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.

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.

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.

Several lines of recent evidence support a role for chromatin in splicing regulation. Here, we show that splicing can also contribute to histone modification, which implies bidirectional communication between epigenetic mechanisms and RNA processing. Genome-wide analysis of histone methylation in human cell lines and mouse primary T cells reveals that intron-containing genes are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3) relative to intronless genes. In intron-containing genes, H3K36me3 marking is proportional to transcriptional activity, whereas in intronless genes, H3K36me3 is always detected at much lower levels. Furthermore, splicing inhibition impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover, the increase of H3K36me3 correlates with the length of the first intron, consistent with the view that splicing enhances H3 methylation. We propose that splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating RNA polymerase II.

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2 Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15 Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c7-type domains (A, B, C and D) with homology to triheme cytochromes c7. In cytochromes c7 all three hemes are bis–His coordinated, whereas in c7-type domains the last heme is His–Met coordinated. The full-length GSU1996 has a 12 nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a “nanowire” of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c7-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c7-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c7-type domains from one multiheme protein were compared.

The aim of this chapter is to provide an overview of the available and emerging molecular diagnostic methods that take advantage of the unique nanoscale properties of nanoparticles (NPs) to increase the sensitivity, detection capabilities, ease of operation, and portability of the biodetection assemblies. The focus will be on noble metal NPs, especially gold NPs, fluorescent NPs, especially quantum dots, and magnetic NPs, the three main players in the development of probes for biological sensing. The chapter is divided into four sections: a first section covering the unique physicochemical properties of NPs of relevance for their utilization in molecular diagnostics; the second section dedicated to applications of NPs in molecular diagnostics by nucleic acid detection; and the third section with major applications of NPs in the area of immunoassays. Finally, a concluding section highlights the most promising advances in the area and presents future perspectives.

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Increasing levels of environmental pollution and destruction of the planet, as
well as the future of societies, have been prominent themes in our time.
Concern about the sustainable future of ecosystems and future generations
brings to each sector of activity the need to define a process of sustainable development
and measures that ought to be implemented.
In the construction sector this is a major issue, since it consumes a great part of
natural resources, and leads to high emissions of pollutants. Likewise, this sector
determines the mode of living of human beings, as well as their comfort and health.
The work herein presented has the purpose to study how, and what systems and
procedures should be implemented to ensure techniques for sustainable construction,
and consequently achieve the principles of sustainable development into its
components: environment, society and economy.
The national reality of professionals will also be referred to, thus contributing to
a practice of sustainable construction, complementary to what currently exists both
national and internationally.
Thus, this study aims to reflect on the importance of certification, systems and
procedures, to ensure compliance of construction with the sustainability goals.

Mining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.