Solid-state NMR analysis of unlabeled fungal cell walls from Aspergillus and Candida species.

Fungal infections cause high mortality in immunocompromised individuals, which has emerged as a significant threat to human health. The efforts devoted to the development of antifungal agents targeting the cell wall polysaccharides have been hindered by our incomplete picture of the assembly and remodeling of fungal cell walls. High-resolution solid-state nuclear magnetic resonance (ss NMR) studies have substantially revised our understanding of the polymorphic structure of polysaccharides and the nanoscale organization of cell walls in Aspergillus fumigatus and multiple other fungi. However, this approach requires 13C/15N-enrichment of the sample being studied, severely restricting its application. Here we employ the dynamic nuclear polarization (DNP) technique to compare the unlabeled cell wall materials of A. fumigatus and C. albicans prepared using both liquid and solid media. For each fungus, we have identified a highly conserved carbohydrate core for the cell walls of conidia and mycelia, and from liquid and solid cultures. Using samples prepared in different media, the recently identified function of α-glucan, which packs with chitin to form the mechanical centers, has been confirmed through conventional ss NMR measurements of polymer dynamics. These timely efforts not only validate the structural principles recently discovered for A. fumigatus cell walls in different morphological stages, but also open up the possibility of extending the current investigation to other fungal materials and cellular systems that are challenging to label.

A 13C three-dimensional DQ-SQ-SQ correlation experiment for high-resolution analysis of complex carbohydrates using solid-state NMR.

Complex carbohydrates are the key components of the protective cell walls of microbial pathogens and the bioenergy reservoir in plants and algae. Structural characterization of these polymorphic molecules requires assistance from multidimensional 13C correlation approaches. To facilitate the analysis of carbohydrate structure using solid-state NMR, we present a three-dimensional (3D) 13C-13C-13C experiment that includes a double-quantum (DQ) dimension and is thus free of the cube's body diagonal. The enhanced resolution supports the unambiguous resonance assignment of many polysaccharides in plant and fungal cell walls using uniformly 13C-labeled cells of spruce and Aspergillus fumigatus. Long-range structural restraints were effectively obtained to revisit our understanding of the spatial organization of plant cellulose microfibrils. The method is widely applicable to the investigations of cellular carbohydrates and carbon-based biomaterials.

Effect of cross polarization radiofrequency phases on signal phase.

Utilizing phases of radio frequency (RF) pulses to manipulate spin dynamics is routine in NMR and MRI, leading to spectacular techniques like phase cycling. In a very different area, cross polarization (CP) also has a long history as part of a vast number of solid-state NMR pulse sequences. However, a detailed study devoted to the effect of CP RF phases on NMR signal, seems not to be readily available. From first principles, we arrive at a simple dependence of NMR signal on arbitrary CP RF phases, for static and MAS conditions, accompanied by experimental verification. In the process, the CP propagator emerges as a product of RF "pulses" and a period of "free precession", conforming to coherence transfer pathway theory. The theoretical expressions may lend confidence for dealing with CP blocks with tunable phases in pulse sequences.

Interlocked benzenes in triangular π-architectures: anchoring groups dictate ion binding and transmission.

Macrocyclic compounds like crown ethers, calixarenes, etc. are well explored in the literature as receptors for alkali metal ions. In most of these studies, the size of the macrocyclic cavity has evolved as the prominent determining criterion for the selective binding of various ions. However, approaches to systematically tailor the ion transport properties via the interplay of topological as well as electronic properties of the hosts are rarely addressed. Herein, we investigate the performance of [2.2.2]PCPs ([2.2.2]paracyclophane and [2.2.2]paracyclophene) and cyclohexaphenylene (CHP) as receptors for the alkali ions, Li+, Na+ and K+. The three macrocycles differ in terms of the groups (ethylene, vinylene and phenylene) anchoring the three benzene rings into triangular three-dimensional architectures, thereby providing opportunities for controlling the topological and the electronic features of the cavities. Based on electronic structure calculations, we predict that PCPs and CHP could be used in conjunction with dehydrobenzoannulenes that possess similar triangular π-architectures in two-dimensions to achieve selective ion transmission. Furthermore, an extended network of CHP, graphenylene, is shown for the first time to be potentially useful in energy storage applications in lithium ion batteries akin to graphyne and graphdiyne. The ion binding properties of graphenylenes would be rather interesting to investigate experimentally for energy applications, particularly in the context of the recent successful synthesis of one of the members of the graphenylene family. Overall, we have attempted to provide a unified description of the cationic interactions with 2D and 3D triangular π-architectures, describing the utility of materials like graphyne, graphdiyne and graphenylene for ion sensing and separation and energy storage applications.

Magnetic alignment and quadrupolar/paramagnetic cross-correlation in complexes of Na with LnDOTP5-.

The observation of a double-quantum filtered signal of quadrupolar nuclei (e.g. (23)Na) in solution has been traditionally interpreted as a sign for anisotropic reorientational motion. Ling and Jerschow (2007) have found that a (23)Na double-quantum signal is observed also in solutions of TmDOTPNa(5). Interference effects between the quadrupolar and the paramagnetic interactions have been reported to lead to the appearance of double-quantum coherences even in the absence of a residual quadrupolar interaction. In addition, such processes lead to differential linebroadening effects between the satellite transitions, akin to effects that are well known for dipolar-CSA cross-correlation. Here, we report experiments on sodium in the presence of LnDOTP compounds, where it is shown that these cross-correlation effects correlate well with the pseudo-contact shift. In addition, anisotropic g-values of the lanthanide compounds in question, can also lead to alignment within the magnetic field, and consequently to the appearance of line splitting and double-quantum coherences. The two competing effects are demonstrated and it is concluded that both cross-correlated relaxation and alignment in the magnetic field must be at work in the systems described here.

Does neuronal damage precede vascular damage in subjects with type 2 diabetes mellitus and having no clinical diabetic retinopathy?

AIM: To investigate the occurrence of neuronal damage, as the earliest change occurring, before the clinical evidence of diabetic retinopathy. METHODS: 70 eyes of subjects with type 2 diabetes mellitus and with no evidence of diabetic retinopathy (cases) and 40 eyes of subjects with no diabetes mellitus (controls) were studied using spectral-domain OCT and microperimetry. The influence of age and gender on the outcome measures was also analyzed. RESULTS: Age- and gender-matched subjects showed a decreased mean retinal nerve fiber layer thickness in cases when compared to the controls (27 vs. 33 µm; p=0.018). Among the cases, subjects between 40 and 45 years of age showed a reduced mean central foveal thickness (175.1 vs. 198.1 µm; p=0.05), mean retinal thickness in the central 6-mm fundus (260.5 vs. 275.3 µm; p=0.006) and mean retinal nerve fiber layer thickness (29 vs. 39 µm; p=0.036) when compared to the controls. However, no differences were noted in the microperimetry outcomes in cases when compared to the controls. The duration of diabetes and the glycemic control did not show any significant changes on the outcome measures in cases, except for a significantly lower mean retinal sensitivity in diabetics with glycosylated hemoglobin values<7% as compared to those with glycosylated hemoglobin≥7% (14.1±2.9 vs. 15.4±1.7 dB; p=0.027). CONCLUSION: The results suggest that there is some evidence of early neuronal damage particularly on spectral-domain OCT, before the clinical evidence of diabetic retinopathy, in subjects with type 2 diabetes mellitus.

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants.

We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110alpha requires (i) an inhibitory contact between the p85 nSH2 domain and the p110alpha helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110alpha. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (tau(c) approximately 12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110alpha C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110alpha, similar to the truncated p85ni-572(STOP). Conversely, wild-type p85ni poorly inhibits p110alphaN345K. Strikingly, the p110alphaN345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110alpha-N345 is not additive with the p85ni-572(STOP) mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572(STOP) mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.

Effectively doubling the magnetic field in spin-1/2-spin-1, HSQC, HDQC, coupled HSQC, and coupled HDQC in solution NMR.

Pulse sequences for spin-1/2-spin-1 pair heteronuclear single quantum correlation (HSQC), heteronuclear double quantum correlation (HDQC), and coupled-HSQC, and coupled-HDQC NMR spectroscopies are outlined, and experimental realization for a (13)C-(2)H pair is demonstrated in solution state. In both the coupled versions, conditions for generation of in-phase and antiphase multiplets in either dimension are arrived at. The patterns and the intensity ratios are explained. The double quantum (2Q) experiments confirm doubling of both the shift frequency and the splitting due to coupling (to spin 1/2) of the 2Q coherence emanating from spin 1. The frequency doubling is equivalent to the corresponding single quantum (1Q) coherence at double the magnetic field strength. The coupling doubling, however, is independent of the magnetic field strength and a signature feature of the 2Q coherence. The ramification of the relative relaxation rates of 1Q and 2Q coherences is discussed.

Mechanism of constitutive phosphoinositide 3-kinase activation by oncogenic mutants of the p85 regulatory subunit.

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.