Physiological Buffer Research Articles

Structural dynamics of channels and transporters by high-speed atomic force microscopy.

Channels and transporters are vital for transmembrane transport of ions and solutes, and also of larger compounds such as lipids and macromolecules. Therefore, they are crucial in many biological processes such as sensing, signal transduction, and the regulation of the distribution of molecules. Dysfunctions of these membrane proteins are associated to numerous diseases, and their interaction with drugs is critical in medicine. Understanding the behavior of channels and transporters requires structural and dynamic information to decipher the molecular mechanisms underlying their function. High-Speed Atomic Force Microscopy (HS-AFM) now allows the study of single transmembrane channels and transporters in action under physiological conditions, i.e., at ambient temperature and pressure, in physiological buffer and in a membrane, and in a most direct, label-free manner. In this chapter, we discuss the HS-AFM sample preparation, application, and data analysis protocols to study the structural and conformational dynamics of membrane-embedded channels and transporters.

Circular dichroism and its uses in biomolecular research - A Review

The higher-order structure of proteins as well as their thermal stability can be determined using the circular dichroism (CD). CD is a common approach for swiftly assessing binding, secondary structure, and folding properties of proteins. In a nutshell, circular dichroism is an absorption spectroscopy technique that employs circularly polarized light to explore structural properties of optically active chiral compounds. Biological molecules, as well as their interactions with metals and other compounds, are studied extensively. Circular dichroism is becoming more widely acknowledged as a useful technique for studying the various conformations taken by proteins and nucleic acids in solution. Because CD is a quantitative approach, it can be used to track protein denaturation and protein-ligand interaction. These CD measures will have two key advantages: they can be performed on small amounts of material in a physiological buffer, and they will provide one of the greatest methods for monitoring any structural changes that occur as a result of changes in environmental conditions. It has proven possible to generate proteins on a big scale for therapeutic reasons utilizing recombinant DNA technology. Circular dichroism is also well-known as a useful method which is used for determining the folding characteristics of proteins. CD is used to see if a purified, produced peptide is either bended or if it has a mutation that impacts its strength and confirmation. The basic steps in getting this CD data, as well as the methodologies for interpreting the spectra in order to predict the protein structure, are summarized in this article. However, many researchers’ value is harmed when they use circular dichroism, either because of poor experimental design or because of insufficient data. The essential steps in getting this CD data, as well as the methodologies for interpreting the spectra in order to predict the protein structure, will be summarized in this article. However, the value of many investigations using circular dichroism is harmed due to insufficient attention to critical components of instrument calibration or sample characterization.

Endothelin-1 potentiates TRPV1-mediated vasoconstriction of human adipose arterioles in a protein kinase C-dependent manner.

The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1. Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed. TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 μM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 μM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1. TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm.

A Brief Review of How to Construct an Enzyme-Based H<sub>2</sub>O<sub>2</sub> Sensor Involved in Nanomaterials

This article briefly reviews how to construct an enzyme based hydrogen peroxide sensor involving nanomaterials, which has the advantages of high efficiency, good sensitivity and selectivity, fast response time and an extended range of linearity with lower detection limit. Glucose biosensor is constructed by immobilizing glucose oxidase enzyme on the polycarbonate membrane and the protective cover is then filled with a physiological phosphate buffer, pH 7.4. The novel blocking hydrophobic membrane which is only permeable to hydrogen peroxide is used to eliminate electrochemical interferences. This constructed enzyme based H2O2 biosensor is miniaturized by the involvement of nanomaterials like carbon nanotubes, platinum nanoparticles and silver nanoparticles and it can achieve the effective microscopic detection of glucose. The introduction of nanomaterials including some pure metals (Ag, Au, Pd, Ni, Pt, and Cu), metal oxide (ZnO and TiO2), bimetallic (Au/Ag and Au/Pt) and carbon (nanotubes and graphene) nanomaterials in the construction of the enzyme based H2O2 biosensor improves its sensitivity and performance by enhancing the enzymatic activity, and allows the introduction of many new signal transduction technologies in biosensors. This review article summarizes the working principles of glucose oxidase based hydrogen peroxide sensor, importance of involving nanomaterials in biosensor manufacturing, basic characteristics and components of a biosensor, generations glucose biosensors, procedure of making hydrogen peroxide based biosensor, synthesis of nanomaterials involved in hydrogen peroxide biosensor, and finally some examples of nanomaterials which intervene in hydrogen peroxide biosensor.

Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

Human Serum Albumin Binds Streptolysin O (SLO) Toxin Produced by Group A Streptococcus and Inhibits Its Cytotoxic and Hemolytic Effects.

The pathogenicity of group A Streptococcus (GAS) is mediated by direct bacterial invasivity and toxin-associated damage. Among the extracellular products, the exotoxin streptolysin O (SLO) is produced by almost all GAS strains. SLO is a pore forming toxin (PFT) hemolitically active and extremely toxic in vivo. Recent evidence suggests that human serum albumin (HSA), the most abundant protein in plasma, is a player in the innate immunity “orchestra.” We previously demonstrated that HSA acts as a physiological buffer, partially neutralizing Clostridioides difficile toxins that reach the bloodstream after being produced in the colon. Here, we report the in vitro and ex vivo capability of HSA to neutralize the cytotoxic and hemolytic effects of SLO. HSA binds SLO with high affinity at a non-conventional site located in domain II, which was previously reported to interact also with C. difficile toxins. HSA:SLO recognition protects HEp-2 and A549 cells from cytotoxic effects and cell membrane permeabilization induced by SLO. Moreover, HSA inhibits the SLO-dependent hemolytic effect in red blood cells isolated from healthy human donors. The recognition of SLO by HSA may have a significant protective role in human serum and sustains the emerging hypothesis that HSA is an important constituent of the innate immunity system.

Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Antimetastatic effects of VOflavonoid complexes on A549 cell line

BackgroundNon-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer and more than 90 % of mortality is due to metastasis-related deaths. Flavonoids are considered nutraceuticals due to the variety of pharmacological properties. In this paper, we studied the effects of baicalin, silibinin, apigenin, luteolin, and its oxidovanadium(IV) cation complexes on the viability, adhesion to fibronectin, invasion, and migration on human lung cancer cell line A549. In addition, in order to complete the study of the interaction of VOflavonoids and bovine serum albumin (BSA), the binding ability of silibinin and VOsil to the protein was evaluated. MethodTo establish the non-cytotoxic concentration range of the tested compounds, the cancer cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cell migration and invasion assays were performed using Boyden chambers and adhesion assay using MTT method. The interaction of compounds with BSA were investigated in physiological buffer (pH = 7.4) by fluorescence spectroscopy. ResultsAll complexes inhibited the metastatic cascade steps to a greater extent than their respective ligands. Likewise, based on binding constant values (Kb) for BSA-silibinin and BSA-VOsil, we can suggest that both compounds can interact with the protein. ConclusionAlthough all the complexes suppressed cell adhesion, invasion and migration, VOlut can be considered as a good candidate to continue the trials because it presented encouraging results as a potential antitumor and antimetastatic agent, and can be transported by BSA.

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

AbstractThe reactivity of platinized ultramicroelectrodes (Pt‐black UMEs) towards superoxide anion O2.−, an unstable Reactive Oxygen Species (ROS), and its relatives, H2O2 and O2, was studied. Voltammetric studies in PBS demonstrate that Pt‐black UMEs provide: i) a well‐resolved reversible redox signature for O2.− detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H2O2 at pH 7.4. The oxygen reduction reaction (ORR) at Pt‐black surfaces solely yields H2O2 (2 electrons/2 H+) at physiological pH. Consequently, Pt‐black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico‐chemical investigations.

Abstract 15019: Differential Effects of Omega-3 Fatty Acid Combinations on the Inhibition of Oxidation Of Human Low-density Lipoproteins in vitro

Background: Omega-3 fatty acids (O3FAs) reduce levels of triglyceride but may also have additional atheroprotective benefits. Randomized trials have suggested potential clinical outcome differences among O3FAs formulations. Oxidative modification of low-density lipoproteins (LDL) contributes to endothelial dysfunction, inflammation, and other aspects of atherogenesis. Individual O3FAs have been shown to inhibit LDL oxidation to varying degrees, but the antioxidant effects of combining O3FAs is unknown. Objective: To compare the dose- and time-dependent antioxidant effects of eicosapentaenoic acid (EPA, 20:5) alone and in combination with either docosapentaenoic acid (DPA, 22:5) or docosahexaenoic acid (DHA, 22:6) in human LDL in vitro . Methods: Human LDL was isolated from healthy subjects and adjusted to a final ApoB concentration of 100 μg/mL in physiologic buffer (PBS) before being incubated with O3FAs at 37°C. The EPA levels in the combinations were fixed at either 2.0, 3.0, or 4.5 μM, while the DPA or DHA levels were set at 0, 0.5, 1.0, and 2.0 μM. Oxidation was initiated by copper sulfate (10 μM) and measured over time by formation of malondialdehyde (MDA), a lipid oxidation product. Results: When combined with EPA, both DPA and DHA increased inhibition of oxidation in a dose- and time-dependent manner, with DPA showing a greater effect and more prolonged inhibition. At high levels of EPA (4.5 μM), DPA showed significantly greater antioxidant activity than equimolar DHA; these differences were more apparent with time. After 8 hours, adding 1.0 μM DPA reduced MDA formation by 61% versus vehicle (3.10 ± 0.42 vs. 7.98 ± 0.56, p <0.001), while adding 1.0 μM DHA only reduced MDA formation by 20% (6.40 ± 1.23, p <0.05). When 2 μM DPA or DHA were added, after 10 hours only DPA still had a significant degree of inhibition versus vehicle (37%; 6.65 ± 0.95 vs. 8.21 ± 0.53, p <0.05). Similar trends were observed in combinations containing 2.0 and 3.0 μM EPA. Conclusion: Adding DPA or DHA to EPA provide dose-dependent incremental antioxidant effects, with DPA providing a larger degree and a longer duration of inhibition. These observations highlight potential differences among O3FAs (and their combinations) in novel mechanisms of atheroprotection.

A Reliable BioFET Immunosensor for Detection of p53 Tumour Suppressor in Physiological-Like Environment.

The concentration of wild-type tumour suppressor p53wt in cells and blood has a clinical significance for early diagnosis of some types of cancer. We developed a disposable, label-free, field-effect transistor-based immunosensor (BioFET), able to detect p53wt in physiological buffer solutions, over a wide concentration range. Microfabricated, high-purity gold electrodes were used as single-use extended gates (EG), which avoid direct interaction between the transistor gate and the biological solution. Debye screening, which normally hampers target charge effect on the FET gate potential and, consequently, on the registered FET drain-source current, at physiological ionic strength, was overcome by incorporating a biomolecule-permeable polymer layer on the EG electrode surface. Determination of an unknown p53wt concentration was obtained by calibrating the variation of the FET threshold voltage versus the target molecule concentration in buffer solution, with a sensitivity of 1.5 ± 0.2 mV/decade. The BioFET specificity was assessed by control experiments with proteins that may unspecifically bind at the EG surface, while 100pM p53wt concentration was established as limit of detection. This work paves the way for fast and highly sensitive tools for p53wt detection in physiological fluids, which deserve much interest in early cancer diagnosis and prognosis.

Nanogap dielectrophoresis combined with buffer exchange for detecting protein binding to trapped bioparticles

We demonstrate a gold nanogap electrode platform that can rapidly create a linear array of biological particles by low-voltage dielectrophoresis (DEP). We further combine microfluidic buffer exchange to introduce protein molecules in high-conductivity solutions while trapping and immobilizing particles. The nanogap between the gold electrodes enables low operating voltages that prevent unwanted Joule heating in high-conductivity buffers. This platform is used to trap bioparticles composed of lipid membranes such as spherical supported lipid bilayers and brain-derived myelin particles, followed by detection of protein binding to specific membrane-bound receptors. We use bioparticles with different sizes, physicochemical properties, and origins to demonstrate a platform that can be used to study a variety of biomolecular interactions. The low-power linear DEP trap combined with microfluidic buffer exchange has potential to enable a portable biosensing platform to rapidly concentrate rare biological particles and perform on-chip binding assays with improved detection limits in physiological buffers.

Chromatographic Separation of Fluoroquinolone Drugs and Drug Degradation Profile Monitoring through Quality-by-Design Concept.

The article reports on the development of an efficient, robust and sensitive HPLC-DAD method for the simultaneous determination of five fluoroquinolone-based antimicrobial drugs, namely ciprofloxacin, moxifloxacin, norfloxacin, ofloxacin and pefloxacin in both aquatic and tablet formulations. The robustness of the high-performance liquid chromatography with diode-array detection (HPLC-DAD) method has been evaluated through the concepts of quality-by-design (QbD) and full factorial design of experiments (DoEs), using a Minitab 17 statistical tool. The proposed method offers sequential separation with well-defined peak shape and resolution, and has also been evaluated by following international council for harmonization (ICH) pharmaceutical guidelines. A linear signal response has been achieved for the target fluoroquinolones (FQ) drugs in the concentration range of 45-20,000ng/mL, with an average correlation coefficient (r2) value of 0.9997, and a data precision and accuracy range of 99.3-100.9%, with an RSD value of ≤0.95%, for hexaplicate measurements. The methodology offers superior sensitivity for the target FQ drugs, with the limit of detection (LD) range of 10-25ng/mL, and the limit of quantification (LQ) range of 51-86ng/mL, respectively. Using the proposed method, the article carries the first of its kind report in studying the degradation profile monitoring and drug assay determination in tablet formulations and under various physiological buffer stress conditions, for pharmaceutical validation.

Effect of pH on the degradation kinetics of a Mg–0.8Ca alloy for orthopedic implants

Abstract One of the promising applications of magnesium and magnesium alloys is their use as biodegradable implants in biomedical applications. The pH around an orthopedic implant greatly affects the degradation kinetics of biodegradable Mg–Ca alloys. At the location of a fracture, local pH changes, and this has to be considered in the optimization of implant materials. In this study, the effect of the pH of a physiological buffer on degradation of a Mg–0.8Ca alloy was studied. The pH of Hank’s balanced salt solution (HBSS) was adjusted to 1.8, 5.3 and 8.1. Degradation of a Mg–0.8Ca implant was tested using immersion test and electrochemical techniques. Immersion tests revealed an initial weight gain for all samples followed by weight loss at extended immersion time. Weight gain was highest at acidic pH (1.8) and lowest at alkaline pH (8.1). This was in agreement with results from electrochemical polarization tests where the degradation rate was highest (7.29 ± 2.2 mm/year) at pH 1.8 and lowest (0.31 ± 0.06 mm/year) in alkaline medium of pH 8.1. The pH of all HBSS buffers except the most acidic (pH 1.8) reached a steady state of ∼pH 10 at the end of the two-month immersion period, independent of the initial pH of the solution. Corrosion products formed on the sample surfaces were investigated by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX) and X-ray diffractometry (XRD), revealing the formation of magnesium and calcium phosphates with distinct morphologies that were different for each of the pH conditions. Thus, pH of physiological buffers has a significant effect on the degradation and corrosion of Mg–Ca alloys used for biomedical applications.

Application of sodium hydrocarbonate solution on purpose of correction acid-base state in patient with comorbid pathology – COPD and community-acquired pneumonia

Background. Community-acquired pneumonia is a frequent complication of chronic obstructive pulmonary disease (COPD), especially in patients with small weight. Respiratory acidosis is a natural manifestation of COPD, which clinically is characterized by dominated obstructive pulmonary ventilation. Respiratory acidosis is a form of acid-base deviation which associated with insufficient excretion of carbon dioxide by the lungs. But in patients with comorbid pathology (COPD and community-acquired pneumonia) in addition to respiratory acidosis also can be evolved a metabolic acidosis (MA) conditioned by tissue hypoxia, intense systemic inflammation with next disturbances in correlation between pro-inflammatory and anti-inflammatory mediators, accelerating catabolic processes. The severity of acid-base deviation in patients with the possibility of developing decompensated acidosis depends not only on the degree of obstructive ventilation disorders, but also increasingly to the gravity of community-acquired pneumonia which causes severe acidotic metabolic changes.
 Objective. To evaluate the expediency of application 4.2 % buffered sodium bicarbonate solution (Soda-buffer) in the case of mixed acidosis in patients with comorbid pathology: COPD which is compounded by community-acquired pneumonia.
 Materials and methods. To all patients with comorbid pathology aged from 18 to 75 were prescribed the drug Soda-buffer manufactured by “Yuria-Pharm” (Ukraine), which contains 42 mg of sodium bicarbonate intravenously at a rate of 1.5 mmol/kg per hour (4.2 % Soda-buffer – 3 ml/kg per hour) under control of blood pH, acid-base and water-electrolyte (water-salt) balance of the body.
 Results and discussion. Qualified treatment of patients with comorbid pathology (COPD and community-acquired pneumonia) should be comprehensive and directed towards the struggle against the manifestations of obstructive ventilation disorders and hypoxemia, lower respiratory tract infection, intense inflammatory process in the lung parenchyma and bronchial tree. The complex treatment includes not only modern antibacterial drugs in combination with systemic glucocorticoids, but also methods of correction of metabolic, hemodynamic and coagulation disorders. With the aim of acidosis correction the most effective way is using infusion solutions which contain sodium bicarbonate. Due to dissociation of sodium bicarbonate has released a bicarbonate anion that binds hydrogen ions to form of carbonic acid, which then decomposes into water and carbon dioxide. In case of severe respiratory insufficiency oxidation of sodium hydrocarbonate can contribute increasing of hypercapnia by the connection with the accumulated CO2. It was found that 4.2 % buffered sodium bicarbonate solution in the comprehensive therapy of patients with comorbid pathology of COPD and community-acquired pneumonia helps to restore acid-base balance, reduce metabolic disorders and improve the clinical condition of patients. In the matter of normalization of the function of external respiration and reduction of the manifestations of respiratory insufficiency, usually substantially reduces not only respiratory, but also MA. Therefore, during correction of concomitant MA by Soda-buffer we should compensate the deficiency of bases not more than half percentage. In situation with rapid balancing of acidosis, particularly in the case of impaired pulmonary ventilation, the rapid release of CO2 may exacerbate cerebral acidosis. It’s a well known fact that small uses of Soda-buffer together with other infusion solutions with an acidic pH are provided a neutralizing agent and prevent the appearance of post-infusion phlebitis after administration of widely used infusion solutions (glucose fluids of different concentrations, chloride solution, ciprofloxacin and some other fluoroquinolones).
 Conclusions. Soda-buffer (4.2 % sodium bicarbonate buffered solution) is an effective infusion agent for the correction of MA in patients with comorbid pathology (COPD and community-acquired pneumonia) in condition of provided effective gas exchange. This solution is a physiological bicarbonate buffer that maintains a constant pH level, prevents abrupt alkalization of the blood and provides a smooth correction of acidosis at the same time with increasing alkaline blood reserves. The drug also increases the excretion of sodium and chlorine ions, osmotic diuresis, alkalizes urine.

Biocompatible fluorescent probe for detecting mitochondrial alkaline phosphatase activity in live cells

Alkaline phosphatase (ALP) is an enzyme that actively plays a significant role in the various metabolic processes by transferring a phosphate group to the protein, nucleic acid, etc. The elevated level of ALP in blood plasma is the hallmark of inflammation/cancer. The hyperactive mitochondria in cancer cells produce an excess of ATP to fulfill the high energy demand. Thus, we have developed a fluorescent probe Mito-Phos for ALP, which can detect phosphatase expression in mitochondria in live cells. The probe Mito-Phos has shown ~15-fold fluorescence intensity increments at 450 nm in the presence of 500 ng/mL of ALP. It takes about 60 min to consume the whole amount of ALP (500 ng/mL) in physiological buffer saline. It can selectively react with ALP even in the presence of other probable cellular reactive components. It is highly biocompatible and nontoxic to the live cells. It has shown ALP expression in a dose-dependent manner by providing concomitant fluorescence images in the blue-channel region. It has localized exclusively in the mitochondria in live cells. The probe Mito-Phos is highly biocompatible with the ability to assess ALP expression in mitochondria in live cells.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

69. Ischemic spinal nerve root injury secondary to herniated lumbar intervertebral disc

BACKGROUND CONTEXT The painful clinical sequelae associated with sciatica secondary to intervertebral disc herniation have been traditionally attributed to mechanical compression of the spinal nerve root. More recent studies have also implicated inflammatory mediators and microvascular changes in the pathogenesis of acute sciatica. The application of nucleus pulposus (NP) to a spinal nerve root has been shown to induce thrombus formation in the associated nerve microvasculature and result in greater injury to the axons in the center than in the periphery, suggesting an ischemic mechanism of injury. The spinal nerve blood flow changes that are associated with nerve dysfunction seen in acute sciatica offer a potential target for therapeutic intervention. The VEGF angiogenic cytokines have been demonstrated to be potent stimulators of the differentiation and function of vascular endothelial cells after injury. PURPOSE To determine the relationship between the severity of spinal nerve root injury in patients with acute sciatica secondary to lumbar disc herniation (AS/LDH) undergoing surgical discectomy and the levels of VEGF-A and VEGF-B in serum, spinal nerve root lavage and disc tissue homogenate. STUDY DESIGN/SETTING ELISA cytokine immunoassay. PATIENT SAMPLE Twenty-four patients with AS/LDH of less than 16 weeks duration, undergoing laminectomy discectomy surgery. OUTCOME MEASURES VEGF-A and VEGF-Bconcentrations (pg/ml) in herniated nucleus pulposus homogenate (HNP-H), spinal nerve root lavagate (SN-L) and serum. METHODS At the time of surgery, the spinal nerve root was lavaged with 5 cc of saline, the extruded disc tissue removed surgically and serum samples were obtained. Disc and lavagate samples were stored in physiological buffer with protease inhibitors at -80° C. The concentrations of VEGF-A and VEGF-Bin disc tissue homogenate, serum and lavagate samples were measured in triplicate using ELISA immunoassay. Research ethics committee approval was obtained. RESULTS In AS/LDH patients, VAS pain and significant spinal nerve root deficit scores (3.0 - 4.0/5) correlated closely with serum and HNP-H tissue levels of VEGF-A and VEGF-B (Pearson r=0.78). In 11/24 patients, elevated VEGF-A and B concentrations correlated to a moderate degree with TNFα and IL-2, 6 and 8 inflammatory cytokine levels (r=0.61). Low concentrations of VEGF and inflammatory cytokine concentrations were seen in all SN-L test samples. CONCLUSIONS In patients with acute sciatica secondary to lumbar disc herniation, the severity of spinal nerve injury appears to be related to a combined mechanism, involving ischemic and inflammatory components. This research study was funded by grants from the National Institutes of Health, Bethesda, USA and from the University of British Columbia Orthopaedic Research Excellence Fund. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Polymer Topology Determines the Formation of Protein Corona on Core-Shell Nanoparticles.

Linear and cyclic poly(2-ethyl-2-oxazoline) (PEOXA) adsorbates provide excellent colloidal stability to superparamagnetic iron oxide nanoparticles (FexOy NPs) within protein-rich media. However, dense shells of linear PEOXA brushes cannot prevent weak but significant attractive interactions with human serum albumin. In contrast, their cyclic PEOXA counterparts quantitatively hinder protein adsorption, as demonstrated by a combination of dynamic light scattering and isothermal titration calorimetry. The cyclic PEOXA brushes generate NP shells that are denser and more compact than their linear counterparts, entirely preventing the formation of a protein corona as well as aggregation, even when the lower critical solution temperature of PEOXA in a physiological buffer is reached.

Investigating the binding measurements of human α-acid glycoprotein with chlorambucil and dacarbazine in the presence of imidazolium based -ionic liquid by affinity capillary electrophoresis

Human alpha (α1)-acid glycoprotein (AGP) is an acute phase protein whose plasma concentration increases several-folds in the presence of various diseases. The variability in AGP plasma concentration is expected to have a huge impact on the drug binding equilibrium. Therefore, a precise measurement of AGP-drug binding is of great demand for drug development. In the current study, an ionic liquid-based aqueous two-phase system combined with affinity capillary electrophoresis (ILATPS/ACE) was utilised in order to improve the accuracy of AGP-drug binding analysis through the measurements of electrophoretic mobilities. The utilisation of ILATPS has shown to have a positive impact on the stability of AGP activity solution during the storage for an extended period of time. In addition, the effect of various alkyl chains (C2-C10) of imidazolium-based ILs with concentrations ranging between 10.00 and 1000.0 μmol L−1 on the AGP binding with the anti-cancer drugs chlorambucil (CHL) and dacarbazine (DAC) was examined by the system developed (ILATPS/ACE). A 100.00 μmol L−1 1-ethyl-3-methylimidazolium chloride (EMImCl) prepared in the physiological buffer conditions containing AGP (5.00–100.00 µmol L−1) has provided an accurate apparent binding constant of 1.99 ± 0.11 and 6.95 ± 0.14 L mmol−1 with CHL and DAC respectively. Apart from the ACE analysis, EMImCl/phosphate buffer solution was found to be a distinguished system that could lengthen the stability of AGP activity for a period of time reaching 90 days during the solution storage at 4.00 °C. This effect is thought to be due to the easy conversion of one-phase EMImCl/phosphate buffer/AGP at the ambient lab temperature into the two-phase solution at refrigerator temperature, 4.00 °C, and vice versa. Therefore, the ILATP/ACE system could be used to enhance the accuracy for other AGP-drug bindings with a fast, easy to use, and cost-effective analysis.