From single-omics to interactomics: How can ligand-induced perturbations modulate single-cell phenotypes?

Cells suffer from perturbations by different stimuli, which, consequently, rise to individual alterations in their profile and function that may end up affecting the tissue as a whole. This is no different if we consider the effect of a therapeutic agent on a biological system. As cells are exposed to external ligands their profile can change at different single-omics levels. Detecting how these changes take place through different sequencing technologies is key to a better understanding of the effects of therapeutic agents. Single-cell RNA-sequencing stands out as one of the most common approaches for cell profiling and perturbation analysis. As a result, single-cell transcriptomics data can be integrated with other omics data sources, such as proteomics and epigenomics data, to clarify the perturbation effects and mechanism at the cell level. Appropriate computational tools are key to process and integrate the available information. This chapter focuses on the recent advances on ligand-induced perturbation and single-cell omics computational tools and algorithms, their current limitations, and how the deluge of data can be used to improve the current process of drug research and development.

Aberrant hippocampal transmission and behavior in mice with a stargazin mutation linked to intellectual disability.

Mutations linked to neurodevelopmental disorders, such as intellectual disability (ID), are frequently found in genes that encode for proteins of the excitatory synapse. Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary proteins that regulate crucial aspects of receptor function. Here, we investigate a mutant form of the TARP family member stargazin, described in an ID patient. Molecular dynamics analyses predicted that the ID-associated stargazin variant, V143L, weakens the overall interface of the AMPAR:stargazin complex and impairs the stability of the complex. Knock-in mice harboring the V143L stargazin mutation manifest cognitive and social deficits and hippocampal synaptic transmission defects, resembling phenotypes displayed by ID patients. In the hippocampus of stargazin V143L mice, CA1 neurons show impaired spine maturation, abnormal synaptic transmission and long-term potentiation specifically in basal dendrites, and synaptic ultrastructural alterations. These data suggest a causal role for mutated stargazin in the pathogenesis of ID and unveil a new role for stargazin in the development and function of hippocampal synapses.

Metas lipídicas: mundo real / Lipid goals: real world

INTRODUÇÃO: Frente à crescente evidência da redução de eventos cardiovasculares relacionados à redução do LDL colesterol (LDL-c), a Sociedade Brasileira de Cardiologia (SBC) propôs em 2017 metas mais agressivas de LDL-c. OBJETIVO: Avaliar em um centro terciário de cardiologia a proporção de pacientes que atingiram metas de LDL-c propostas pela Atualização da Diretriz Brasileira de Dislipidemias e Prevenção da Aterosclerose da SBC conforme estratificação de risco cardiovascular. METODOLOGIA: Foram analisados 2180 pacientes consecutivos em controle ambulatorial quanto à fatores de risco cardiovascular e terapia medicamentosa vigente. Conforme a diretriz, foram classificados em risco baixo, intermediário, alto e muito alto com metas de LDL-c < 130, 100, 70 e 50 mg/dL, respectivamente. RESULTADOS: A média de idade foi de 65 anos, sendo 53% dos pacientes do sexo feminino. Do total, 1225 (56.2%) eram de risco muito alto, 900 (41.3%) alto, 50 (2.3%) intermediário e 5 (0.2%) de baixo risco. Trezentos e noventa e nove pacientes (18.3%) atingiram as metas de LDL-c estabelecidas pela diretriz, sendo 11.1%, 26.2%, 46% e 80% de cada faixa de risco, respectivamente. Destes, 74.5% dos pacientes de muito alto risco, 56.2% de alto risco, 86% de risco intermediário e 40% de baixo risco estavam em uso de estatinas na intensidade e doses preconizadas pela diretriz. Apenas 148 (6.8%) pacientes não usavam estatina. (AU)

Estamos conseguindo atingir as metas atuais de LDL colesterol nos pacientes de muito alto risco cardiovascular? Quantos destes poderiam se beneficiar de inibidores da PCSK9 pelos critérios FOURIER/ODYSSEY e NICE? / Are we meeting current LDL cholesterol targets in patients at very high cardiovascular risk? How many of these could benefit from PCSK9 inhibitors by the FOURIER / ODYSSEY and NICE criteria?

INTRODUÇÃO: Com o aumento de evidências que a diminuição do LDL colesterol (LDL) se relaciona com a diminuição de eventos cardiovasculares, diretrizes de diferentes partes do mundo objetivam menores metas de LDL através da estratificação de risco cardiovascular. A inibição dá para proteína converta-se subtilisina/kexina tipo 9 (PCSK9) reduz os níveis de LDL em até 60%, com subsequente diminuição em desfechos cardiovasculares. OBJETIVO: Nossa meta foi avaliar em um centro terciário de cardiologia a proporção de pacientes de muito alto risco cardiovascular que atingiram a meta de LDL < 50mg/dL atualmente proposto pela diretriz da Sociedade Brasileira de Cardiologia. Adicionalmente, nós averiguamos o número de pacientes que estavam recebendo terapia adequada com estatinas e quantos destes poderiam se beneficiar de inibidores da PCSK9 pelos critérios FOURIER/ODYSSEY e pelas recomendações propostas pelo National Institute for Health and Excellence (NICE). METODOLOGIA: Foram rastreados 2180 pacientes consecutivos de março de 2018 a fevereiro de 2019 para fatores de risco cardiovascular, níveis de colesterol e terapia medicamentosa vigente. Em seguida, foram estratificados conforme o risco cardiovascular, sendo avaliada a adequação à terapia com estatinas recomendada. Em seguida, avaliamos quantos dos pacientes de muito alto risco, que estavam em uso de estatinas de alta intensidade, apresentavam níveis de LDL utilizados para inclusão nos estudos FOURIER/ODYSSEY (≥ 70mg/dL) e recomendados pelo NICE (≥ 140mg/dL) para a introdução de inibidores da PCSK9. RESULTADOS: Dos 2180 pacientes avaliados, 1125 (56.2%) pacientes eram de muito alto risco cardiovascular. Destes pacientes, 136 (11.1%) apresentavam LDL < 50mg/dL, estando 320 (26.1%) pacientes adicionais com LDL < 70mg/dL. Quando avaliado o tratamento com estatinas vigente, 913 (74.5%) pacientes estavam recebendo estatinas de alta intensidade. Destes, 617 (65.9%) teriam indicação de introdução de inibidores da PCSK9 pelos critérios FOURIER/ODYSSEY e 88 (9.4%) pelas recomendações do NICE. CONCLUSÕES: Com metas progressivamente menores de LDL, a busca por níveis ideais de LDL é um desafio para a prática clínica atual. Por mais que pacientes estejam recebendo a terapia recomendada com estatinas, permanece a dificuldade em atingir metas ideais, principalmente no grupo de pacientes de maior risco. Esses pacientes se beneficiariam da inibição da PCSK9, sendo o critério NICE, uma estratégia mais custo-efetiva, ainda aplicável em uma proporção substancial de pacientes. (AU)

Hemangioma cavernoso cardíaco: o relato de um tumor raro / Cardiac cavernous hemangioma: the report of a rare tumor

Os tumores cardíacos são raros e podem ser divididos em primários ou secundários de acordo com sua origem. O acometimento do coração por metástases de outros tumores é o cenário mais comum. Quanto aos tumores primários, em média 75% são benignos e 25% são malignos. A maioria dos pacientes é assintomática até o surgimento de alterações hemodinâmicas ou invasão de estruturas. As manifestações clínicas variam desde intolerância ao exercício, dispneia e dor torácica até síncope e morte súbita. A suspeita diagnóstica tem crescido graças aos avanços na ecocardiografia, mas o padrão ouro para a definição ainda é a biópsia. É importante destacar que os avanços tecnológicos em estudos tomográficos e de ressonância magnética têm contribuído para maior detalhamento das lesões e consequentemente para o diagnóstico diferencial. O tratamento pode ser conservador nos casos assintomáticos e descobertos incidentalmente ou cirúrgico nos casos sintomáticos. Este relato trata de uma paciente do sexo feminino, 53 anos, hipertensa, diabética e tabagista em investigação de dor torácica. Ela foi submetida inicialmente à cintilografia de perfusão miocárdica com dipiridamol que constatou hipocaptação em paredes anterior e septal. Diante disso, solicitou-se coronariografia, a qual evidenciou constrição no terço distal da artéria descendente anterior, associada à imagem radiopaca em topografia justa-cardíaca anterior, sugerindo compressão extrínseca. Um ecocardiograma transtorácico foi realizado e identificou imagem hiperecogênica de aspecto esponjoso e heterogêneo, bem delimitada e com halo hiperecóico, situando-se adjacente às paredes anterior e anterolateral do ventrículo esquerdo. O estudo com Doppler sugeriu fluxo em seu interior. A ressonância magnética do coração destacou câmaras cardíacas de dimensões preservadas, função biventricular dentro da normalidade, ausência de fibrose miocárdica e volumosa massa pericárdica. Após discussão do caso entre as equipes responsáveis e orientação da paciente sobre os resultados, indicou-se a ressecção da massa associada à biópsia. A paciente foi operada no dia 29 de janeiro de 2019, apresentando boa evolução pós-operatória e obteve alta da terapia intensiva no 2º dia pós-operatório em boas condições clínicas. O estudo de anatomia patológica da peça cirúrgica demonstrou Hemangioma Cavernoso Cardíaco. (AU)

Photodegradation of pharmaceutical persistent pollutants using hydroxyapatite-based materials.

Pharmaceutical persistent pollutants pose a serious threat to the environment. The aim of this study was to use, for the first time, hydroxyapatite-based biomaterials as photocatalysts to degrade micropollutants. Diclofenac and fluoxetine were selected for these initial tests. Hydroxyapatite (Ca10(PO4)(OH)2, HAp) is one of the most commonly used biomaterials/bioceramics, being a major constituent of bone. In this work sustainable HAp-based materials of marine origin, obtained from cod fish bones, were used; these photocatalysts were previously fully studied and characterised. Both single-phase HAp and HAp-titania multicomponent materials (1 wt% TiO2) were employed as UV light photocatalysts, the latter showing better performance, indicated by higher degradation rates of both compounds. The HAp-titania photocatalyst showed excellent degradation of both persistent pollutants, the maximum degradation performance being 100% for fluoxetine and 92% for diclofenac, with pollutant and photocatalyst concentrations of 2 ppm and 4 g/L, respectively. Variations in features such as pollutant and photocatalyst concentrations were investigated, and results showed that generally fluoxetine was degraded more easily than diclofenac. The photocatalyst's crystallinity was not affected by the photodegradation reaction; indeed the material exhibited good photostability, as the degradation rate did not decrease when the material was reused. Tests were also performed using actual treated wastewater; the photocatalyst was still effective, even if with lower efficiency (-20% and -4% for diclofenac and fluoxetine, respectively). TOC analysis showed high but incomplete mineralisation of the pollutants (maximum 60% and 80% for DCF and FXT, respectively).

Dynamic structure of NGF and proNGF complexed with p75NTR: pro-peptide effect.

Crystallographic structures of NGF/p75NTR and proNGF/p75NTR were previously obtained in 2:1 and 2:2 stoichiometries, respectively. However, evidence shows that both stoichiometries can occur for mature neurotrophins and pro-neurotrophins. We used Molecular Dynamics (MD) simulations to examine the energetic and structural characteristics of these two complete systems as well as the uncomplexed forms of NGF and understand how these could translate in a new view of different biological outcomes. Here, we show that one chain at the 2:2 proNGF complex seems to be preferentially lost creating a 2:1 structure able to interact with sortilin. We also demonstrated that the structure of the neurotrophin dimers is not pre-established and suffers large structural modifications upon p75NTR binding. Moreover, our data suggests an elegant explanation for the dual role of NGF in neuronal cell death and survival, where different stoichiometries induce conformational changes that might be the basis for the different biological outcomes observed with the mature and proforms of neurotrophins.

Evolution of drug resistance: insight on TEM ß-lactamases structure and activity and ß-lactam antibiotics.

Since the discovery of the first penicillin bacterial resistance to ß-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of ß-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. ß-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the ß-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM ß-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

Ligand-induced structural changes in TEM-1 probed by molecular dynamics and relative binding free energy calculations.

The TEM family of enzymes has had a crucial impact on the pharmaceutical industry due to their important role in antibiotic resistance. Even with the latest technologies in structural biology and genomics, no 3D structure of a TEM-1/antibiotic complex is known previous to acylation. Therefore, the comprehension of their capability in acylate antibiotics is based on the protein macromolecular structure uncomplexed. In this work, molecular docking, molecular dynamic simulations, and relative free energy calculations were applied in order to get a comprehensive and thorough analysis of TEM-1/ampicillin and TEM-1/amoxicillin complexes. We described the complexes and analyzed the effect of ligand binding on the overall structure. We clearly demonstrate that the key residues involved in the stability of the ligand (hot-spots) vary with the nature of the ligand. Structural effects such as (i) the distances between interfacial residues (Ser70-Oγ and Lys73-Nζ, Lys73-Nζ and Ser130-Oγ, and Ser70-Oγ-Ser130-Oγ), (ii) side chain rotamer variation (Tyr105 and Glu240), and (iii) the presence of conserved waters can be also influenced by ligand binding. This study supports the hypothesis that TEM-1 suffers structural modifications upon ligand binding.

Extending the applicability of the O-ring theory to protein-DNA complexes.

Many biological processes depend on protein-based interactions, which are governed by central regions with higher binding affinities, the hot-spots. The O-ring theory or the "Water Exclusion" hypothesis states that the more deeply buried central regions are surrounded by areas, the null-spots, whose role would be to shelter the hot-spots from the bulk solvent. Although this theory is well-established for protein-protein interfaces, its applicability to other protein interfaces remains unclear. Our goal was to verify its applicability to protein-DNA interfaces. We performed Molecular Dynamics simulations in explicit solvent of several protein-DNA complexes and measured a variety of solvent accessible surface area (SASA) features, as well as, radial distribution functions of hot-spots and null-spots. Our aim was to test the influence of water in their coordination sphere. Our results show that hot-spots tend to have fewer water molecules in their neighborhood when compared to null-spots, and higher values of ΔSASA, which confirms their occlusion from solvent. This study provides evidence in support of the O-ring theory with its applicability to a new type of protein-based interface: protein-DNA.

Understanding the importance of the aromatic amino-acid residues as hot-spots.

Protein-protein interactions (PPI) are crucial for the establishment of life. However, its basic principles are still elusive and the recognition process is yet to be understood. It is important to look at the biomolecular structural space as a whole, in order to understand the principles behind conformation-function relationships. Since the application of an alanine scanning mutagenesis (ASM) study to the growth hormone it was demonstrated that only a small subset of residues at a protein-protein interface is essential for binding - the hot-spots (HS). Aromatic residues are some of the most typical HS at a protein-protein interface. To investigate the structural role of the interfacial aromatic residues in protein-protein interactions, we performed Molecular Dynamic (MD) simulations of protein-protein complexes in a water environment and calculated a variety of physical-chemical characteristics. ASM studies of single residues and of dimers or high-order clusters were performed to check for cooperativity within aromatic residues. Major differences were found between the behavior of non-HS aromatic residues and HS aromatic residues that can be used to design drugs to block the critical interactions or to predict major interactions at protein-protein complexes.

Linalool and methyl chavicol present basil (Ocimum sp. ) cultivated in Brazil / Linalol e metil-chavicol presentes em manjericão (Ocimum sp. ) cultivados no Brasil

In Brazil, Ocimum species are commonly known as aromatic and restorative herbs. The present research aimed to study the chemical composition of the essential oils of fresh and dry basil (Ocimum sp) leaves obtained by hydrodistillation and analyzed by GC-FID and GC-MS. The obtained yield was 0.70% for dry leaves and 0.26% for fresh leaves. The major compounds were: linalool (29.50-32.26%) and methyl chavicol (36.81-41.62%). Eucalyptol could also be detected (9.99-7.68%). The oil from dry leaves presented a more complex chemical composition. This study serves to contribute to the knowledge of medicinal plants occurring in Brazil.

No Brasil, as espécies de Ocimum são conhecidas como ervas aromáticas e restaurativas. Nesse trabalho foi estudado a composição química dos óleos essenciais das folhas frescas e secas de manjericão (Ocimum sp) obtido por hidrodestilação e analisados por CG-FID e CG-EM. Os teores encontrados foram de 0,70% para as folhas secas e 0,26% para as folhas frescas. Os componentes majoritários foram: Linalol (29,50-32,26%) e metil-chavicol (36,81-41,62%). Eucaliptol também foi detectado (9,99-7,68%). O óleo das folhas secas apresentou composição química mais complexa. Esse trabalho contribui para o conhecimento das plantas medicinais de ocorrência no Brasil.

Enoyl reductases as targets for the development of anti-tubercular and anti-malarial agents.

Tuberculosis (TB) and Malaria are neglected diseases, which continue to be major causes of morbidity and mortality worldwide, killing together around 5 million people each year. Mycolic acids, the hallmark of mycobacteria, are high-molecular-weight alpha-alkyl, beta-hydroxy fatty acids. Biochemical and genetic experimental data have shown that the product of the M. tuberculosis inhA structural gene (InhA) is the primary target of isoniazid mode of action, the most prescribed anti-tubercular agent. InhA was identified as an NADH-dependent enoyl-ACP(CoA) reductase specific for long-chain enoyl thioesters and is a member of the Type II fatty acid biosynthesis system, which elongates acyl fatty acid precursors of mycolic acids. M. tuberculosis and P. falciparum enoyl reductases are targets for the development of anti-tubercular and antimalarial agents. Here we present a brief description of the mechanism of action of, and resistance to, isoniazid. In addition, data on inhibition of mycobacterial and plasmodial enoyl reductases by triclosan are presented. We also describe recent efforts to develop inhibitors of M. tuberculosis and P. falciparum enoyl reductase enzyme activity.

Detailed microscopic study of the full zipA:FtsZ interface.

Protein-protein interaction networks are very important for a wide range of biological processes. Crystallographic structures and mutational studies have generated a large number of information that allowed the discovery of energetically important determinants of specificity at intermolecular protein interfaces and the understanding of the structural and energetic characteristics of the binding hot spots. In this study we have used the improved MMPB/SA (molecular mechanics/Poisson-Boltzmann surface area) approach that combining molecular mechanics and continuum solvent permits to calculate the free energy differences upon alanine mutation. For a better understanding of the binding determinants of the complex formed between the FtsZ fragment and ZipA we extended the alanine scanning mutagenesis study to all interfacial residues of this complex. As a result, we present new mutations that allowed the discovery of residues for which the binding free energy differences upon alanine mutation are higher than 2.0 kcal/mol. We also observed the formation of a hydrophobic pocket with a high warm spot spatial complementarity between FtsZ and ZipA. Small molecules could be designed to bind to these amino acid residues hindering the binding of FtsZ to ZipA. Hence, these mutational data can be used to design new drugs to control more efficiently bacterial infections.

New designs for MRI contrast agents.

New designs for Magnetic Resonance Imaging contrast agents are presented. Essentially, they all are host-guest inclusion complexes between y-cyclodextrins and polyazamacrocycles of gadolinium (III) ion. Substitutions have been made to the host to optimise the host-guest association. Molecular mechanics calculations have been performed, using the UFF force field for metals, to decide on the suitability of the substitutions, and to evaluate the host-guest energies of association. Interesting general conclusions have been obtained, concerning the improvement of Magnetic Resonance Imaging contrast agents; namely, a set of rational methodologies have been deduced to improve the association between the gadolinium (III) chelates and the cyclodextrins, and their efficiency is demonstrated with a large set of substituted complexes, opening new doors to increase the diagnostic capabilities of Magnetic Resonance Imaging.