SpotLight Proteomics Identifies Variable Sequences of Blood Antibodies Specific Against Deamidated Human Serum Albumin.

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in the blood of patients with Alzheimer's disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+ binding.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Thermal effects and drugs competition on the palmitate binding capacity of human serum albumin.

Human serum albumin (HSA) is the most abundant plasma protein of the circulatory system. It is a multidomain, multifunctional protein that, combining diverse affinities and wide specificity, binds, stores, and transports a variety of biological compounds, pharmacores, and fatty acids. HSA is finding increasing uses in drug-delivery due to its ability to carry functionalized ligands and prodrugs. All this raises the question of competition for binding sites occupancy in case of multiple ligands, which in turn influences the protein structure/dynamic/function relationship and also has an impact on the biomedical applications. In this work, the effects of interactive binding of palmitic acid (PA), warfarin (War) and ibuprofen (Ibu) on the thermal stability of HSA were studied using DSC, ATR-FTIR, and EPR. PA is a high-affinity physiological ligand, while the two drugs are widely used for their anticoagulant (War) and anti-inflammatory (Ibu) efficacy, and are exogenous compounds that accommodate in the deputed drug site DS1 and DS2, respectively overlapping with some of the fatty acid binding sites. The results indicate that HSA acquires the highest thermal stability when it is fully saturated with PA. The binding of this physiological ligand does not hamper the binding of War or Ibu to the native state of the protein. In addition, the three ligands bind simultaneously, suggesting a synergic cooperative influence due to allosteric effects. The increased thermal stability subsequent to binary and multiple ligands binding moderates protein aggregation propensity and restricts protein dynamics. The biophysics findings provide interesting features about protein stability, aggregation, and dynamics in interaction with multiple ligands and are relevant in drug-delivery.

Anticancer effects of simvastatin-loaded albumin nanoparticles on monolayer and spheroid models of breast cancer.

Breast cancer is a prominent cause of death among women and is distinguished by a high occurrence of metastasis. From this perspective, apart from conventional therapies, several alternative approaches have been researched and explored in recent years, including the utilization of nano-albumin and statin medications like simvastatin. The objective of this study was to prepare albumin nanoparticles incorporating simvastatin by the self-assembly method and evaluate their impact on breast cancer metastasis and apoptosis. The data showed the prepared nanoparticles have a diameter of 185 ± 24nm and a drug loading capacity of 8.85 %. The findings exhibit improved release in a lysosomal-like environment and under acidic pH conditions. MTT data showed that nanoparticles do not exhibit a dose-dependent effect on cells. Additionally, the results from MTT, flow cytometry, and qPCR analyses demonstrated that nanoparticles have a greater inhibitory and lethal effect on MDA-MB-231 cells compared to normal simvastatin. And cause cells to accumulate in the G0/G1 phase, initiating apoptotic pathways by inhibiting cell cycle progression. Nanoparticles containing simvastatin can prevent cell invasion and migration in both monolayer and spheroid models, as compared to simvastatin alone, at microscopic levels and in gene expression. The obtained data clearly showed that, compared to simvastatin, nanoparticles containing simvastatin demonstrated significant efficacy in suppressing the growth, proliferation, invasion, and migration of cancer cells in monolayer (2D) and spheroid (3D) models.

Hierarchical-Bioinspired MOFs Enhanced Electromagnetic Wave Absorption.

Proteins exhibit complex and diverse multi-dimensional structures, along with a wide range of functional groups capable of binding metal ions. By harnessing the unique characteristics of proteins, it is possible to enhance the synthesis of metal-organic frameworks (MOFs) and modify their morphology. Here, the utilization of biomineralized bovine serum albumin (BSA) protein as a template for synthesizing Mil-100 with superior microwave absorption (MA) properties is investigated. The multi-dimensional structure and abundant functional groups of biomineralized BSA protein make it an ideal candidate for guiding the synthesis of Mil-100 with intricate network structures. The BSA@Mil-100 synthesized using this method exhibits exceptional uniformity and monodispersity of nanocrystals. The findings suggest that the BSA protein template significantly influences the regulation of nanocrystal and microstructure formation of Mil-100, resulting in a highly uniform and monodisperse structure. Notably, the synthesized 2-BSA@Mil-100 demonstrates a high reflection loss value of -58 dB at 8.85 GHz, along with a maximum effective absorption bandwidth value of 6.79 GHz, spanning from 6.01 to 12.8 GHz. Overall, this study highlights the potential of utilizing BSA protein as a template for MOF synthesis, offering an effective strategy for the design and development of high-performance MA materials.

Tissue-Adaptive BSA Hydrogel with Dual Release of PTX and bFGF Promotes Spinal Cord Injury Repair via Glial Scar Inhibition and Axon Regeneration.

Spinal cord injury (SCI) is a severe clinical disease usually accompanied by activated glial scar, neuronal axon rupture, and disabled motor function. To mimic the microenvironment of the SCI injury site, a hydrogel system with a comparable mechanical property to the spinal cord is desirable. Therefore, a novel elastic bovine serum albumin (BSA) hydrogel is fabricated with excellent adhesive, injectable, and biocompatible properties. The hydrogel is used to deliver paclitaxel (PTX) together with basic fibroblast growth factor (bFGF) to inhibit glial scar formation as well as promote axon regeneration and motor function for SCI repair. Due to the specific interaction of BSA with both drugs, bFGF, and PTX can be controllably released from the hydrogel system to achieve an effective concentration at the wound site during the SCI regeneration process. Moreover, benefiting from the combination of PTX and bFGF, this bFGF/PTX@BSA system significantly aided axon repair by promoting the elongation of axons across the glial scar with reduced reactive astrocyte secretion. In addition, remarkable anti-apoptosis of nerve cells is evident with the bFGF/PTX@BSA system. Subsequently, this multi-functionalized drug system significantly improved the motor function of the rats after SCI. These results reveal that bFGF/PTX@BSA is an ideal functionalized material for nerve repair in SCI.

Immunological Quantitation of the Glycation Site Lysine-414 in Serum Albumin in Human Plasma Samples by Indirect ELISA Using Highly Specific Monoclonal Antibodies.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance.

Transport of Zinc-Phthalocyanine to Cancer Cells Using Myoglobin-Albumin Fusion Protein for Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive approach to cancer treatment, wherein cell death is initiated by singlet oxygen (1O2) production via energy transfer from excited photosensitizers to ground-state O2. Effective clinical photosensitizers necessitate water solubility for in vivo administration. Hydrophobic dyes, such as phthalocyanines, cannot be used directly as photosensitizers. Herein, we synthesized a myoglobin-(human serum albumin) fusion protein reconstituted with zinc-phthalocyanine (ZnPc), termed ZnPcMb-HSA. The photophysical properties of ZnPcMb-HSA closely resemble those of ZnPc-substituted Mb. Notably, ZnPc dissociates from ZnPcMb-HSA and selectively accumulates within cancer cells, while the protein components remain extracellular. Treatment of four distinct cell lines with ZnPcMb-HSA, followed by red-light irradiation, effectively induced apoptosis. The half-maximal inhibitory concentrations (IC50) against these cancer cell lines ranged between 0.1-0.5 µM. Reconstituted Mb-HSA emerges as a promising carrier for transporting various water-insoluble porphyrinoid photosensitizer to target cancer cells in PDT applications.

Investigation of steric hindrance effect on the interactions between four alkaloids and HSA by isothermal titration calorimetry and molecular docking.

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA.

Exploring the interaction of a potent anti-cancer drug Selumetinib with bovine serum albumin: Spectral and computational attributes.

The binding of drugs to plasma proteins determines its fate within the physiological system, hence profound understanding of its interaction within the bloodstream is important to understand its pharmacodynamics and pharmacokinetics and thereby its therapeutic potential. In this regard, our work delineates the mechanism of interaction of Selumetinib (SEL), a potent anti-cancer drug showing excellent effect against multiple solid tumors, with plasma protein bovine serum albumin (BSA), using methods such as absorption, steady-state fluorescence, time-resolved, fluorescence resonance energy transfer, Fourier transform infrared spectra (FTIR), circular dichroism (CD), synchronous and 3D-fluorescence, salt fluorescence, molecular docking and molecular dynamic simulations. The BSA fluorescence intensity was quenched with increasing concentration of SEL which indicates interactions of SEL with BSA. Stern-Volmer quenching analysis and lifetime studies indicate the involvement of dynamic quenching. However, some contributions from the static quenching mechanism could not be ruled out unambiguously. The association constant was found to be 5.34 × 105 M-1 and it has a single binding site. The Förster distance (r) indicated probable energy transmission between the BSA and SEL. The positive entropy changes and enthalpy change indicate that the main interacting forces are hydrophobic forces, also evidenced by the results of molecular modeling studies. Conformation change in protein framework was revealed from FTIR, synchronous and 3D fluorescence and CD studies. Competitive binding experiments as well as docking studies suggest that SEL attaches itself to site I (subdomain IIA) of BSA where warfarin binds. Molecular dynamic simulations indicate the stability of the SEL-BSA complex. The association energy between BSA and SEL is affected in the presence of different metals differently.

Biophysical and structural characterization of tetramethrin serum protein complex and its toxicological implications.

Tetramethrin (TMT) is a commonly used insecticide and has a carcinogenic and neurodegenerative effect on humans. The binding mechanism and toxicological implications of TMT to human serum albumin (HSA) were examined in this study employing a combination of biophysical and computational methods indicating moderate binding affinity and potential hepato and renal toxicity. Fluorescence quenching experiments showed that TMT binds to HSA with a moderate affinity, and the binding process was spontaneous and predominantly enthalpy-driven. Circular dichroism spectroscopy revealed that TMT binding did not induce any significant conformational changes in HSA, resulting in no changes in its alpha-helix content. The binding site and modalities of TMT interactions with HSA as computed by molecular docking and molecular dynamics simulations revealed that it binds to Sudlow site II of HSA via hydrophobic interactions through its dimethylcyclopropane carboxylate methyl propanyl group. The structural dynamics of TMT induce proper fit into the binding site creating increased and stabilizing interactions. Additionally, molecular mechanics-Poisson Boltzmann surface area calculations also indicated that non-polar and van der Waals were found to be the major contributors to the high binding free energy of the complex. Quantum mechanics (QM) revealed the conformational energies of the binding confirmation and the degree of deviation from the global minimum energy conformation of TMT. The results of this study provide a comprehensive understanding of the binding mechanism of TMT with HSA, which is important for evaluating the toxicity of this insecticide in humans.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites.

BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated. METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS. RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated. CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Lower Serum Albumin Level: A Prospective Risk Predictor of Hepatocellular Carcinoma in Patients With Chronic Hepatitis B Virus Infection.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours in China, at high annual incidence and mortality. Chronic hepatitis B virus infection (CHB) is considered as a leading cause to bring about HCC in China. Serum albumin (ALB) level has been adopted to verify its risk with HCC development as a combination variable with other factors. However, the predictive value of a single ALB level on HBV-related HCC risk remained unclear. The aim of this study was to evaluate the prediction ability of serum ALB concentration on the risk of HBV-related HCC development. A prospectively enrolled clinical cohort compromising 2932 cases of CHB patients with at least 1-year exclusion window was selected to explore the predictive role of serum ALB level on incident HCC risk. Baseline clinical data including host characters and laboratory test were collected at the initial period of hospitalisation. The hazard ratio of ALB level associated with HCC development was assessed by Cox proportional hazards regression model using univariate and multivariate analyses. We evaluated the discrimination accuracy of ALB level in predicting HCC development by receiver operating characteristic (ROC) curves. Dose-dependent and time-dependent effects of ALB level on HCC risk prediction were demonstrated, respectively, using a restricted cubic spline and a Fine and Grey competing risk model. Referred to patients with higher ALB level, those with lower ALB level exhibited significantly increased risk of HCC development after adjustment for host variables (dichotomised analyses: hazard ratio = 3.12, 95% confidence interval 1.63-5.97, p = 8.23 × 10-4, plog-rank = 5.97 × 10-4; tertile analyses: hazard ratio = 2.07, 95% confidence interval 1.63-2.64, p = 3.77 × 10-9, plog-rank < 2.00 × 10-16; quartile analyses: hazard ratio = 2.10, 95% confidence interval 1.56-2.84, p = 9.87 × 10-7, plog-rank < 2.00 × 10-16). There was a statistically increasing trend on HCC risk which was found following by the decrease of ALB level (ptrend < 0.0001). Similar findings were present by the Kaplan-Meier analysis, cumulative incidences of HCC development were significantly higher in patients with lower ALB levels, with the p value obtained from log-rank test were all < 0.0001. The result of dose-dependent effect showed hazard ratio (HR) value of HCC risk was gradually decreasing as the increasing of ALB level, with non-linear correlation being statistically significant (Wald χ2 = 20.59, p = 0.000). HR value in lower ALB level remained persistently prominent by fluctuating around 2.73 in the whole follow-up time by adjusting for host variables. Sub-cohort analysis by ROC revealed that the discrimination ability of the ALB model was performed better than Child-Pugh (C-P) model in both cohort of patients with 1-year (area under curve [AUC] 0.762 vs. 0.720) and 2-year exclusion window (AUC 0.768 vs. 0.728). The AUC added by ALB level was demonstrated significantly from host model to full model. Lower ALB level was significantly associated with an increased risk of HBV-related HCC and could provide extra useful clinical utility to other host features, which might be a promising non-invasive indicator for surveillance on HCC development.

Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237 nm. These aggregates experienced a dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8 mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68 mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

Predictors for the Development of Thromboembolic Events in Cancer Patients Treated with Bevacizumab, Ramucirumab, and Aflibercept: A Single-Institution Retrospective Analysis.

INTRODUCTION: The risk of thromboembolic events developing limits the dose of antiangiogenic agents, thereby reducing their efficacy. This retrospective study therefore sought to identify predictors for the development of antiangiogenic agent-induced thromboembolic events and to elucidate whether differences in the likelihood of thromboembolic events exist between different antiangiogenic agents or cancer types, to guide future strategies for optimizing safety, efficacy, and quality of life in patients receiving chemotherapy. METHODS: This study retrospectively investigated 468 cancer patients who received chemotherapy with bevacizumab, ramucirumab, or aflibercept at our outpatient chemotherapy center between December 2016 and April 2022. Variables related to the development of thromboembolic events were extracted from the medical records, and multivariate logistic regression analysis was performed to identify predictors for the development of thromboembolic events. The Wilcoxon/Kruskal-Wallis test was used to detect significant differences between groups. RESULTS: Significant factors included serum albumin level (odds ratio [OR] = 0.363, 95% confidence interval [CI] = 0.193-0.685; p = 0.0017) and diabetes mellitus (OR = 5.356, 95% CI = 1.711-16.769; p = 0.0039). Renin-angiotensin system inhibitors (OR = 0.307) had low OR, although it was not significant. No difference in the development of thromboembolic events was evident between cancer types (p = 0.0781), but differences were identified between the three antiangiogenic agents (p = 0.0132). Ramucirumab was associated with a lower likelihood of thromboembolic events. CONCLUSION: Serum albumin level and diabetes mellitus were identified as significant predictors for the development of antiangiogenic agent-induced thromboembolic events. In addition, the likelihood of thromboembolic events did not differ between cancer types but differed between antiangiogenic agents.

Superactivity of Bimetallic CuFeO2 Submicron Particles towards Selective Proteolysis, Depending on their Surface Hydroxyls.

Nano bimetallic oxides as nanoproteases have the great advantages in the heterogeneous hydrolysis of proteins. Here, we report that bimetallic delafossite CuFeO2 submicron particles (CuFeO2 SMPs) display a high protease activity towards selective cleavage of peptide bond involving hydrophobic residue at 25 °C. CuFeO2 SMPs have excellent regeneration performance with high structural stability. The strong Lewis acidity of Fe3+ and the strong nucleophilicity of Cu+ bound hydroxyl groups are both necessary for the high protease activity of CuFeO2 SMPs. Low-valent metal ion has a great advantage in that low-valent Cu+ bound hydroxyl has strong nucleophilicity, resulting in promotion of protein hydrolysis via high-efficient bimetallic catalysis. This study provides evidence that the protease activity of CuFeO2 SMPs depends on metal ion-bound hydroxyls on their surface. Our findings highlight that the valence of metal ions in artificial protease and their surface hydroxyls are two important factors that determine their catalytic efficiency.

Quantitation of oxidized and reduced albumin in mammals. An intriguing analytical question.

Oxidized albumin is considered a short-term biomarker of oxidative stress and its measurement in blood contributes to evaluate the impact of diseases, drugs, dialytic treatments, physical activity, environmental contaminants etc. on the red-ox balance of humans as well as of other mammalians. Nevertheless, the most common methods for quantifying the oxidized and reduced albumins are costly and time-consuming. Furthermore, there is a dearth of information regarding the proper ways to store human serum or plasma samples in order to prevent inaccurate quantification of these various albumin forms. This paper explores these aspects and proposes a few spectrophotometric assay procedures which make the quantitation of oxidized and reduced albumin very fast, precise and un-expensive in various mammals.

Measurement of changes in serum-based inflammatory indicators to monitor response to nivolumab monotherapy in advanced gastric cancer: a multicenter retrospective study.

BACKGROUND: Nonresectable gastric cancer develops rapidly; thus, monitoring disease progression especially in patients receiving nivolumab as late-line therapy is important. Biomarkers may facilitate the evaluation of nivolumab treatment response. Herein, we assessed the utility of serum-based inflammatory indicators for evaluating tumor response to nivolumab. METHODS: This multicenter retrospective cohort study included 111 patients treated with nivolumab monotherapy for nonresectable advanced or recurrent gastric cancer from October 2017 to October 2021. We measured changes in the C-reactive protein (CRP)-to-albumin ratio (CAR), platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR) in serum from baseline to after the fourth administration of nivolumab. Furthermore, we calculated the area under the receiver operating characteristic curves (AUC ROCs) for CAR, PLR, and NLR to identify the optimal cutoff values for treatment response. We also investigated the relationship between clinicopathologic factors and disease control (complete response, partial response, and stable disease) using the chi-squared test. RESULTS: The overall response rate (complete and partial response) was 11.7%, and the disease control rate was 44.1%. The median overall survival (OS) was 14.0 (95% CI 10.7‒19.2) months, and the median progression-free survival (PFS) was 4.1 (95% CI 3.0‒5.9) months. The AUC ROCs for CAR, PLR, and NLR before nivolumab monotherapy for patients with progressive disease (PD) were 0.574 (95% CI, 0.461‒0.687), 0.528 (95% CI, 0.418‒0.637), and 0.511 (95% CI, 0.401‒0.620), respectively. The values for changes in CAR, PLR, and NLR were 0.766 (95% CI, 0.666‒0.865), 0.707 (95% CI, 0.607‒0.807), and 0.660 (95% CI 0.556‒0.765), respectively. The cutoff values for the treatment response were 3.0, 1.3, and 1.4 for CAR, PLR, and NLR, respectively. The PFS and OS were significantly longer when the treatment response values for changes in CAR, PLR, and NLR were below these cutoff values (CAR: OS, p < 0.0001 and PFS, p < 0.0001; PLR: OS, p = 0.0289 and PFS, p = 0.0302; and NLR: OS, p = 0.0077 and PFS, p = 0.0044). CONCLUSIONS: Measurement of the changes in CAR, PLR, and NLR could provide a simple, prompt, noninvasive method to evaluate response to nivolumab monotherapy. TRIAL REGISTRATION: This study is registered with number K2023006.

Separation of recombinant erythropoietin and human serum albumin without the use of sophisticated equipment.

A number of drugs based on recombinant erythropoietin contain human serum albumin as an auxiliary component. The presence of this protein hinders the proper control of the drug quality in accordance with the requirements of regulating agencies. We propose the novel method for separation of recombinant erythropoietin (epoetin beta) and human serum albumin. It is based on the subsequent use of hydrophobic sorbent and anion exchange resin placed in gravity flow columns (without the use of spin-columns). The proposed approach makes it possible to concentrate and purify the preparations containing the epoetin beta both at high and at minimal concentrations (the ratio of the amount of albumin and erythropoietin in the used preparations can reach 125:1). The average yield of epoetin beta after the use of hydrophobic sorbent and anion exchange resin was 75% and 97%, respectively. It was shown that the determined conditions of sample preparation had no affect on the content of the epoetin beta in the product.