Xylonucleic acid: synthesis,structure, and orthogonal pairing properties

There is a common interest for studying xeno-nucleic acid systems in the fields of synthetic biology and the origin of life, in particular, those with an engineered backbone and possessing novel properties. Along this line, we have investigated xylonucleic acid (XyloNA) containing a potentially prebiotic xylose sugar (a 3′-epimer of ribose) in its backbone. Herein, we report for the first time the synthesis of four XyloNA nucleotide building blocks and the assembly of XyloNA oligonucleotides containing all the natural nucleobases. A detailed investigation of pairing and structural properties of XyloNAs in comparison to DNA/RNA has been performed by thermal UV-melting, CD, and solution state NMR spectroscopic studies. XyloNA has been shown to be an orthogonal self-pairing system which adopts a slightly right-handed extended helical geometry. Our study on one hand, provides understanding for superior structure-function (-pairing) properties of DNA/RNA over XyloNA for selection as an informational polymer in the prebiotic context, while on the other hand, finds potential of XyloNA as an orthogonal genetic system for application in synthetic biology.     

Affect and Cognition in Attitude Formation toward Familiar and Unfamiliar Attitude Objects

At large attitudes are built on earlier experience with the attitude object. If earlier experiences are not available, as is the case for unfamiliar attitude objects such as new technologies, no stored evaluations exist. Yet, people are still somehow able to construct attitudes on the spot. Depending on the familiarity of the attitude object, attitudes may find their basis more in affect or cognition. The current paper investigates differences in reliance on affect or cognition in attitude formation toward familiar and unfamiliar realistic attitude objects. In addition, individual differences in reliance on affect (high faith in intuition) or cognition (high need for cognition) are taken into account. In an experimental survey among Dutch consumers (N = 1870), we show that, for unfamiliar realistic attitude objects, people rely more on affect than cognition. For familiar attitude objects where both affective and cognitive evaluations are available, high need for cognition leads to more reliance on cognition, and high faith in intuition leads to more reliance on affect, reflecting the influence of individually preferred thinking style. For people with high need for cognition, cognition has a higher influence on overall attitude for both familiar and unfamiliar realistic attitude objects. On the other hand, affect is important for people with high faith in intuition for both familiar and unfamiliar attitude objects and for people with low faith in intuition for unfamiliar attitude objects; this shows that preferred thinking style is less influential for unfamiliar objects. By comparing attitude formation for familiar and unfamiliar realistic attitude objects, this research contributes to understanding situations in which affect or cognition is the better predictor of overall attitudes.     

Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.     

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.     

1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone ¹⁵N, ¹³C and ¹H chemical shift and ¹³Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.     

Importance of N2-Fixation on the Productivity at the North-Western Azores Current/Front System,and the Abundance of Diazotrophic Unicellular Cyanobacteria

To understand the impact of the northwestern Azores Current Front (NW-AzC/AzF) system on HCO₃⁻-and N₂-fixation activities and unicellular diazotrophic cyanobacteria (UCYN) distribution, we combined geochemical and biological approaches from the oligotrophic surface to upper mesopelagic waters. N₂-fixation was observed to sustain 45–85% of the HCO₃⁻-fixation in the picoplanktonic fraction performing 47% of the total C-fixation at the deep chlorophyll maximum north and south of the AzF. N₂-fixation rates as high as 10.9 μmol N m^-3 d^-1 and surface nitrate δ¹⁵N as low as 2.7‰ were found in the warm (18–24°C), most saline (36.5–37.0) and least productive waters south of the AzF, where UCYN were the least abundant. However, picoplanktonic UCYN abundances up to 55 cells mL^-1 were found at 45–200m depths in the coolest nutrient-rich waters north of the AzF. In this area, N₂-fixation rates up to 4.5 μmol N m^-3 d^-1 were detected, associated with depth-integrated H¹³CO₃⁻-fixation rates at least 50% higher than observed south of the AzF. The numerous eddies generated at the NW-AzC/AzF seem to enhance exchanges of plankton between water masses, as well as vertical and horizontal diapycnal diffusion of nutrients, whose increase probably enhances the growth of diazotrophs and the productivity of C-fixers.     

Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment

In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.     

Adaptation of the fungal parasite Zygorhizidium planktonicum during 200 generations of growth on homogeneous and heterogeneous populations of its host, the diatom Asterionella formosa

We followed adaptation of the chytrid parasite Zygorhizidium planktonicum during 200 generations of growth on its host, the freshwater diatom Asterionella formosa, in a serial passage experiment. Evolution of parasite fitness was assessed both on a homogenous and heterogeneous host population, consisting of respectively a single new and ten different new host strains. These 10 host strains were genetically different and also varied in their initial susceptibility to the parasite. Parasite fitness increased significantly and rapidly on the new, genetically homogenous host population, but remained unaltered during 200 generations of growth on the heterogeneous host population. Enhanced parasite fitness was the result of faster and more efficient transmission, resulting in higher values of R0 (number of secondary infections). Consequently, parasites that evolved within the uniclonal host population infected significantly more of these hosts than did their ancestors. We thus provide experimental evidence for the widely held view that host genetic diversity restricts evolution of parasites and moderates their harmful effects. Genetically uniform host populations are not only at increased risk from fungal epidemics because they all share the same susceptibility, but also because new parasite strains are able to adapt quickly to new host environments and to improve their fitness.