We have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive-negative, hydrophobic-hydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl-, Br-, [SCN](-), methane-[C1SO3](-) and ethanesulfonates [C2SO3](-), than for anions with a relatively stable hydration shell, positively hydrated-lactate [Lac](-), butylsulfonate [C4SO3](-) and acetate [Ac](-). Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein-water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e. g. [Ac](-)) to those with negatively hydrated groups (e. g. [C1SO3](-)) and the prevailing hydrophobic hydration (e. g. [C4SO3](-)). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.

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The close structural similarity between the two cyclooxygenase (COXs) isoforms and the absence of selective inhibitors without side effects continues to stimulate the development of novel approaches towards selective anti-inflammatory drugs. In the present study a small library of new indolic compounds involving two different substitutions patterns at the indole scaffold was synthesized. In order to establish a relation between the spatial distribution of known functional groups related with inhibitory activity, two substitution patterns were explored: one with substituents at N-1, C-3, C-5 positions and another at C-2, C-3 and C5 positions. Accordingly, indole positions C-5, C-3 and N-1 were substituted with: sulfonamide or methylsulfone at C-5, p-halo-benzyl group at C-3, and an alkyl chain with a trifluoromethyl group at N-1. Alternatively, a p-halo-benzyl group was introduced at C-2, leaving the indolic nitrogen free. Inhibitory studies were performed and the activity results obtained against both COXs isoforms were rationalized based on docking and NMR studies. Docking studies show that dialkyation at C-2 and C-3 favors a binding with an orientation similar to that of the known selective inhibitor SC-558. From the tested compounds, this substitution pattern is correlated with the highest inhibitory activity and selectivity: 70% COX-2 inhibition at 50 M, and low COX-1 inhibition (18±9%). Additionally, Saturation Transfer Difference NMR experiments reveal different interaction patterns with both COXs isoforms that may be related with different orientations of the sulfonamide group in the binding pocket. Despite the moderated inhibitory activities found, this study represents an innovative approach towards COXs inhibitory activity rationalization and to the design of anti-inflammatory drugs.

Light has always fascinated mankind and since the beginning of recorded history it has been both a subject of research and a tool for investigation of other phenomena. Today, with the advent of nanotechnology, the use of light has reached its own dimension where light-matter interactions take place at wavelength and subwavelength scales and where the physical/chemical nature of nanostructures controls the interactions. This is the field of nanophotonics which allows for the exploration and manipulation of light in and around nanostructures, single molecules, and molecular complexes. What is more is the use of nanophotonics in biomolecular interactionsnanobiophotonicshas prompt for a plethora of molecular diagnostics and therapeutics making use of the remarkable nanoscale properties. In this paper, we shall focus on the uses of nanobiophotonics for molecular diagnostics involving specific sequence characterization of nucleic acids and for gene delivery systems of relevance for therapy strategies. The use of nanobiophotonics for the combined diagnostics/therapeutics (theranostics) will also be addressed, with particular focus on those systems enabling the development of safer, more efficient, and specific platforms. Finally, the translation of nanophotonics for theranostics into the clinical setting will be discussed.

Nanotechnology has prompted new and improved materials for biomedical applications with particular emphasis in therapy and diagnostics. Special interest has been directed at providing enhanced molecular therapeutics for cancer, where conventional approaches do not effectively differentiate between cancerous and normal cells; that is, they lack specificity. This normally causes systemic toxicity and severe and adverse side effects with concomitant loss of quality of life. Because of their small size, nanoparticles can readily interact with biomolecules both at surface and inside cells, yielding better signals and target specificity for diagnostics and therapeutics. This way, a variety of nanoparticles with the possibility of diversified modification with biomolecules have been investigated for biomedical applications including their use in highly sensitive imaging assays, thermal ablation, and radiotherapy enhancement as well as drug and gene delivery and silencing. Here, we review the available noble metal nanoparticles for cancer therapy, with particular focus on those already being translated into clinical settings.

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This paper describes for the first time the intimate molecular details of the association between a platinated oligonucleotide and a zinc-finger peptide. Site-specific platination of the guanine in a ss hexanucleotide gave {[Pt(dien)d(5’-TACGCC-3’)], Pt(dien)(6-mer)}, II, characterized by mass spectrometry and 1H-NMR spectroscopy. The work extends the study of platinum-nucleobase complex-zinc finger interactions using small molecules such as [Pt(dien)(9-EtGua)]2+, I . The structure of the (34-52) C-terminal finger of the HIV nucleocapsid protein HIVNCp7 (ZF1) was characterized by 1H-NMR spectroscopy and compared with that of the N-terminal single finger and the 2-finger “intact” NCp7. Interaction of II with ZF1 results in significant changes in comparison to the “free” uncomplexed hexanucleotide – the major shifts occur for Trp37 resonances are broadened and shifted upfield and other major shifts are for Gln45 (H21, H3, Q), Met46 (NH, H2), Lys47 (NH, Q) and Glu50 (H2, H3). The Zn-Cys/His chemical shifts show only marginal deviations. The solution structure of ZF1, the 6-mer/ZF1 and II/ZF1 adducts were calculated from the NOESY-derived distance constraints. The DNA position in II/ZF1 is completely different than in the absence of platinum. Major differences are the appearance of new Met46-Cyt6H5 and Trp37-Cyt5H5 contacts but severe weakening of the Trp37-Gua4 contact, attributed to the steric effects caused by Gua4 platination, accompanied by a change in the position of the aromatic ring. The results demonstrate the feasibility of targetting specific ZF motifs with DNA-tethered coordination compounds, such as Pt compounds and Co-macrocycles – with implications for drug targetting and indeed the intimate mechansims of DNA repair of platinated DNA.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p = 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p = 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 +/- 3.0%) than control (39.1 +/- 2.2%) and 40 ng/ml rDpl (39.8 +/- 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

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Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars’ structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-D-galactose (G) and 3,6-anhydro-α-L-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAEopt agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and Mv obtained for the MAEopt sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products.

This work reports a novel strategy to prepare affinity composite membranes using supercritical fluid technology. By blending molecularly imprinted polymeric particles with PMMA, a porous hybrid structure with affinity to the template molecule, bisphenol A, was prepared using a supercritical carbon dioxide (scCO2)-assisted method. Membranes were characterized in terms of morphology, mechanical performance and transport properties. The ability of the polymers and hybrid membranes to adsorb bisphenol A was tested in aqueous solutions and fitted to a linearized Langmuir equation, showing that adsorption takes place at homogeneous affinity binding sites within the imprinted surface. Filtration experiments showed that the imprinted hybrid membrane was able to adsorb higher amounts of template even in non-equilibrium dynamic binding conditions. The hybridization of the \{PMMA\} membrane herein reported conveys two important improvements over neat \{PMMA\} membrane: it introduced molecular affinity towards the template molecule and significantly increased the permeability of the porous structures, which are key parameters in processes that involve membranes. This technique could expand the applications of polymeric beads powders and enhance the efficiency of the membrane's transport properties. Our work presents a new method to confer affinity to a porous structure by immobilization of imprinted polymers, combining polymer synthesis and membrane formation using supercritical fluid technology.

Molecularly imprinted polymeric particles with molecular recognition towards Bisphenol A (BPA) were synthesized for the first time using the semi-covalent imprinting approach in supercritical carbon dioxide (scCO2). The material{'}s affinity to BPA was achieved by co-polymerizing ethylene glycol dimethacrylate (EGDMA) with a template-containing monomer{,} Bisphenol A dimethacrylate (BPADM) in scCO2. Bisphenol A is then cleaved from the polymeric matrix by hydrolysis with tetrabutylammonium hydroxide (n-Bu4OH) also in a supercritical environment{,} taking advantage of the high diffusivity of scCO2. The selectivity of the molecular imprinted polymer (MIP) was assessed by evaluating its capability to bind BPA in comparison with progesterone and [small alpha]-ethinylestradiol. In addition{,} the cross-linked particles were used to prepare a PMMA-based hybrid imprinted membrane by a scCO2-assisted phase inversion method. Results show that the incorporation of MIP particles was able to confer molecular affinity to BPA to the membrane and that at dynamic conditions of filtration{,} this imprinted porous structure was able to adsorb a higher amount of BPA than the corresponding non-imprinted hybrid membrane. Our work represents a valuable greener alternative to conventional methods{,} for the synthesis of affinity materials which are able to maintain molecular recognition properties in water.

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