Pouring SIRAH on NAMD.

Molecular dynamics (MD) simulations provide an invaluable platform for exploring the dynamics of complex biomolecular systems at atomic resolution. However, compatibility issues between force fields and MD software engines can limit the interoperability and transferability of simulations. This work demonstrates the successful use of the coarse-grained SIRAH force field on the widely used NAMD MD engine across a range of increasingly complex biomolecular systems. By leveraging NAMD's ability to read AMBER input files, SIRAH simulations can be run seamlessly on NAMD, including its recently released GPU-accelerated version, NAMD3. The benchmark systems demonstrate consistent results across AMBER, NAMD2, and NAMD3. Thus, these data highlight the enhanced simulation throughput achievable on GPU-accelerated desktop computers using all three engines along with SIRAH. Overall, this study expands the range of the SIRAH force field by utilizing advanced GPU computing resources and high-performance supercomputing facilities, which are particularly effective with NAMD.

New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

Exploring the Use of a Lipopeptide in Dipalmitoylphosphatidylcholine Monolayers for Enhanced Detection of Glyphosate in Aqueous Environments.

The growing reliance on pesticides for pest management in agriculture highlights the need for new analytical methods to detect these substances in food and water. Our research introduces a SPRWG-(C18H37) lipopeptide (LP) as a functional analog of acetylcholinesterase (AChE) for glyphosate detection in environmental samples using phosphatidylcholine (PC) monolayers. This LP, containing hydrophilic amino acids linked to an 18-carbon aliphatic chain, alters lipid assembly properties, leading to a more flexible system. Changes included reduced molecular area and peak pressure in Langmuir adsorption isotherms. Small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) analyses provided insights into the LP's structural organization within the membrane and its interaction with glyphosate (PNG). Structural and geometric parameters, as derived from in silico molecular dynamics simulations (MD), substantiated the impact of LP on the monolayer structure and the interaction with PNG. Notably, the presence of the LP and glyphosate increased charge transfer resistance, indicating strong adherence of the monolayer to the indium tin oxide (ITO) surface and effective pesticide interaction. A calibration curve for glyphosate concentration adjustment revealed a detection limit (LOD) of 24 nmol L-1, showcasing the high sensitivity of this electrochemical biosensor. This LOD is significantly lower than that of a similar colorimetric biosensor in aqueous media with a detection limit of approximately 0.3 µmol L-1. Such an improvement in sensitivity likely stems from adding a polar residue to the amino acid chain of the LP.

Antioxidant Capacity and NF-kB-Mediated Anti-Inflammatory Activity of Six Red Uruguayan Grape Pomaces.

Grape pomaces have a wide and diverse antioxidant phenolics composition. Six Uruguayan red grape pomaces were evaluated in their phenolics composition, antioxidant capacity, and anti-inflammatory properties. Not only radical scavenging methods as DPPH· and ABTS·+ were employed but also ORAC and FRAP analyses were applied to assess the antioxidant potency of the extracts. The antioxidant reactivity of all extracts against hydroxyl radicals was assessed with ESR. The phenol profile of the most bioactive extract was analyzed by HPLC-MS, and a set of 57 structures were determined. To investigate the potential anti-inflammatory activity of the extracts, Nuclear Factor kappa-B (NF-κB) modulation was evaluated in the human colon cancer reporter cell line (HT-29-NF-κB-hrGFP). Our results suggest that Tannat grapes pomaces have higher phenolic content and antioxidant capacity compared to Cabernet Franc. These extracts inhibited TNF-alpha mediated NF-κB activation and IL-8 production when added to reporter cells. A molecular docking study was carried out to rationalize the experimental results allowing us to propose the proactive interaction between the NF-κB, the grape extracts phenols, and their putative anti-inflammatory bioactivity. The present findings show that red grape pomace constitutes a sustainable source of phenolic compounds, which may be valuable for pharmaceutical, cosmetic, and food industry applications.

Identification of potential biological target for trypanocidal sesquiterpene lactones derivatives.

Sesquiterpene lactones are natural products of the Asteraceae family that have shown trypanocidal activity against Trypanosoma cruzi, even exceeding the effectiveness of drugs used in the treatment of American trypanosomiasis. However, there is no agreement on their mechanism of action and their specificity to interact with parasite proteins. For this reason, we aimed to find biological targets that can interact with these compounds by reverse virtual screening with ligand pharmacophores and putative binding sites and the use of bioinformatic databases. Therefore, 41 possible biological targets were found, and four of them (with crystallized proteins), interfering directly or indirectly in the trypanosomatid redox system, were studied in detail. As a first approach, we focused on the study of trypanothione reductase, and protein-ligand interaction fingerprint analyses were performed to find binding site determinants that promote a possible inhibition of the enzyme. This study contributes to the understanding of one of the putative mechanisms of action of sesquiterpene lactones on one of the numerous suggested targets.Communicated by Ramaswamy H. Sarma.

A Computer Simulation Insight into the Formation of Apocarotenoids: Study of the Carotenoid Oxygenases BCO1 and BCO2 and Their Interaction with Putative Substrates.

Carotenoids are isoprenoid pigments, and sources of vitamin A in humans. The first metabolic pathway for their synthesis is mediated by the enzymes ß,ß-carotene-15,15'-dioxygenase (BCO1) and ß,ß-carotene-9',10'-dioxygenase (BCO2), which cleave carotenoids into smaller compounds, called apocarotenoids. The objective of this study is to gain insight into the interaction of BCO1 and BCO2 with carotenoids, adding structural diversity and importance in the agro-food and/or health sectors. Homology modeling of BCO1 and BCO2, and the molecular dynamics of complexes with all carotenoids were performed. Interaction energy and structures were analyzed. For both enzymes, the general structure is conserved with a seven beta-sheet structure, and the ß-carotene is positioned at an optimal distance from the catalytic center. Fe2+ forms in an octahedral coordination sphere with four perfectly conserved histidine residues. BCO1 finds stability in a structure in which the ß-carotene is positioned ready for enzymatic catalysis at the 15-15' bond, and BCO2 in positioning the bond to be cleaved (C9-C10) close to the active site. In BCO1 the carotenoids interact with only seven residues with aromatic rings, while the interaction of BCO2 is much more varied in terms of the type of interaction, with more residues of different chemical natures.

Modelling of the Citrus CCD4 Family Members: In Silico Analysis of Membrane Binding and Substrate Preference.

Coloring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of ß-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein-ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme.

Predictive value of plasma cytokines for acute kidney injury following lung resection surgery: prospective observational study / Valor preditivo das citocinas plasmáticas para lesão renal aguda após cirurgia de ressecção pulmonar: estudo observacional prospectivo

Abstract Background and objectives: Patients undergoing lung resection surgery are at risk of developing postoperative acute kidney injury. Determination of cytokine levels allows the detection of an early inflammatory response. We investigated any temporal relationship among perioperative inflammatory status and development of acute kidney injury after lung resection surgery. Furthermore, we evaluated the impact of acute kidney injury on outcome and analyzed the feasibility of cytokines to predict acute kidney injury. Methods: We prospectively analyzed 174 patients scheduled for elective lung resection surgery with intra-operative periods of one-lung ventilation periods. Fiberoptic broncho-alveolar lavage was performed in each lung before and after one-lung ventilation periods for cytokine analysis. As well, cytokine levels were measured from arterial blood samples at five time points. acute kidney injury was diagnosed within 48 h of surgery based on acute kidney injury criteria. We analyzed the association between acute kidney injury and cardiopulmonary complications, length of intensive care unit and hospital stays, intensive care unit re-admission, and short-term and long-term mortality. Results: The incidence of acute kidney injury in our study was 6.9% (12/174). Acute kidney injury patients showed higher plasma cytokine levels after surgery but differences in alveolar cytokines were not detected. Although no patient required renal replacement therapy, acute kidney injury patients had higher incidence of cardiopulmonary complications and increased overall mortality. Plasma interleukin-6 at 6 h was the most predictive cytokine of acute kidney injury (cut-off point at 4.89 pg.mL-1). Conclusions: Increased postoperative plasma cytokine levels are associated with acute kidney injury after lung resection surgery in our study, which worsens the prognosis. Plasma interleukin-6 may be used as an early indicator for patients at risk of developing acute kidney injury after lung resection surgery.

Resumo Justificativa e objetivos: Os pacientes submetidos à cirurgia de ressecção pulmonar apresentam risco de desenvolver lesão renal aguda pós-operatória. A determinação dos níveis de citocinas permite detectar uma resposta inflamatória precoce. Investigamos a relação temporal entre o estado inflamatório perioperatório e o desenvolvimento de lesão renal aguda após cirurgia de ressecção pulmonar. Além disso, avaliamos o impacto da lesão renal aguda no desfecho e analisamos a viabilidade das citocinas para prever este tipo de lesão. Métodos: No total, foram analisados prospectivamente 174 pacientes agendados para cirurgia eletiva de ressecção pulmonar com períodos intraoperatórios de ventilação monopulmonar. Lavado bronco-alveolar com fibra óptica foi realizado em cada pulmão antes e após os períodos de ventilação monopulmonar para análise das citocinas. Os níveis de citocina foram medidos a partir de amostras de sangue arterial em cinco momentos. A lesão renal aguda foi diagnosticada dentro de 48 horas após a cirurgia, com base nos critérios para sua verificação. Analisamos a associação entre lesão renal aguda e complicações cardiopulmonares, tempo de internação em unidade de terapia intensiva e de internação hospitalar, reinternação em unidade de terapia intensiva e mortalidade a curto e longo prazos. Resultados: A incidência de lesão renal aguda no estudo foi de 6,9% (12/174). Os pacientes com lesão renal aguda apresentaram níveis mais altos de citocinas plasmáticas após a cirurgia, mas não foram detectadas diferenças nas citocinas alveolares. Embora nenhum paciente tenha precisado de terapia renal substitutiva, os com lesão renal aguda apresentaram maior incidência de complicações cardiopulmonares e aumento da mortalidade geral. A interleucina-6 plasmática em seis horas foi a citocina mais preditiva de lesão renal aguda (ponto de corte em 4,89 pg.mL-1). Conclusões: O aumento dos níveis plasmáticos de citocinas no pós-operatório está associado à lesão renal aguda após cirurgia de ressecção pulmonar no estudo, o que piora o prognóstico. A interleucina-6 plasmática pode ser usada como um indicador precoce para pacientes com risco de desenvolver lesão renal aguda após cirurgia de ressecção pulmonar.

[Predictive value of plasma cytokines for acute kidney injury following lung resection surgery: prospective observational study]. / Valor preditivo das citocinas plasmáticas para lesão renal aguda após cirurgia de ressecção pulmonar: estudo observacional prospectivo.

BACKGROUND AND OBJECTIVES: Patients undergoing lung resection surgery are at risk of developing postoperative acute kidney injury. Determination of cytokine levels allows the detection of an early inflammatory response. We investigated any temporal relationship among perioperative inflammatory status and development of acute kidney injury after lung resection surgery. Furthermore, we evaluated the impact of acute kidney injury on outcome and analyzed the feasibility of cytokines to predict acute kidney injury. METHODS: We prospectively analyzed 174 patients scheduled for elective lung resection surgery with intra-operative periods of one-lung ventilation. Fiberoptic broncho-alveolar lavage was performed in each lung before and after one-lung ventilation periods for cytokine analysis. As well, cytokine levels were measured from arterial blood samples at five time points. Acute kidney injury was diagnosed within 48h of surgery based estabilished criteria for its diagnosis. We analyzed the association between acute kidney injury and cardiopulmonary complications, length of intensive care unit and hospital stays, intensive care unit re-admission, and short-term and long-term mortality. RESULTS: The incidence of acute kidney injury in our study was 6.9% (12/174). Acute kidney injury patients showed higher plasma cytokine levels after surgery, but differences in alveolar cytokines were not detected. Although no patient required renal replacement therapy, acute kidney injury patients had higher incidence of cardiopulmonary complications and increased overall mortality. Plasma interleukin-6 at 6h was the most predictive cytokine of acute kidney injury (cut-off point at 4.89pg.mL-1). CONCLUSIONS: Increased postoperative plasma cytokine levels are associated with acute kidney injury after lung resection surgery in our study, which worsens the prognosis. Plasma interleukin-6 may be used as an early indicator for patients at risk of developing acute kidney injury after lung resection surgery.

An in silico study of the citrus dioxygenases CCD4 family substrates.

The coloration of Citrus fruits is related with the concentration of carotenoids, isoprenoid pigments of 40 carbon atoms (C40). Rodrigo et al. and Ma et al. reported a CCD4-type citrus dioxygenase responsible for the generation of C30 apocarotenoids providing a reddish-orange pigmentation to the peel of many mandarins and oranges. Among them, CCD4b was the first case described of a dioxygenase that cleaves carotenoids C40 in the double bond 7', 8' or 7, 8, generating ß-citraurin or 8-ß-apocarotenal. Here we report the three-dimensional structures of CCD4a and CCD4b, modeled by sequence homology (2BIW) and validated by molecular dynamics (MD). Docking calculations were performed in CCD4a and CCD4b structures with thousands of rotated initial carotenoid conformations and all the possible poses in the active site were found. The interaction energy was measured by means of ASE scoring, Amber99 refinement and London ΔG rescoring. For the case of CCD4a model, the results showed London ΔG score of -19, -17 and -15 kcal/mol for zeaxanthin, ß-cryptoxanthin and ß-carotene, respectively. The same sequence in the estimated interaction strength for the three ligands was obtained using MD. The interaction energy of CCD4b indicated that, in agreement with experimental data, zeaxanthin and ß-cryptoxanthin could be cleaved by the enzyme, ß- and α-carotene have chances to be oxidized and lycopene has not good interaction energy to be predicted as substrate. These findings will be discussed considering the potential in vivo substrates and products, and the physiological role in Citrus fruits. Communicated by Ramaswamy H. Sarma.

Combining Mass Spectrometry and X-Ray Crystallography for Analyzing Native-Like Membrane Protein Lipid Complexes.

Membrane proteins represent a challenging family of macromolecules, particularly related to the methodology aimed at characterizing their three-dimensional structure. This is mostly due to their amphipathic nature as well as requirements of ligand bindings to stabilize or control their function. Recently, Mass Spectrometry (MS) has become an important tool to identify the overall stoichiometry of native-like membrane proteins complexed to ligand bindings as well as to provide insights into the transport mechanism across the membrane, with complementary information coming from X-ray crystallography. This perspective article emphasizes MS findings coupled with X-ray crystallography in several membrane protein lipid complexes, in particular transporters, ion channels and molecular machines, with an overview of techniques that allows a more thorough structural interpretation of the results, which can help us to unravel hidden mysteries on the membrane protein function.