Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer

DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.     

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 3. A new highly electron-deficient octacationic macrocycle: tetrakis-2,3-[5,6-di{2-(N-methyl)pyridiniumyl}pyrazino]porphyrazine, [(2-Mepy)8TPyzPzH(2)]8+

A new octacationic macrocycle, tetrakis-2,3-[5,6-di{2-(N-methyl)pyridiniumyl}pyrazino]porphyrazine, was obtained in its hydrated form as the water-soluble iodide salt. This compound, abbreviated as [(2-Mepy)(8)TPyzPzH(2)](I(8)).8H(2)O (2-Mepy = 2(N-methyl)pyridiniumyl moiety), was obtained by demetalation of the corresponding Mg(II) complex, [(2-Mepy)(8)TPyzPzMg(H(2)O)](I(8)).5H(2)O, which in turn was prepared from its corresponding neutral hydrated species tetrakis-2,3-[5,6-di(2-pyridyl)pyrazino]porphyrazinato(monoaquo)magnesium(II), [Py(8)TPyzPzMg(H(2)O)].4H(2)O, by reaction with CH(3)I in N,N-dimethylformamide. The quaternization reactions by using CH(3)I or methyl p-toluenesulfonate were also conducted on the monomeric precursor 2,3-dicyano-5,6-di(2-pyridyl)-1,4-pyrazine, [(CN)(2)Py(2)Pyz], with formation of the monoquaternized ion [(CN)(2)Py(2-Mepy)Pyz](+) neutralized by iodide and p-toluenesulfonate anions. Single-crystal X-ray work allowed elucidation of the structure of the two salt-like species. The diquaternized ion [(CN)(2)(2-Mepy)(2)Pyz](2+) could also be obtained as a p-toluenesulfonate salt, but attempts at direct macrocyclization of this dicationic species were unsuccessful. The iodide salt [(2-Mepy)(8)TPyzPzH(2)](I(8)).8H(2)O is water-soluble, with different solubilities depending on the range of pH explored. It was established that the macrocycle [(2-Mepy)(8)TPyzPzH(2)](8+) undergoes facile deprotonation and behaves as a strong acid. Aggregation phenomena are observed for both the octacation [(2-Mepy)(8)TPyzPzH(2)](8+) and its corresponding centrally deprotonated species [(2-Mepy)(8)TPyzPz](6+). Nevertheless, both cationic moieties exist in their monomeric form under specific experimental conditions. UV-visible monitored titrations with NaOH provide information about the type of protonation/deprotonation equilibria which are complicated by the occurrence of aggregation phenomena.     

Synthesis and 13C-NMR study of pyrazolo[4,5-c] and pyrazolo[4,3-c benzazepines. III

The synthesis of pyrazolo[4,5-c][1]benzazepin-10-ones 10 and 13 and pyrazolo[4,3-c][1]benzazepin-10-ones 11 and 12 is reported. The structure of compounds 10-13 and that of their parent compounds 2-5 ensues from a ¹³C nmr study.     

Theory of oxides surfaces, interfaces and supported nano-clusters

Oxides surfaces and thin films are finding continuous new technological applications and represent an important class of systems in materials science. Today we assist to a considerable effort to characterize the surfaces and the interfaces of oxide materials at an atomistic level. The intense experimental activity in this field has stimulated a parallel computational activity based on high-quality first principle calculations. In this review we focus our attention on the properties of oxide surfaces, and we describe the main factors that contribute to determine their behaviour: (1) nature of the bonding and electronic structure of the oxide; (2) surface morphology and defectivity; (3) doping and functionalization; (4) redox properties; (5) nano-dimensionality (e.g. in ultra-thin films). We also show how each of these parameters can affect the properties of supported metal atoms and nano-particles.     

Effective method to compute vibrationally resolved optical spectra of large molecules at finite temperature in the gas phase and in solution

The authors present a new method for the computation of vibrationally resolved optical spectra of large molecules, including the Duschinsky rotation of the normal modes and the effect of thermal excitation. The method automatically selects the relevant vibronic contributions to the spectrum, independently of their frequency, and it is able to provide fully converged spectra with moderate computational times, both in vacuo and in solution. By describing the electronic states in the frame of the density functional theory and its time-dependent extension, they computed the room temperature absorption spectra of coumarin C153 and trans-stilbene in cyclohexane and the phosphorescence spectrum of porphyrazine in gas phase, showing that the method is fast and efficient. The comparison with experiment for trans-stilbene and coumarin C153 is very satisfactory, confirming the progress made toward a reliable method for the computation and interpretation for the optical spectra of large molecules.     

Antiviral properties of lactoferrin--a natural immunity molecule

Lactoferrin, a multifunctional iron binding glycoprotein, plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Lactoferrin's iron withholding ability is related to inhibition of microbial growth as well as to modulation of motility, aggregation and biofilm formation of pathogenic bacteria. Independently of iron binding capability, lactoferrin interacts with microbial, viral and cell surfaces thus inhibiting microbial and viral adhesion and entry into host cells. Lactoferrin can be considered not only a primary defense factor against mucosal infections, but also a polyvalent regulator which interacts in viral infectious processes. Its antiviral activity, demonstrated against both enveloped and naked viruses, lies in the early phase of infection, thus preventing entry of virus in the host cell. This activity is exerted by binding to heparan sulphate glycosaminoglycan cell receptors, or viral particles or both. Despite the antiviral effect of lactoferrin, widely demonstrated in vitro studies, few clinical trials have been carried out and the related mechanism of action is still under debate. The nuclear localization of lactoferrin in different epithelial human cells suggests that lactoferrin exerts its antiviral effect not only in the early phase of surface interaction virus-cell, but also intracellularly. The capability of lactoferrin to exert a potent antiviral activity, through its binding to host cells and/or viral particles, and its nuclear localization strengthens the idea that lactoferrin is an important brick in the mucosal wall, effective against viral attacks and it could be usefully applied as novel strategy for treatment of viral infections.     

Solid phase DNA extraction on PDMS and direct amplification

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.     

Size effect on the fluorescence properties of dansyl-doped silica nanoparticles

We present here the study of the photophysical properties of new dye-doped silica nanoparticles (DDNs) bearing dansyl fluorescent derivatives covalently linked to the silica matrix. The described experimental evidences show how the different location of the chromophores induces great changes in their photophysical behavior, suggesting that fluorophores located near the surface of the nanoparticles have a very different behavior with respect to the internal molecules. These latter ones, in fact, are shielded from the solvent and have a strong blue emission, while those at the periphery interact with the solvent and show a weaker red-shifted emission. As a consequence, the fluorescence properties of these nanoparticles are an average between the characteristics of the two different families of dyes. The relative amount of fluorophores located in the two compartments can be controlled simply by changing the size since, from our results, the thickness of the solvent permeable layer is not relevantly affected by the diameter of the nanoparticles. It is noteworthy that the fluorophores located in the outer shell exhibit very peculiar features: they are sensitive and interact with small molecules such as solvent molecules but, at the same time, they are not accessible to big receptor species such as beta-cyclodextrins. Such results indicate that most of the solvent-sensitive dansyl moieties are located within pores large enough to only accommodate solvent but not big molecules as cyclodextrins, giving precious insight on the morphology of the nanoparticles.     

On the One-Dimensional Transition State Theory and the Relation between Statistical and Deterministic Oscillation Frequencies of Anharmonic Energy Wells

The transition state theory allows the development of approximated models useful to study the non-equilibrium evolution of systems undergoing transformations between two states (e.g., chemical reactions). In a simplified 1D setting, the characteristic rate constants are typically written in terms of a temperature-dependent characteristic oscillation frequency ${\nu }_s$, describing the exploration of the phase space. As a particular case, this statistical oscillation frequency ${\nu }_s$ can be defined for an arbitrary convex potential energy well. This value is compared here with the deterministic oscillation frequency ${\nu }_d$ of the corresponding anharmonic oscillator. It is proved that there is a universal relationship between statistical and deterministic frequencies, which is the same for classical and relativistic mechanics. The independence of this relationship from the adopted physical laws gives it an interesting thermodynamic and pedagogical meaning. Several examples clarify the meaning of this relationship from both physical and mathematical viewpoints.