deleterious self-interactions. To overcome this, cells have a system, in which Hsp70 and Hsp90 play a central role to aid protein folding and avoid misfolding. The topics of this review include the biophysical tools used for monitoring protein-ligand and protein-protein interactions and also some important results related to the study of molecular chaperones and heat shock proteins (Hsp), with a focus on the Hsp70/Hsp90 network. The biophysical tools and their use to probe the conformation and interaction of Hsp70 and Hsp90 are briefly reviewed.

Many studies have provided observational data on the association of obesity and thyroid cancers, but only few of them propose mechanisms that would permit a better understanding of the causal molecular mechanisms of this association. Considering that there is an increasing incidence of both obesity and thyroid cancers, we need to summarize and link recent studies in order to characterize and understand the contribution of obesity-related factors that might affect thyroid cancer development and progression. Adipose tissue is involved in many vital processes, including insulin sensitivity, angiogenesis, regulation of energy balance, activation of the complement system, and responses such as inflammation. Although these processes have their own molecular pathways, they involve the same molecules through which obesity and adipose tissue might exert their roles in carcinogenesis, not only affecting MAPK and PI3K or even insulin pathways, but also recruiting local inflammatory responses that could result in disease formation and progression. This review describes five important issues that might explain the link between excessive weight and thyroid cancer: thyroid hormones, insulin resistance, adipokines, inflammation, and sexual hormones., (© 2014 Society for Endocrinology.)

The peroxisome proliferator-activated receptors (PPARs) regulate genes involved in lipid and carbohydrate metabolism, and are targets of drugs approved for human use. Whereas the crystallographic structure of the complex of full length PPARγ and RXRα is known, structural alterations induced by heterodimer formation and DNA contacts are not well understood. Herein, we report a small-angle X-ray scattering analysis of the oligomeric state of hPPARγ alone and in the presence of retinoid X receptor (RXR). The results reveal that, in contrast with other studied nuclear receptors, which predominantly form dimers in solution, hPPARγ remains in the monomeric form by itself but forms heterodimers with hRXRα. The low-resolution models of hPPARγ/RXRα complexes predict significant changes in opening angle between heterodimerization partners (LBD) and extended and asymmetric shape of the dimer (LBD-DBD) as compared with X-ray structure of the full-length receptor bound to DNA. These differences between our SAXS models and the high-resolution crystallographic structure might suggest that there are different conformations of functional heterodimer complex in solution. Accordingly, hydrogen/deuterium exchange experiments reveal that the heterodimer binding to DNA promotes more compact and less solvent-accessible conformation of the receptor complex.

Peroxisome proliferator activated receptors (PPARs δ, α and γ) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

This paper reports the first case of coenurosis in the State of Mato Grosso do Sul, Brazil. This disease is caused by the larval stage of the tapeworm Taenia multiceps (Leske, 1780). The animal in which the disease was diagnosed was an 18-month-old ewe from an endemic area of Southern Brazil as an imported animal among a group of 30 sheep. The clinic-pathological condition was that commonly found in herbivores affected by the disease, especially sheep. Apathy, nystagmus, intermittent blindness, circling and pressing head against obstacles were the neurological signs reported. The necropsy showed that a brain lesion in the subcortex of the right hemisphere was a bladder-like cyst measuring 4 cm in diameter filled with a translucent fluid with a large number of white spherules (protoscolices) floating. In addition to the identification of the Coenurus cerebralis protoscolices, the brain tissue lesion was histopathologically described.

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-D-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.

The clinical picture of dengue is characterized by a maximum duration of 14 days despite frequent complaints of longer symptoms. This study evaluated the occurrence of persistent symptoms (> 14 days) and its impact on daily life. A hundred eighteen patients were interviewed, and the main symptoms at diagnosis were mialgia (98.3%), fever (97.5%) and weakness (95.8%). The presence of at least a persistent symptom was related by 77 (65.2%) individuals of wich 10 (8.5%) described it as intense and lasting for 30 days or more. The most persistent symptoms mentioned were weakness (58 cases), hiporexia (49) and sleepiness (40), occurring mostly in women, with odds ratio: 5.4 (IC95%: 2.3-12.3). A significant association between the persistence of the symptoms and the history of extra expenses (p = 0,02) was found, as well as a delay to return to normal activities (p < 0.001). Thus, it was verified that dengue presented a relevant impact on every day life, even after 14 days, a fact wich was associated with the presence of persistent symptoms of the illness.

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics simulations of the TRalpha and TRbeta LBDs in the absence and in the presence of the natural ligand Triac. The simulations show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary structure elements, while the structure remains essentially compact, resembling a molten globule state. This differs from most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TRalpha and TRbeta subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H11, and the interaction of the region between H1 and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

Among the benign tumors of the esophagus, the discovery of a granular cell tumor is exceptional. Although endoscopy permits recognition of this tumor and supports its benign nature, only histologic examination of the endoscopic biopsy specimen permits one to determine its precise nature. The endoscopic appearance of esophageal granular cell tumor ranges from a plaque-like thickening of the mucosa to a polypoid mass. We describe here seven cases of granular cell tumor of the esophagus diagnosed by histologic examination of endoscopic biopsy specimens. We believe the actual incidence of this tumor in this location might be greater if every esophagus were given as much attention at endoscopic examination and at autopsy.