Interactions of human serum albumin with phosphate and Tris buffers: impact on paclitaxel binding and nanoparticles self-assembly.

AIM: To investigate the conformational changes in human serum albumin (HSA) caused by chemical (CD) and thermal denaturation (TD) at pH 7.4 and 9.9, crucial for designing controlled drug delivery systems with paclitaxel (PTX). METHODS: Experimental and computational methods, including differential scanning calorimetry (DSC), UV-Vis and intrinsic fluorescence spectroscopy, mean diameter, polydispersity index (PDI), ζ-potential, encapsulation efficiency (EE), in vitro release and protein docking studies were conducted to study the HSA denaturation and nanoparticles (NPs) preparation. RESULTS: TD at pH 7.4 produced smaller NPs (287.1 ± 12.9 nm) than CD at pH 7.4 with NPs (584.2 ± 47.7 nm). TD at pH 9.9 exhibited high EE (97.3 ± 0.2%w/w) with rapid PTX release (50% within 1h), whereas at pH 7.4 (96.4 ± 2.1%w/w), release only 40%. ζ-potentials were around -30 mV. CONCLUSION: Buffer type and pH significantly influence NP properties. TD in PBS at pH 7.4, provided optimal conditions for a stable and efficient drug delivery system.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

Chiral separation of propranolol by electrokinetic chromatography using nanodiamonds and human serum albumin as a pseudo-stationary phase in river water.

Propranolol is currently considered as an emerging contaminant in water bodies. In this study, R- and S-propranolol were determined in river samples by electrokinetic chromatography (EKC) using nanodiamonds (NDs) and human serum albumin (HSA) as a pseudo-stationary phase in order to achieve enantioseparation. Previously, river samples were preconcentrated using a column filled with Amberlite® IR-120 and Dowex® 50WX8 resins. The setting up of influential factors such as temperature, voltage, pH, and HSA and NDs concentration is accurately described along this manuscript. A multivariate study and optimization was carried out to obtain the enantioseparation of propranolol (Rs = 2.91), which was reached under the following experimental conditions: voltage of 16 kV, temperature of 16°C, phosphate buffer pH 9.5, NDs of 0.20%, and HSA of 15 µmol l-1 . The recoveries of analytes under optimal conditions were higher than 98%. The limits of detection were 0.85 µg l-1 for R- and S-propranolol. The method was applied to real samples, and the obtained results in three different water sources studied were 1.02, 0.59, and 0.30 µg l-1 for the R-enantiomer and 0.99, 0.54, and 0.28 µg l-1 for the S-enantiomer. The accuracy of the proposed methodology (including bias and precision) has allowed us to propose it as a successful tool for the control of water quality.

Hollow-fiber liquid phase microextraction for determination of fluoxetine in human serum by nano-liquid chromatography coupled to high resolution mass spectrometry.

Therapeutic drug monitoring (TDM) is a personalized care tool based on the determination of a target drug concentration in human serum. An antidepressant drug of interest for such investigations is fluoxetine (FXT), due to a severe impact of genetic polymorphisms on its metabolism. A bioanalytical method employed for TDM purposes must exhibit satisfactory selectivity and detectability, which becomes more difficult due to highly complex biological matrices. In this study, a highly selective bioanalytical method for the determination of FXT in human serum is proposed, which provides excellent clean-up efficiency based on a low cost hollow fiber liquid-phase microextraction (HF-LPME) sample preparation step and nano-liquid chromatography coupled to high-resolution mass spectrometry (nano-LC-HRMS). HF-LPME was performed using a two-phase "U" configuration, with 6 cm fiber, 20 µL of 1-octanol acting as supported liquid membrane, and ammonium hydroxide (pH 10) as the donor phase with NaCl (10 % m/v) and methanol (5 % v/v) as additives, requiring only 250 µL of the sample. The procedure was conducted for 30 min under a 750 rpm stirring rate. Gradient elution was carried out employing an acetonitrile-water as mobile phase, the composition of 30:70 to 100:00 (v/v) for 15 min, using formic acid 0.1 % (v/v) as an additive. MS1 was acquired in an Orbitrap mass analyzer, while MS2 was acquired in a linear trap quadrupole. Satisfactory linearity (Pearson's r = 0.99709) was obtained for a concentration range of 0.02 to 2.5 µg mL-1, which is compatible with the therapeutic and toxic range for FXT. The developed method presents adequate precision (1.61 to 7.45 %) and accuracy (95 to 114 %) and allows the dilution of high concentration samples in a 1:4 ratio (v/v), enabling its application for forensic serum samples. To our knowledge, this is the first study reporting a method based on HF-LPME and nano-LC-HRMS with any analytical purpose, especially with a TDM focus.

Albúmina en cirrosis: perspectiva inflamatoria / Albumin in cirrhosis: inflammatory perspective

La albúmina sérica humana es la proteína más abundante en el plasma, su estructura molecular le confiere estabilidad, pero también flexibilidad para ligar y transportar un amplio rango de moléculas. Su función oncótica es la propiedad más reconocida que la lleva a introducirse en la terapéutica médica como un expansor de volumen. Sin embargo, en los últimos años se le han adicionado funciones con carácter antioxidante, inmunomodulador y de estabilización endotelial, que hacen presumir que su impacto terapéutico está más allá de sus funciones volumétricas. En los últimos años, específicamente en la cirrosis y la falla hepática aguda sobre crónica, se ha tenido un cambio en el paradigma fisiológico, desde una perspectiva netamente hemodinámica hacia una perspectiva inflamatoria, en donde las funciones oncóticas y no oncóticas de la albúmina están alteradas y tienen un carácter pronóstico en estas entidades. Este conocimiento creciente, desde una perspectiva inflamatoria, hace que se fortalezca el uso terapéutico de la albúmina sérica humana desde las indicaciones tradicionales como prevención de la disfunción circulatoria posparacentesis, prevención y tratamiento de lesión renal aguda, hasta las discusiones para administración a largo plazo en pacientes cirróticos con ascitis.

Human serum albumin is the most abundant protein in plasma, with a molecular structure that provides stability while also allowing flexibility to bind and transport a wide range of molecules. Its oncotic function is the most recognized property, leading to its introduction in medical therapy as a volume expander. However, in recent years, additional functions with antioxidant, immunomodulatory, and endothelial stabilization properties have been identified, suggesting that its therapeutic impact extends beyond its volumetric functions. Specifically, in cirrhosis and acute-on-chronic liver failure, there has been a shift in the pathophysiological paradigm from a purely hemodynamic perspective to an inflammatory perspective, where both oncotic and non-oncotic functions of albumin are altered and have prognostic significance in these conditions. This growing understanding from an inflammatory perspective strengthens the therapeutic use of human serum albumin, not only for traditional indications such as the prevention of post-paracentesis circulatory disfunction, prevention and treatment of acute kidney injury, but also for discussions regarding long-term administration in cirrhotic patients with ascites.

Bi-coloured enhanced luminescence imaging by targeted switch on/off laser MEF coupling for synthetic biosensing of nanostructured human serum albumin.

In this communication luminescent bioconjugated human serum albumin nanostructures (HSA NPs) with tiny ultraluminescent gold core-shell silica nanoparticles (Au@SiO2-Fl) were designed with enhanced bi-coloured luminescence properties. The HSA NPs were obtained from Human Serum Albumin free (HSA free) through the desolvation method, and Au@SiO2-Fl, through modified Turkevich and Störber methods. In this manner, porous HSA Nanostructures of 150.0-200 nm and Au@SiO2-Fl 45.0 nm final diameters were obtained. Both methodologies and structures were conjugated to obtain modified Nanocomposites based on tiny gold cores of 15 nm surrounded with well spatial Nanostructured architectures of HSA (d15 Au@SiO2-Fl-HSA) that generated variable nanopatterns depending on the modified methodology of synthesis applied within colloidal dispersions. Therefore, three methodologies of non-covalent conjugation were developed. In optimal conditions, through Transmission Electronic Microscopy (TEM), well resolved multilayered nano-architectures with a size 190.0-200 nm in average with variable contrast depending of the focused nanomaterial within the nanocomposite were shown. Optimized nanoarchitecture was based on a template tiny gold core-shell surrounded by nanostructured HSA NPs (d15 Au@SiO2-Fl-HSA). In this manner, the NanoImaging generated by laser fluorescence microscopy permitted to record improved optical properties and functionalities, such as: (i) enhanced ultraluminescent d15 Au@SiO2-Fl-HSA composites in comparison to individual components based on Metal Enhanced Fluorescence (MEF); (ii) diminished photobleaching; (iii) higher dispersibility; (iv) higher resolution of single bright nano-emitters of 210.0 nm sizes; and (v) enhanced bi-coloured Bio-MEF coupling with potential non-classical light delivery towards other non-optical active biostructures for varied applications. The characterization of these nanocomposites allowed the comparison, evaluation and discussion focused on new properties generated and functionalities based on the incorporation of different types of tuneable materials. In this context, the biocompatibility, Cargo confined spaces, protein-based materials, optical transparent could be highlighted, as well as optical active materials. Thus, the potential applications of nanotechnology to both nanomedicine and nano-pharmaceutics were discussed.

Análise de custos de pacientes cirróticos descompensados recebendo albumina a longo prazo versus tratamento médico padrão nos sistemas de saúde público e privado do Brasil / Albumin versus standard medical treatment in brazilian public and private healthcare systems

ABSTRACT Background: Cirrhosis is one of the final stages of chronic liver disease. Common causes of cirrhosis include alcoholism and viral hepatitis infections. Cirrhosis can progress from an asymptomatic, compensated phase to decompensation and the appearance of overt symptoms. There is no specific treatment for decompensated cirrhosis. The ANSWER trial positioned long-term albumin infusions as a potential treatment for patients with cirrhosis and uncomplicated ascites. Objective: This study assesses the economic impact of albumin infusions following the ANSWER trial regimen in Brazilian patients with decompensated cirrhosis from the public and private healthcare systems perspectives. Methods: The incremental cost per patient per year was calculated for standard medical treatment (SMT) plus long-term albumin infusions versus SMT alone. Costs of diuretics and albumin were obtained from Banco de Preços em Saúde and the Drug Market Regulation Chamber. Costs for complication and procedures were gathered from the published literature. Costs were transformed to 2021 Brazilian reals (BRL). Incidences of clinical complications and treatments were gathered from the ANSWER trial. Univariate sensitivity analysis was performed by increasing and decreasing all inputs by 20%. Results: The cost per patient per year was 118,759 BRL and 189,675 BRL lower for patients treated with SMT and albumin (compared to SMT only) for the public and private healthcare systems, respectively. The additional cost of albumin was offset by reduced complications and treatments (149,526 BRL and 249,572 BRL, respectively). The univariate sensitivity analysis showed cost savings for both healthcare systems in all the scenarios assessed. Conclusion: This economic analysis suggests that, if the ANSWER trial clinical outcomes translate into real-world effectiveness, addition of albumin infusions to SMT in patients with decompensated cirrhosis may lead to cost savings for the public and private healthcare systems in Brazil.

RESUMO Contexto: A cirrose representa o estágio final da doença hepática crônica. Causas comuns de cirrose incluem alcoolismo e infecções por hepatite viral. A cirrose pode progredir de uma fase compensada assintomática para descompensação e aparecimento de sintomas evidentes. Não há tratamento específico para cirrose descompensada. O estudo ANSWER demonstrou que a administração de albumina a longo prazo pode representar um potencial tratamento para pacientes com cirrose e ascite não complicada. Objetivo: Nosso estudo avalia o impacto econômico da administração de albumina a longo prazo seguindo o protocolo do estudo ANSWER em pacientes brasileiros com cirrose descompensada, sob a perspectiva dos sistemas de saúde público e privado. Métodos: O custo incremental por paciente por ano foi calculado para o tratamento médico padrão (SMT) associado a administração de albumina a longo prazo comparado a SMT apenas. Os custos de diuréticos e albumina foram obtidos no Banco de Preços em Saúde e na Câmara de Regulação do Mercado de Medicamentos. Os custos de complicações e procedimentos foram coletados da literatura publicada. Os custos foram transformados em Reais de 2021 (BRL). As incidências de complicações clínicas e tratamentos foram coletadas do estudo ANSWER. Uma análise de sensibilidade univariada foi realizada aumentando e diminuindo todas as variáveis em 20%. Resultados: O custo por paciente por ano foi de R$ 118.759 e R$ 189.675 menor para pacientes tratados com SMT e albumina (comparado apenas com SMT) para os sistemas de saúde público e privado, respectivamente. O custo adicional da albumina foi compensado pela redução de complicações e tratamentos (149.526 BRL e 249.572 BRL, respectivamente). A análise de sensibilidade univariada mostrou redução de custos para ambos os sistemas de saúde em todos os cenários avaliados. Conclusão: Esta análise econômica sugere que, se os resultados clínicos do estudo ANSWER se confirmarem no mundo real, a administração de albumina associada ao SMT em pacientes com cirrose descompensada pode levar a redução de custos para os sistemas de saúde público e privado no Brasil.

Direct Oral FXa Inhibitors Binding to Human Serum Albumin: Spectroscopic, Calorimetric, and Computational Studies.

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 104 M-1. However, the ITC studies reported significantly different binding constants (103 M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

Molecular diagnosis of abdominal angiostrongyliasis by PCR using serum samples.

Abdominal angiostrongyliasis (AA) is a zoonotic disease caused by the nematode Angiostrongylus costaricensis, which is endemic in southern Brazil. Humans become infected by ingesting third-stage (L3) larvae and are considered accidental hosts since neither eggs nor first-stage (L1) larvae are found in feces. The definitive diagnosis can be made by histopathologic examination of surgical specimens or intestinal biopsies. The present study assessed the use of PCR to carry out the molecular detection of AA from serum samples. A total of 62 human serum samples were divided into three groups: (i) 28 serum samples from human patients with presumptive histopathological diagnosis of AA; (ii) 23 serum samples from individuals with unknown serology for AA; (iii) 11 serum samples from patients that suffered from different parasitosis were included. The serum samples were initially tested by in-house indirect ELISA and then by PCR. A total of 14 samples were positive by ELISA, and 6 were positive by PCR. Six samples that were negative by ELISA were positive by PCR. Amplicons were sequenced, and Angiostrongylus DNA was confirmed. We conclude that PCR amplification can be used to confirm Angiostrongylus DNA in serum, which is especially important in cases where antibody levels are too low to be detected. It may also serve as a useful target for survey studies.

Development of melatonin-loaded, human-serum-albumin nanoparticles formulations using different methods of preparation for ophthalmic administration.

The use of proteins such as human serum albumin (HSA) to form nanometric systems seems very promising since they are non-toxic, biodegradable and have no antigenic activity. This molecule is ideal to transport insoluble drugs such as melatonin (Mel), which has antiapoptotic and antioxidant properties and appears promising for the treatment of neurodegenerative eye diseases. The objective of this study was to obtain nanoparticulate systems loaded with Mel, improving the conventional desolvation method. Systems were stabilised using two different strategies: one through the use of Eudragit S100 as a cross-linking agent and the other through thermal stabilisation. The systems thus obtained (Np-HSA-Eu-Mel and Np-HSA-Mel, respectively) were characterised and compared in terms of physicochemical and pharmacotechnical parameters. Whitish colloidal dispersions of nanometric size (≈170 nm), spherical shape, and monodisperse population were obtained. Besides, the pH was close to neutrality reaching 20 % drug encapsulation whereas the process performance was higher than 80 %. In FT-IR studies, thermal analysis and X-ray diffraction (XRD), the incorporation of the drug in the cavities of the nanoparticles could be evidenced. Regarding the physical stability of nanoparticles, for Np-HSA-Eu-Mel instability was observed at pH > 7. However, Np-HSA-Mel was able to remain stable at different pH levels. Mel release from these systems was consequently affected, where the former released faster than the active compared to the last. On the other hand, it was observed that the drying process (lyophilization in this case) applied to the nanoparticles suspensions does not affect their original properties after redispersion over a three months period. Likewise, the formulation did not produce irritation when administered topically, whereas when administered subconjunctivally, only slight irritation was observed 24 h after administration. According to the result of this study, the Np-HSA-Mel formulation could achieve advantageous properties as a vehicle for the transport of insoluble drugs for the treatment of neurodegenerative diseases at the ocular level.

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

Protein Adsorption onto Modified Porous Silica by Single and Binary Human Serum Protein Solutions.

Typical porous silica (SBA-15) has been modified with pore expander agent (1,3,5-trimethylbenzene) and fluoride-species to diminish the length of the channels to obtain materials with different textural properties, varying the Si/Zr molar ratio between 20 and 5. These porous materials were characterized by X-ray Diffraction (XRD), N2 adsorption/desorption isotherms at -196 °C and X-ray Photoelectron Spectroscopy (XPS), obtaining adsorbent with a surface area between 420-337 m2 g-1 and an average pore diameter with a maximum between 20-25 nm. These materials were studied in the adsorption of human blood serum proteins (human serum albumin-HSA and immunoglobulin G-IgG). Generally, the incorporation of small proportions was favorable for proteins adsorption. The adsorption data revealed that the maximum adsorption capacity was reached close to the pI. The batch purification experiments in binary human serum solutions showed that Si sample has considerable adsorption for IgG while HSA adsorption is relatively low, so it is possible its separation.

Self-assembled micelles of the (lipo) glycopeptides, teicoplanin, as taxane nanocarriers.

The use of nanoparticles is one of the strategies currently studied to minimize the toxicity and lack of tissue specificity of many cancer drugs used in chemotherapy. In this research the physicochemical and biological behavior of a novel self-assembled nanostructure of the antibiotic Teicoplanin (Teico) was characterized as a nanocarrier system for solubilizing highly hydrophobic drugs like Paclitaxel (Ptx) in aqueous media. The Teico micelles were loaded with Ptx in DMSO or PEG-400. The interaction between the loaded micelles and Albumin human serum albumin (HSA) was then studied by size exclusion chromatography. Transmission electron microscopy, dynamic light scattering and high-resolution liquid chromatography were also used to characterize the physicochemical and structural properties of the micelles to form the Teico/Ptx and Teico/Ptx/HSA micelles. Cellular uptake of Ptx was evaluated by fluorescent microscopy. Thein vitrocytotoxicity of the complexes was studied on Hep-2 tumor cells, by a Crystal Violet assay. Teico cosolvent-free micelles can solubilize up to 20 mg.ml-1of Ptx dissolved in PEG, increasing four times the solubility of Ptx in water compared to Abraxane, and 20 000 times the intrinsic solubility of Ptx in water. In addition, Teico/Ptx micelles binds spontaneously HSA through hydrophobic interaction. Teico and Teico/HSA micelles as a Ptx transporter does not affect its release or biological activity. Therefore, Teico/Ptx or Teico/Ptx/HSA complexes appear as new alternatives for transporting larger amounts of hydrophobic drugs that offer advantages, turning it an interesting option for further study.

Human serum albumin-resveratrol complex formation: Effect of the phenolic chemical structure on the kinetic and thermodynamic parameters of the interactions.

The thermodynamics and kinetics of binding between human serum albumin (HSA) and resveratrol (RES) or its analog (RESAn1) were investigated by surface plasmon resonance (SPR). The binding constant and the kinetic constants of association and dissociation indicated that RESAn1 has higher affinity toward HSA than does RES. The formation of these complexes was entropically driven ( [Formula: see text] , [Formula: see text]  KJ mol-1). However, for both polyphenols, the activation energy (Eact) of association (a) of free molecules was higher than that for dissociation (d) of the stable complex ( [Formula: see text]  KJ mol-1), and the rate of association was faster than that of dissociation since the activation Gibbs free energy (ΔG‡) was lower for the former (ΔGaHSA-RES‡â‰…54.73,ΔGdHSA-RES‡â‰…73.83,ΔGaHSA-RESAn1‡â‰…54.14,ΔGdHSA-RESAn1‡â‰…73.97 KJ mol-1). This study showed that small differences in the structure of polyphenols such as RES and RESAn1 influenced the thermodynamics and kinetics of the complex formation with HSA.

Studies on the interaction between HSA and new halogenated metformin derivatives: influence of lipophilic groups in the binding ability.

In the type II diabetes mellitus, Metformin hydrochloride is recommended as a common FAD approved drug. Synthesis of novel metformin series has been widely explored, mainly due to its biological importance and to improve their pharmacokinetic profile. Generally, human serum albumin (HSA) is the main protein used to study drug viability in vitro analysis. Thus, the present study reports the synthesis of three new halogenated metformin derivatives (MFCl, MFBr and MFCF3) and its interaction toward HSA by multiple spectroscopic techniques (UV-Vis, circular dichroism, steady-state, time-resolved and synchronous fluorescence), combined to computational methods (molecular docking and quantum chemical calculation). The interaction between each halogenated metformin derivative and HSA is spontaneous (ΔG°<0), entropically driven (ΔS°>0), moderate (Ka and Kb ≈ 104 M-1) and occurs preferentially in the subdomain IIA (close to Trp-214 residue). Molecular docking results suggested hydrogen bonding, van der Waals and hydrophobic interactions as the main binding forces. Quantum chemical calculations suggested imino groups as the most intense electrostatic negative potentials, while the positive electrostatic potential is located at the hydrogen atoms on N,N-dimethyl and the phenyl systems which can help the hydrophobic interactions. [Formula: see text]Communicated by Ramaswamy H. Sarma.

Enzyme-Linked Aptamer Assay (ELAA) for Detection of Toxoplasma ROP18 Protein in Human Serum.

Toxoplasma gondii engenders the common parasitic disease toxoplasmosis in almost all warm-blooded animals. Being a critical secretory protein, ROP18 is a major virulence factor of Toxoplasma. There are no reports about ROP18 detection in human serum samples with different clinical manifestations. New aptamers against ROP18 protein were developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX). An Enzyme-Linked Aptamer Assay (ELAA) platform was developed using SELEX-derived aptamers, namely AP001 and AP002. The ELAA was used to evaluate total antigen from T. gondii RH strain (RH Ag) and recombinant protein of ROP18 (rROP18). The results showed that the ELAA presented higher affinity and specificity to RH Ag and rROP18, compared to negative controls. Detection limit of rROP18 protein in serum samples was measured by standard addition method, achieving a lower concentration of 1.56 µg/mL. Moreover, 62 seropositive samples with different clinical manifestations of toxoplasmosis and 20 seronegative samples were tested. A significant association between ELAA test positive for human serum samples and severe congenital toxoplasmosis was found (p = 0.006). Development and testing of aptamers-based assays opens a window for low-cost and rapid tests looking for biomarkers and improves our understanding about the role of ROP18 protein on the pathogenesis of human toxoplasmosis.

Evaluation of Novel Chalcone-Thiosemicarbazones Derivatives as Potential Anti-Leishmania amazonensis Agents and Its HSA Binding Studies.

A series of seven chalcone-thiosemicarbazones (5a-5g) were synthesized and evaluated as potential new drugs (anti-leishmanial effect). Although four of the chalcone-thiosemicarbazones are already known, none of them or any compound in this class has been previously investigated for their effects on parasites of the Leishmania genus. The compounds were prepared in satisfactory yields (40-75%) and these compounds were evaluated against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis after 48 h of culture. The half maximal inhibitory concentration (IC50) values of the intracellular amastigotes were determined to be in the range of 3.40 to 5.95 µM for all compounds assayed. The selectivity index showed value of 15.05 for 5a, whereas pentamidine (reference drug) was more toxic in our model (SI = 2.32). Furthermore, to understand the preliminary relationship between the anti-leishmanial activity of the chalcone-thiosemicarbazones, their electronic (σ), steric (MR) and lipophilicity (π) properties were correlated, and the results indicated that moieties with electronic withdrawing effects increase the anti-leishmanial activity. The preliminary pharmacokinetic evaluation of one of the most active compound (5e) was studied via interaction to human serum albumin (HSA) using multiple spectroscopic techniques combined with molecular docking. The results of antiparasitic effects against L. amazonensis revealed the chalcone-thiosemicarbazone class to be novel prototypes for drug development against leishmaniasis.

In vitro tyrosinase, acetylcholinesterase, and HSA evaluation of dioxidovanadium (V) complexes: An experimental and theoretical approach.

The present study reports the biological evaluation of vanadium(V) complexes (1-3) against three different proteins: tyrosinase, acetylcholinesterase (AChE), and human serum albumin (HSA), which were studied by spectroscopic techniques and molecular docking. Despite the synthesis and characterization of complexes 1 and 2 having already previously described, complex 3 is a novel dioxidovanadium(V) derivative. Complex 1 can activate both tyrosinase and AChE enzymes in about 11.5 and 47.0%, respectively. On the other hand, complexes 2 and 3 inhibited the same enzymes (1.30 and 46.0% for tyrosinase and 20.0 and 21.9% for AChE, respectively). Molecular docking calculations suggested that the presence of the hydroxyl group in complex 1 is essential to activate tyrosinase enzymes. According to theoretical analysis, hydrogen bonding, van der Waals, and hydrophobic forces are the main binding interactions for each V(V) complex and AChE. Moreover, the interaction between HSA and vanadium(V) complexes occurs via ground-state association, being only enthalpically driven for complexes 1 and 2 and entropically and enthalpically driven for complex 3. The interaction is spontaneous for all samples and the binding modes do not perturb significantly the secondary and surface structures of the albumin. As there are few reported cases in the literature that explore vanadium complexes against these three proteins, the present results may contribute to future studies by offering different scaffolds to design new vanadium(V) complexes in the hyperpigmentation process and Alzheimer's disease.