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Determination of filtration and consolidation properties of protein crystal suspensions using analytical photocentrifuges with low volume samples
摘要： Proteins and in particular pharmaceutical proteins like monoclonal antibodies are expensive products. Protein crystallization is an interesting alternative process step to the common purification and formulation of such proteins. The solid-liquid separation of proteins however, is a challenging task because the proteins are sensitive to mechanical stress, which could lead to crystal breakage and often are only available in small amounts for research and development. In this study a newly developed filtration cell for the LUMiSizer ? centrifuge is used to analyze the filtration behavior with low volume samples. Isometric and needle shaped lysozyme crystals serve as model protein crystals. The needle shaped crystals showed about twice as high compressibility and much higher cake resistance than the isometric lysozyme crystals. After the filtration at maximum pressure in the centrifuge the mean particle diameter decreased compared to the unstressed median diameter. With the filtration cell the cake height and filter cake compression can be monitored in-situ and the filter cake resistance and solid volume content can be calculated with one experiment using 200 to 300 μl sample.
关键词： Centrifugal filtration,Cake filtration,Protein crystals
更新于2025-09-23 15:23:52
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Photon Counting - Fundamentals and Applications || Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies
摘要： The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.
关键词： biophysical techniques,STORM,nanoscopy,STED,protein dynamics,palm,spectroscopy,molecular brightness,fluorescence fluctuation
更新于2025-09-23 15:23:52
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Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination
摘要： In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H+] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562 nm was linear in the range pH = 4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30 μM. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.
关键词： Glutathione transferase,Biosensor,α-Endosulfan,Pesticides determination,Protein engineering
更新于2025-09-23 15:23:52
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Hyperspectral imaging for non-destructive prediction of total nitrogen concentration in almond kernels
摘要： There is increasing awareness of the need to consume high-quality foods because of health concerns. Food safety and health awareness campaigns have provided an impetus for non-destructive and real-time methods for food quality assessment. Total nitrogen is used as an indicator of crude protein content in foods and we examined the potential of hyperspectral imaging to predict total nitrogen concentration in four brands of almonds purchased from commercial retailers. A hyperspectral imaging system in the wavelength range 400-1000 nm was used in the study. A partial linear squares regression (PLSR) model was developed, which predicted total nitrogen concentration with a determination coefficient (R2 p) of 0.82 and a root mean error square of calibration (RMSEC) of 0.16. These results indicated that hyperspectral imaging has great potential to predict total nitrogen concentration of almond kernels.
关键词： food quality,nuts,crude protein,nutritional composition,rapid assessment,almond
更新于2025-09-23 15:23:52
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Observation of Site-Resolved Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater
摘要： Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, i.e. localized heating, is required for their investigation. Here, we solve this problem by the site-specific incorporation of the non-canonical amino acid β-(1-azulenyl)-L-alanine (AzAla) via genetic code expansion. Being an exception to Kasha′s rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation it serves as a fluorescent label. We endowed PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. Using ultrafast IR spectroscopy, we could indeed observe VET from the incorporation sites in the protein to a bound peptide ligand on a picosecond timescale. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role for function in a wide range of proteins.
关键词： protein modification,energy transfer,non-canonical amino acid,time-resolved spectroscopy,mutagenesis
更新于2025-09-23 15:23:52
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Analysis of Golgi pH in CHO cells using ratiometric pH-sensitive fluorescent proteins
摘要： Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable CHO cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase and galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4 – 6.5, trans-Golgi: 5.9 – 6.0, and cytoplasm: 7.1 – 7.2). The intracellular pH was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through intracellular pH control.
关键词： cis-Golgi,trans-Golgi,ratiometric-pH sensitive protein,CHO cells,intracellular pH
更新于2025-09-23 15:23:52
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Does the wavelength dependent photoisomerization process of the p?coumaric acid come out from the electronic state dependent pathways?
摘要： Similar to the anion photoactive yellow protein (PYP) chromophore, the neutral form of the PYP chromophore was also found to exhibit a the wavelength-dependent photoisomerization quantum yield. The isomerization quantum yield increases with the increasing excitation energy on the S1 state, while decreases when being excited to the S2 state. Does this wavelength dependent product yield come out from the specific reaction pathways of the S1 and S2 states? This would mean that, the relaxation pathway of the S2 state is distinct from that of the S1 state and does not involve twisting motion. Does it break Kasha's rule by exhibiting a direct transition from the S2 state to the ground state? The underlying mechanism needs further in. In this article, we employed the on-the-fly dynamics simulations and static electronic structure calculations to reveal the deactivation mechanism of the neutral form of the PYP chromophore. Our results indicated that the C_C twisting motion dominates the S1 state decay process. In contrast, for the decay process of the S2 state, an ultrafast transition from the S2 to the S1 state through a planar conical intersection is observed, and the excess energy activates a new reaction channel to the ground state characterized by a puckering distortion of the ring. This pathway competes with the photoisomerization channel. No direct transition from S2 to S0 is observed, hence Kasha's rule is valid for this process. Our calcualtions can provide a reasonable explanation of the wavelength-dependent isomerization quantum yield of neutral PYP chromophore, and we hope it can provide theoretical foundations for comparing the effect of protonation state on the dynamcal behaviors of PYP chromophore.
关键词： Wavelength dependent,Photoisomerization,Fluorescent protein,Nonadiabatic process
更新于2025-09-23 15:23:52
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An infrared IgG immunoassay based on the use of a nanocomposite consisting of silica coated Fe3O4 superparticles
摘要： A reliable, rapid and ultrasensitive immunoassay is described for determination of immunoglobulin G (IgG). It is making use of biofunctional magnetite (Fe3O4) superparticles coated with SiO2 and serving as an infrared (IR) probe. The unique IR fingerprint signals originating from the transverse and longitudinal phonon modes, respectively, of the asymmetric stretching of the Si–O–Si bridges display a satisfactory resistance to optical interference from the environment. The adoption of Fe3O4 superparticles instead of Fe3O4 nanoparticles as the magnetic core warrants a controllable structure and a strong magnetic response. This facilitates the efficient purification of the probes and the alleviation of the interfacial resistance between the liquid-solid interfaces by using a magnet. The gold-coated substrate was used to immobilize goat-anti-human IgG. The analyte (human IgG) was incubated with the IR probes, and then captured by the substrate immobilized antibody with the assistance of an external magnetic field. The integral area of the IR absorption band between 1250 cm?1 – 900 cm?1 was chosen for quantitative assay. The limit of detection is 95 fM, which is two orders of magnitude better than that without the magnetic field. The assay time was shortened from 2 h to 1 min. High selectivity, specificity, and long-term stability of the immunoassay were achieved. The performance of the assay when analyzing blood samples confirmed the practicability of the method.
关键词： Molecular vibration,IR spectroscopy,Core-shell,Sandwich immunoassay,Self-assembly,Protein,Blood,Superparamagnetism,Magnetic beads
更新于2025-09-23 15:23:52
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Pharmacological interference of adrenergic receptor signaling preserve photoreceptors after retinal detachment through inhibition of oxidative stress and inflammation
摘要： Background and Purpose: The current strategy is not sufficient to halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of adrenergic receptor (AR)-targeting pharmaceuticals, the α1-AR antagonist doxazosin or the α2-AR agonist guanabenz, against photoreceptor cell death in RD. Experimental Approach: Brown-Norway rats were created with experimental RD by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with ELISA. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic administration of doxazosin, guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD compared to vehicle controls. Key Results: Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Systemic administration of doxazosin or guanabenz significantly alleviated the RD-induced production of ROS and proinflammatory cytokines, including IL-1β and MCP-1, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. Conclusions and Implications: Our findings point to adrenergic receptors as novel therapeutic targets for photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.
关键词： G protein-coupled receptors,adrenergic receptors,retinal detachment,photoreceptor neuroprotection,NADPH oxidase
更新于2025-09-23 15:23:52
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New light on neurotransmitter-gated receptors: Optical approaches for controlling physiological function
摘要： Neurotransmitter-gated receptors contribute to synaptic transmission and modulation in many ways. Considering glutamate receptors as an example, it becomes clear that these receptor families are highly diverse and that it is experimentally challenging to disentangle the different functional contributions of closely related receptor subtypes. Pharmacological and genetic methods are now complemented by optogenetic approaches, which allow for controlling receptor signaling with light. Using glutamate receptors as an example, I summarize how tethered photoswitchable ligands can be used to control individual receptor subtypes with high spatial and temporal precision, and in specific cells. These, and similarly exciting approaches, offer new possibilities for probing the function of individual receptors in the nervous system.
关键词： optogenetics,ligand-gated ion channels,optopharmacology,G protein-coupled receptors,glutamate receptors
更新于2025-09-23 15:23:52