Collective Nature of Orbital Excitations in Layered Cuprates in the Absence of Apical Oxygens.

We have investigated the 3d orbital excitations in CaCuO_{2} (CCO), Nd_{2}CuO_{4} (NCO), and La_{2}CuO_{4} (LCO) using high-resolution resonant inelastic x-ray scattering. In LCO they behave as well-localized excitations, similarly to several other cuprates. On the contrary, in CCO and NCO the d_{xy} orbital clearly disperses, pointing to a collective character of this excitation (orbiton) in compounds without apical oxygen. We ascribe the origin of the dispersion as stemming from a substantial next-nearest-neighbor (NNN) orbital superexchange. Such an exchange leads to the liberation of the orbiton from its coupling to magnons, which is associated with the orbiton hopping between nearest neighbor copper sites. Finally, we show that the exceptionally large NNN orbital superexchange can be traced back to the absence of apical oxygens suppressing the charge transfer energy.

Weak signal extraction enabled by deep neural network denoising of diffraction data.

The removal or cancellation of noise has wide-spread applications in imaging and acoustics. In applications in everyday life, such as image restoration, denoising may even include generative aspects, which are unfaithful to the ground truth. For scientific use, however, denoising must reproduce the ground truth accurately. Denoising scientific data is further challenged by unknown noise profiles. In fact, such data will often include noise from multiple distinct sources, which substantially reduces the applicability of simulation-based approaches. Here we show how scientific data can be denoised by using a deep convolutional neural network such that weak signals appear with quantitative accuracy. In particular, we study X-ray diffraction and resonant X-ray scattering data recorded on crystalline materials. We demonstrate that weak signals stemming from charge ordering, insignificant in the noisy data, become visible and accurate in the denoised data. This success is enabled by supervised training of a deep neural network with pairs of measured low- and high-noise data. We additionally show that using artificial noise does not yield such quantitatively accurate results. Our approach thus illustrates a practical strategy for noise filtering that can be applied to challenging acquisition problems.

Signature of quantum criticality in cuprates by charge density fluctuations.

The universality of the strange metal phase in many quantum materials is often attributed to the presence of a quantum critical point (QCP), a zero-temperature phase transition ruled by quantum fluctuations. In cuprates, where superconductivity hinders direct QCP observation, indirect evidence comes from the identification of fluctuations compatible with the strange metal phase. Here we show that the recently discovered charge density fluctuations (CDF) possess the right properties to be associated to a quantum phase transition. Using resonant x-ray scattering, we studied the CDF in two families of cuprate superconductors across a wide doping range (up to p = 0.22). At p* ≈ 0.19, the putative QCP, the CDF intensity peaks, and the characteristic energy Δ is minimum, marking a wedge-shaped region in the phase diagram indicative of a quantum critical behavior, albeit with anomalies. These findings strengthen the role of charge order in explaining strange metal phenomenology and provide insights into high-temperature superconductivity.

Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity, SAR, and preclinical evaluation.

Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals, satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-aminoquinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-resistant tuberculosis treatments.

Sonographic early findings in a case of bladder schistosomiasis.

Urinary schistosomiasis is a tropical infection with a high endemicity in the developing countries and is included in the list of "Neglected Tropical Diseases". It is caused by a parasitic worm, Schistosoma haematobium, and it has come into the spotlight as a major cause of urogenital disease. Furthermore, it is linked to bladder cancer and it is a predisposing factor for HIV/AIDS. In this case, we describe a bladder schistosomal disease in a young African boy with persistent macroscopic hematuria and its ultrasound diagnostic bladder imaging.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme.

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.

Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 µg/well, T = 37 °C) was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP KM = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP KM = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.

Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase.

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with ß-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.

The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.

Discovery of new inhibitors of Mycobacterium tuberculosis InhA enzyme using virtual screening and a 3D-pharmacophore-based approach.

Mycobacterium tuberculosis InhA (MtInhA) is an attractive enzyme to drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In this work, two different virtual-ligand-screening approaches were applied in order to identify new InhA inhibitors' candidates from a library of ligands selected from the ZINC database. First, a 3-D pharmacophore model was built based on 36 available MtInhA crystal structures. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules able to satisfy the binding features of MtInhA substrate-binding cavity. The second approach consisted of using four well established docking programs, with different search algorithms, to compare the binding mode and score of the selected molecules from the aforementioned library. After detailed analyses of the results, six ligands were selected for in vitro analysis. Three of these molecules presented a satisfactory inhibitory activity with IC50 values ranging from 24 (±2) µM to 83 (±5) µM. The best compound presented an uncompetitive inhibition mode to NADH and 2-trans-dodecenoyl-CoA substrates, with Ki values of 24 (±3) µM and 20 (±2) µM, respectively. These molecules were not yet described as antituberculars or as InhA inhibitors, making its novelty interesting to start efforts on ligand optimization in order to identify new effective drugs against tuberculosis having InhA as a target. More studies are underway to dissect the discovered uncompetitive inhibitor interactions with MtInhA.

Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme.

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation.

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

A música enquanto sonho a trilha sonora enquanto elemento onírico em De olhos bem fechados, de Stanley Kubrick / The music as a dream: the soundtrack as an oneiric element at Eyes wide shut, by Stanley Kubrick

Baseado no livro Traumnovelle (Breve romance de sonho) de Arthur Schnitzler, o diretor Stanley Kubrick filma em 1999 seu último filme, Eyes wide shut (De olhos bem fechados). Para caracterizar cinematograficamente os diversos planos narrativos, Kubrick utiliza a música como elemento principal em suas caracterizações, em especial a diferença entre sonho e realidade, utilizando diferentes músicas e estilos musicais, tais como jazz, música oriental e contemporânea.(AU)

Based on Traumnovelle (Dream Story) by Arthur Schnitzler, the movie director Stanley Kubrick filmed in 1999 his last work, Eyes wide shut. To cinematographically characterize the various narrative plans, Kubrick uses music as the main element, especially to set the differences between dream and reality buy using different music styles, such as jazz, eastern and contemporary music.(AU)

Desenvolvimento e aplicações de um novo instrumento para estimulação do barorreflexo / Development and application of a new device for barorreflex stimulation

Objetivos: Desenvolver e discutir as possíveis aplicações de um método para estimulação barorreceptora, através de um novo aparelho que permite a realização da manobra de Valsalva de forma automatizada e não-assistida. Métodos: Um manômetro digital foi projetado e desenvolvido pelo Centro de Microgravidade/ FENG-PUCPR para monitorar a pressão intratorácica exercida durante a expiração forçada ou Manobra de Valsalva. Resultados: O equipamento, denominado de Equipamento para Manobra de Valsalva, é constituído de cinco partes principais, sendo elas: um transdutor de pressão (sensor de pressão e amplificador de sinais), um mostrador de caracteres, um mostrador em barra de Diodo Emissor de Luz e um micro-controlador. Testes preliminares indicaram que este novo equipamento permite que um indivíduo realize a manobra de Valsalva de forma correta e sem qualquer assistência durante o procedimento. Conclusões: O aparelho desenvolvido é de fácil manuseio e visualização, leve, portátil e de baixo custo.

O efeito da escopolamina na performance mental durante simulação de microgravidade / The effect of scopolamine on mental performance during microgravity simulation

A otimização da performance mental e a minimização dos erros podem salvar vidas e reduzir custos de missões aeroespaciais. Usando o teste de performance mental computadorizada Manikin Test, pôde-se avaliar aspectos importantes da performance cognitiva sob a influência da escopolamina, medicamento largamente utilizado para prevenir ou minimizar os efeitos da Doença da Locomoção Espacial, durante uma simulação de microgravidade. Para simular os efeitos fisiológicos da microgravidade na Terra, utilizou-se a inversão postural, onde o indivíduo é deitado sobre um leito e a parte do seu corpo fica abaixo da parte inferior (6º negativos ou – 6º HDT, head-down tilt). Realizou-se um estudo duplo-cego, randomizado cruzado, no qual foi avaliada a performance mental de 15 voluntários durante simulação de microgravidade com e sem o uso de escopolamina 0,45mg. Constatou-se que, apesar da ocorrência de efeitos adversos inerentes à escopolamina, não houve diminuição da performance mental dos voluntários. Assim mais estudos são necessários para elucidar os reais efeitos da escopolamina sobre a performance mental durante a simulação de microgravidade. Assim, será possível a criação de medidas medicamentosas efetivas na prevenção e/ou na minimização dos sintomas provocados pela Doença da Locomoção Espacial.

A avaliação da interação de uma dieta controlada com a escopolamina na prevenção da desorientação espacial / Na evaluation of the interaction of a controlled diet with scopolamine on the prevention of spatial disorientation

Este estudo objetivou avaliar o melhor horário de administração da escopolamina, medicamento utilizado na prevenção dos sinais e sintomas da desorientação espacial, e a interação de uma dieta controlada sobre sua ação. Foram realizados testes na cadeira rotatória desenvolvida no Laboratório de Microgravidade/ PUCRS, em 10 voluntários saudáveis. Cada voluntário participou de dois dias de teste, randomizados, um depois de 12h de jejum e no outro após a ingestão de uma dieta padronizada, recebendo, em ambos os dias, a dose oral de 0,45 mg de escopolamina. O teste rotatório foi realizado 60 min, 90 min, 120 min após administração deste medicamento. A sintomatologia da desorientação espacial e os efeitos colaterais do fármaco foram avaliados através do questionário de Graybiel e da avaliação clínica do voluntário durante e após os testes de rotação na cadeira. Os resultados indicaram que não foi possível determinar o melhor horário de administração da escopolamina. No teste realizado a 60 min, a interação com a dieta aumentou o tempo de rotação na cadeira (p = 0,0002). Uma amostra maior e a dosagem séria da escopolamina são necessários para um melhor entendimento de sua ação, seu horário de administração e sua interação com diferentes alimentos.