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.     

Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA-protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.     

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.     

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.     

Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi‐quantitative approaches to their interpretation.     

Small photoactivatable molecules for controlled fluorescence activation in living cells

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.     

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.