Process monitoring of virus-like particle reassembly by diafiltration with UV/Vis spectroscopy and light scattering

Virus-like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross-flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on-line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine-132. VLP formation was verified by off-line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate-related assembly inhibition and showed that off-line size-exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial-Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF.     

Serendipitous syntheses of Rh(H)2Cl(PRPh2)3 complexes, and their crystal structures, where R = Me, Cy (cyclohexyl)

Reactions of RhCl(cod)(THP) (cod = 1,5-cyclooctadiene; THP = P(CH₂OH)₃) with PMePh₂ or PCyPh₂ (Cy = cyclohexyl) in acetone/MeOH solution under H₂ surprisingly form the complexes cis, mer-Rh(H)₂Cl(PRPh₂)₃ (R = Me or Cy); both complexes are characterized by crystallography (the first structures in which the hydride ligands of such dihydrido-chloro-trisphosphine complexes have been located), and by detailed ¹H and ³¹P NMR spectroscopy. The key role of the THP in the observed chemistry is discussed.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

不同发育时期樟子松（Pinus sylvestris L. var. mongolica Litv.）的电阻抗参数与抗寒性的关系

采用电阻抗图谱（EIS）法和电导（EL）法对不同发育时期的樟子松（Pinus sylvestris L. var. mongolica Litv.）茎和针叶进行了抗寒性测定,试图通过比较两种方法测定抗寒性结果的相关性,找到适合冷冻处理后樟子松抗寒性测定和不经冷冻处理估测抗寒性的EIS参数,完善EIS法测定抗寒性。以8年生樟子松苗为试材,在抗寒锻炼阶段（10月份）和脱锻炼阶段（3月份）分别取样进行EIS和EL测定。结果表明,EIS法胞外电阻率（re）与EL法测定的樟子松抗寒性相关性较高（R2=0.97）,但比EL法求出的抗寒性高。针叶的细胞膜时间恒量（τm）和茎的弛豫时间（τ1）随冷冻温度变化与re表现相似的S曲线,相关分析表明,re（茎和针叶）与τ1（茎）和τm（针叶）的变化有较好的相关性（R2=0.74～0.84）。经Logistic方程拟合,EIS的τm（针叶）和τ1（茎）法与EIS（re）法、EL法测定的樟子松抗寒性相关性也较高（R2=0.88～0.91）,说明针叶τm和茎τ1也可以作为计算抗寒性的参数。另外,8年生樟子松两个发育时期（10月和3月份）未经冷冻的针叶τm与茎的τ2随抗寒性的增强而显著增加,表明不经过冷冻处理样本用τ2（茎）和τm（针叶）估计樟子松抗寒性是很有前途的方法。     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Efficiency Boost in All-Small-Molecule Organic Solar Cells: Insights from the Re-Ordering Kinetics

Achieving high-performance in all-small-molecule organic solar cells (ASM-OSCs) significantly relies on precise nanoscale phase separation through domain size manipulation in the active layer. Nonetheless, for ASM-OSC systems, forging a clear connection between the tuning of domain size and the intricacies of phase separation proves to be a formidable challenge. This study investigates the intricate interplay between domain size adjustment and the creation of optimal phase separation morphology, crucial for ASM-OSCs’ performance. It is demonstrated that exceptional phase separation in ASM-OSCs’ active layer is achieved by meticulously controlling the continuity and uniformity of domains via re-packing process. A series of halogen-substituted solvents (Fluorobenzene, Chlorobenzene, Bromobenzene, and Iodobenzene) is adopted to tune the re-packing kinetics, the ASM-OSCs treated with CB exhibited an impressive 16.2% power conversion efficiency (PCE). The PCE enhancement can be attributed to the gradual crystallization process, promoting a smoothly interconnected and uniformly distributed domain size. This, in turn, leads to a favorable phase separation morphology, enhanced charge transfer, extended carrier lifetime, and consequently, reduced recombination of free charges. The findings emphasize the pivotal role of re-packing kinetics in achieving optimal phase separation in ASM-OSCs, offering valuable insights for designing high-performance ASM-OSCs fabrication strategies.     

Interaction of a flavone loaded on surface-modified dextran-spooled superparamagnetic nanoparticles with β-cyclodextrin and DNA

Flavones are biologically active compounds obtained mainly from plant sources. Pharmaceutically important compounds can be delivered to the physiological target by loading them in carriers like cyclodextrins and magnetic nanoparticles. Herein, the binding of 6-methoxyflavone to β-cyclodextrin and DNA is studied using UV–visible absorption and fluorescence spectroscopy. The loading of 6-methoxyflavone onto a magnetic nanoparticles is employed. β-cyclodextrin encapsulates the 6-methoxyflavone to form an inclusion complex. β-cyclodextrin also used to draw forth 6-methoxyflavone loaded onto a magnetic nanoparticles. The morphology, magnetic property and the crystallite size of the nanoparticles are studied using scanning electron microscopy, vibrating sample magnetometry and X-ray diffraction techniques, respectively. The binding of the drug-loaded magnetic nanoparticles to DNA shows that the compound is accessible to DNA and available mostly on the surface of the nanoparticles despite a modified dextan polymer supposedly encapsulates the flavone.     

Curium(III) complexation with pyoverdins secreted by a groundwater strain of Pseudomonas fluorescens

Pyoverdins, bacterial siderophores produced by ubiquitous fluorescent Pseudomonas species, have great potential to bind and thus transport actinides in the environment. Therefore, the influence of pyoverdins secreted by microbes on the migration processes of actinides must be taken into account in strategies for the risk assessment of potential nuclear waste disposal sites. The unknown interaction between curium(III) and the pyoverdins released by Pseudomonas fluorescens (CCUG 32456) isolated from the granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden, is the subject of this paper. The interaction between soluble species of curium(III) and pyoverdins was studied at trace curium(III) concentrations (3 × 10^?7 M) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). Three Cm³⁺—P. fluorescens (CCUG 32456) pyoverdin species, M_pH_qL_r, could be identified from the fluorescence emission spectra, CmH₂L⁺, CmHL, and CmL^?, having peak maxima at 601, 607, and 611 nm, respectively. The large formation constants, log β₁₂₁ = 32.50 ± 0.06, log β₁₁₁ = 27.40 ± 0.11, and log β₁₀₁ = 19.30 ± 0.17, compared to those of other chelating agents illustrate the unique complexation properties of pyoverdin-type siderophores. An indirect excitation mechanism for the curium(III) fluorescence was observed in the presence of the pyoverdin molecules.     

Copper induced oxidation of serotonin: analysis of products and toxicity

Serotonin is a major neurotransmitter that controls many functions, ranging from mood and behaviour through to sleep and motor functions. The non-enzymatic oxidation of serotonin is of significant importance as some oxidation products are considered to be neurotoxic. An interaction between copper and serotonin has been suggested by symptoms observed in a number of neurodegenerative diseases such as Wilson's and Prion diseases. Using PC12 cells as a model of neuronal cells, we show that the interaction between copper and serotonin is toxic to undifferentiated cells. The toxicity is largely due to reactive oxygen species as cell death is significantly reduced in the presence of the antioxidant mannitol. Differentiation of the PC12 cells also confers resistance to the oxidative process. In vitro oxidation of serotonin by copper results in the eventual formation of a coloured pigment, thought to be a melanin-like polymeric species. Using spectroscopic methods we provide evidence for the formation of a single intermediate product. This dimeric intermediate was identified and characterized as 5,5'-dihydroxy-4,4'-bitryptamine. These results indicate that copper structurally alters serotonin and this process may play a role in copper related neurodegenerative diseases.