Facile electrochemical affinity measurements of small and large molecules.

A novel miniaturized sensor for electrochemical detection that contains graphene- and gold nanoparticles was functionalized with proteins. Using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) it was possible to observe and quantify interactions of molecules with these proteins. The protein binders included carbohydrate ligands as small as carbohydrates up to COVID-19 spike protein variants engaged in protein-protein interactions. The system uses off-the-shelf sensors combined with an affordable potentiostat and yet is sensitive enough for small ligand binding.

Biomarkers of the alcohol hangover state: Ethyl glucuronide (EtG) and ethyl sulfate (EtS).

INTRODUCTION: The aim of this study was to investigate the usefulness of ethyl glucuronide (EtG) and ethyl sulfate (EtS) as biomarkers of the hangover state. METHODS: Thirty-sixhealthy social drinkers participated in this study, being of naturalistic design. Eighteen participants experience regular hangovers (the hangover group), whereas the other 18 claim to not experience a hangover (the hangover-immune group). On a control day (alcohol-free) day and a post-alcohol day, urine EtG and EtS concentrations were determined and hangover severity assessed. RESULTS: Urinary EtG and EtS concentrations were significantly increased on post-alcohol day compared to the control day (p = .0001). Both EtG and EtS concentrations did not significantly correlate with the overall hangover score, nor with the estimated peak blood alcohol concentrations and number of alcoholic drinks. EtG correlated significantly only with the individual hangover symptom "headache" (p = .033; r = .403). No significant correlations were found with the EtG to EtS ratio. EtG and EtS concentrations significantly correlated with urine ethanol concentrations. CONCLUSIONS: Although urine EtG and EtS concentration did not significantly correlate to estimated peak blood alcohol concentrations or the number of alcoholic drinks consumed, a significant correlation was found with urine ethanol concentration. However, urine EtG and EtS concentrations did not significantly correlate with overall hangover severity.

Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping.

Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6min. The method was validated from 0.05 to 5µgmL(-1) CAF and 0.025-2.5µgmL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions <13.5%. The limits of detection were 0.16 and 0.63 ngmL(-1) for PX and CAF, respectively. PX/CAF concentration ratios from volunteers were 0.26-1.09 with mean ratios of 0.78±0.26 and 0.38±0.10 for regular and light/non-coffee drinkers, respectively.

Thickness and morphology of polyelectrolyte coatings on silica surfaces before and after protein exposure studied by atomic force microscopy.

Analyte-wall interaction is a significant problem in capillary electrophoresis (CE) as it may compromise separation efficiencies and migration time repeatability. In CE, self-assembled polyelectrolyte multilayer films of Polybrene (PB) and dextran sulfate (DS) or poly(vinylsulfonic acid) (PVS) have been used to coat the capillary inner wall and thereby prevent analyte adsorption. In this study, atomic force microscopy (AFM) was employed to investigate the layer thickness and surface morphology of monolayer (PB), bilayer, (PB-DS and PB-PVS), and trilayer (PB-DS-PB and PB-PVS-PB) coatings on glass surfaces. AFM nanoshaving experiments providing height distributions demonstrated that the coating procedures led to average layer thicknesses between 1 nm (PB) and 5 nm (PB-DS-PB), suggesting the individual polyelectrolytes adhere flat on the silica surface. Investigation of the surface morphology of the different coatings by AFM revealed that the PB coating does not completely cover the silica surface, whereas full coverage was observed for the trilayer coatings. The DS-containing coatings appeared on average 1 nm thicker than the corresponding PVS-containing coatings, which could be attributed to the molecular structure of the anionic polymers applied. Upon exposure to the basic protein cytochrome c, AFM measurements showed an increase of the layer thickness for bare (3.1 nm) and PB-DS-coated (4.6 nm) silica, indicating substantial protein adsorption. In contrast, a very small or no increase of the layer thickness was observed for the PB and PB-DS-PB coatings, demonstrating their effectiveness against protein adsorption. The AFM results are consistent with earlier obtained CE data obtained for proteins using the same polyelectrolyte coatings.

Photocytotoxicity of mTHPC (temoporfin) loaded polymeric micelles mediated by lipase catalyzed degradation.

PURPOSE: To study the in vitro photocytotoxicity and cellular uptake of biodegradable polymeric micelles loaded with the photosensitizer mTHPC, including the effect of lipase-catalyzed micelle degradation. METHODS: Micelles of mPEG750-b-oligo(epsilon-caprolactone)5 (mPEG750-b-OCL5) with a hydroxyl (OH), benzoyl (Bz) or naphthoyl (Np) end group were formed and loaded with mTHPC by the film hydration method. The cellular uptake of the loaded micelles, and their photocytotoxicity on human neck squamous carcinoma cells in the absence and presence of lipase were compared with free and liposomal mTHPC (Fospeg). RESULTS: Micelles composed of mPEG750-b-OCL5 with benzoyl and naphtoyl end groups had the highest loading capacity up to 30% (w/w), likely due to pi-pi interactions between the aromatic end group and the photosensitizer. MTHPC-loaded benzoylated micelles (0.5 mg/mL polymer) did not display photocytotoxicity or any mTHPC-uptake by the cells, in contrast to free and liposomal mTHPC. After dilution of the micelles below the critical aggregation concentration (CAC), or after micelle degradation by lipase, photocytotoxicity and cellular uptake of mTHPC were restored. CONCLUSION: The high loading capacity of the micelles, the high stability of mTHPC-loaded micelles above the CAC, and the lipase-induced release of the photosensitizer makes these micelles very promising carriers for photodynamic therapy in vivo.

Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells.

Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of alpha(v)-integrins. Here, we show that the overexpression of alpha(v)-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding ("tethering") and uptake ("tickling") of lactadherin (also termed MFG-E8)--opsonized particles. Phosphatidylserine (PS)--exposing multilamellar vesicles, "aged" erythrocytes, and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonization in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides--mimicking lactadherin opsonization--we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro, this was confirmed by cytochalasin D preincubation. When RGD-erythrocytes were administered intravenously in tumor-bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonized large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.

Enzyme-induced shedding of a poly(amino acid)-coating triggers contents release from dioleoyl phosphatidylethanolamine liposomes.

The enzymatically degradable poly(amino acid)-lipid conjugate poly(hydroxyethyl l-glutamine)-N-succinyl-dioctadecylamine (PHEG-DODASuc) has been shown to effectively prolong liposome circulation times. In this paper, we investigated whether PHEG-DODASuc can stabilize liposomes composed of the fusogenic, non-bilayer-forming lipid dioleoyl phosphatidylethanolamine (DOPE). Moreover, we evaluated the release of an entrapped compound after enzyme-induced shedding of the PHEG-coating, interbilayer contact and membrane destabilizing phase changes. Contents release was monitored using the fluorescent model compound calcein. Liposome destabilization and lipid mixing was studied by dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and cryogenic-temperature transmission electron microscopy (cryo-TEM). It was shown that PHEG-DODASuc is able to stabilize DOPE-based liposomes and that contents release can be triggered by shedding of the PHEG-coating.

Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle.

The purpose of this research was to develop a stable bladder instillation of EO-9 for the treatment of superficial bladder cancer. First, stability and dissolution studies were performed. Subsequently, the freeze-drying process was optimized by determination of the freeze-drying characteristics of the selected cosolvent/water system and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze-drying process on crystallinity and morphology of the freeze-dried product was determined with x-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. This study revealed that tert-butyl alcohol (TBA) can be used to both dramatically improve the solubility and stability of EO-9 and to shorten the freeze-drying cycle by increasing the sublimation rate. During freeze drying, 3 TBA crystals were found: TBA hydrate-ice crystals, crystals of TBA hydrate, and a third crystal, probably composed of TBA hydrate crystals containing approximately 90% to 95% TBA. Furthermore, it was shown that crystallization of TBA hydrate was inhibited in the presence of both sodium bicarbonate (NaHCO3) and mannitol. Addition of an annealing step resulted in a minor increase in the crystallinity of the freeze-dried product and formation of the delta-polymorph of mannitol. A stable bladder instillation was obtained after reconstitution of the freeze-dried product (containing 8 mg of EO-9, 20 mg of NaHCO3, and 50 mg of mannitol per vial) to 20 mL with a reconstitution solution composed of propylene glycol/water for injection (WfI)/NaHCO3/sodium edetate 60%/40%/2%/0.02% vol/vol/wt/wt, followed by dilution with WfI to a final volume of 40 mL.

Self-assembly of recombinant amphiphilic oligopeptides into vesicles.

The aim of the present study was to design amphiphilic oligopeptides that can self-assemble into vesicular structures. The ratio of hydrophilic to hydrophobic block length was varied, and peptides were designed to have a hydrophobic tail in which the bulkiness of the amino acid side groups increases toward the hydrophilic domain (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu(2/7)-COOH). These peptides were recombinantly produced in bacteria as an alternative to solid-phase synthesis. We demonstrate with different complementary techniques (dynamic and static light scattering, tryptophan fluorescence anisotropy, and electron microscopy) that these amphiphilic peptides spontaneously form vesicles with a radius of approximately 60 nm and a low polydispersity when dispersed in aqueous solution at neutral pH. Morphology and size of the vesicles were relatively insensitive to the variations in hydrophilic block length. Exposure to acidic pH resulted in formation of visible aggregates, which could be fully reversed to vesicles upon pH neutralization. In addition, it was demonstrated that water-soluble molecules can be entrapped inside these peptide vesicles. Such peptide vesicles may find applications as biodegradable drug delivery systems with a pH-dependent release profile.

Membrane activity of the phospholipase C-delta1 pleckstrin homology (PH) domain.

PH-PLCdelta1 [the PH domain (pleckstrin homology domain) of PLCdelta1 (phospholipase C-delta1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCdelta1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCdelta1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCdelta1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCdelta1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCdelta1 cannot. PH-PLCdelta1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCdelta1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCdelta1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report.

The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function.

Physiological and proteomic approaches to studying prefertilization events in the human.

This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa. Characterization of cell physiology at the single-cell level must be a future aim, including the study of ion channel expression and function by patch clamping. Proteomic experiments are aimed at identifying defects in protein expression in specific subgroups of men, e.g. those with globozoospermia. A better understanding of prefertilization events will allow the development of non-assisted reproductive therapy, drug-based treatments for male infertility.