The myth of antibiotic spider silk.

Spider silk is frequently attributed antimicrobial properties. This notion is based on studies reporting antimicrobial activity (AMA) of spider silk; however, close inspection of these studies reveals that the evidence is conflicting, and at best anecdotal. We performed a systematic study of antimicrobial properties of different silk types from seven species across the spider phylogeny. We found no evidence of AMA of silk in direct contact and disc diffusion assays against Gram-negative Escherichia coli and Pseudomonas putida, and the Gram-positive Bacillus subtilis. Furthermore, staining experiments and fluorescence microscopy showed the presence of live bacteria on silk surfaces indicating no antimicrobial effect on direct contact. A critical evaluation of the literature reveals that published tests of AMA are scarce and that all the studies claiming positive results are compromised by methodological shortcomings. Our analysis demonstrates that the common notion that spider silk is antimicrobial is not supported by empirical data.

Single-crystal NMR spectroscopy.

Single-crystal (SC) NMR spectroscopy is a solid-state NMR method that has been used since the early days of NMR to study the magnitude and orientation of tensorial nuclear spin interactions in solids. This review first presents the field of SC NMR instrumentation, then provides a survey of software for analysis of SC NMR data, and finally it highlights selected applications of SC NMR in various fields of research. The aim of the last part is not to provide a complete review of all SC NMR literature but to provide examples that demonstrate interesting applications of SC NMR.

Optimization of pulses with low bandwidth for improved excitation of multiple-quantum coherences in NMR of quadrupolar nuclei.

We discuss the commonly encountered problem when optimizing nuclear magnetic resonance (NMR) pulses using optimal control that the otherwise very precise NMR theory does not provide as excellent agreement with experiments. We hypothesize that this disagreement is due to phase transients in the pulse due to abrupt phase and amplitude changes resulting in a large bandwidth. We apply the gradient optimization using parametrization algorithm that gives high fidelity pulses with a low bandwidth compared to the typical gradient ascent pulse engineering pulses. Our results obtain a better agreement between experiments and simulations supporting our hypothesis and solution to the problem.

Core Scientific Dataset Model: A lightweight and portable model and file format for multi-dimensional scientific data.

The Core Scientific Dataset (CSD) model with JavaScript Object Notation (JSON) serialization is presented as a lightweight, portable, and versatile standard for intra- and interdisciplinary scientific data exchange. This model supports datasets with a p-component dependent variable, {U0, …, Uq, …, Up-1}, discretely sampled at M unique points in a d-dimensional independent variable (X0, …, Xk, …, Xd-1) space. Moreover, this sampling is over an orthogonal grid, regular or rectilinear, where the principal coordinate axes of the grid are the independent variables. It can also hold correlated datasets assuming the different physical quantities (dependent variables) are sampled on the same orthogonal grid of independent variables. The model encapsulates the dependent variables' sampled data values and the minimum metadata needed to accurately represent this data in an appropriate coordinate system of independent variables. The CSD model can serve as a re-usable building block in the development of more sophisticated portable scientific dataset file standards.

Single-spin vector analysis of strongly coupled nuclei in TOCSY NMR experiments.

This paper presents a new way to represent the effect of complex radio-frequency (rf) pulse sequences on J-coupled nuclear spin systems. The model uses a vector representation of the single-spin interactions (chemical-shift and rf interactions) and provides a simple route to gain analytical insight into multipulse NMR experiments. The single-spin Hamiltonian is expressed in an interaction representation as Fourier components. These Fourier components are combined for the two spins to establish the averaged coupling term of the Hamiltonian. This effective Hamiltonian is fast to calculate as only single-spin rotations are used and followed by simple summation of numbers for reconstruction of given coupling interactions. The present method is used to gain analytical insight into the performance of the J-coupling transfer sequence DIPSI-2 through two figures of merit (FOM) providing useful information for optimization of such pulse sequences. The first FOM (ΞAB) reports the efficiency of the desired total correlation spectroscopy transfer and should be as large as possible, while the second (ΞHet) reports the potential leakage of coherence to a heteronuclear spin and should be as small as possible.

Analytical Evaluation of Low-Field 31P NMR Spectroscopy for Lipid Analysis.

We investigate the potential of 31P NMR with simple, maintenance-free benchtop spectrometers to probe phospholipids in complex mixtures. 31P NMR-based lipidomics has become an important topic in a wide range of applications in food- and health-sciences, and the continuous improvements of compact, maintenance- and cryogen-free instruments opens new opportunities for NMR routine analyses. A prior milestone is the evaluation of the analytical performance provided by 31P NMR at low magnetic field. To address this, we assess the ability of state-of-the-art benchtop NMR spectrometers to detect, identify, and quantify several types of phospholipids in mixtures. Relying on heteronuclear cross-polarization experiments, phospholipids can be detected in 2 h with a limit of detection of 0.5 mM at 1 T and 0.2 mM at 2 T, while the headgroups of phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidyl-glycerol (PG) can be unambiguously assigned based on 2D 1H-31P total correlated spectroscopy (TOCSY) spectra. Furthermore, two quantitative methods to obtain absolute concentrations are proposed and discussed, and the performance is evaluated regarding precision and accuracy.

Reduced TCA cycle rates at high hydrostatic pressure hinder hydrocarbon degradation and obligate oil degraders in natural, deep-sea microbial communities.

Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO2 production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea.

Structure of Phospholipid Mixed Micelles (Bicelles) Studied by Small-Angle X-ray Scattering.

Mixed phospholipid micelles (bicelles) are widely applied in nuclear magnetic resonance (NMR) studies of membrane proteins in solution, as they can solubilize these proteins and provide a membrane-like environment. In this work, the structure of bicelles of dihexanoyl phosphatidyl choline (DHPC) and dimyristoyl phosphatidyl choline (DMPC) at different ratios was determined by small-angle X-ray scattering (SAXS) at 37 °C. Samples with concentrations as applied for NMR measurements with 28 wt % lipids were diluted to avoid concentration effects in the SAXS data. The DMPC/DHPC ratio within the bicelles was kept constant by diluting with solutions of finite DHPC concentrations, where the concentration of free DHPC is the same as in the original solution. Absolute-scale modeling of the SAXS data using molecular and concentration constraints reveals a relatively complex set of morphologies of the lipid aggregates as a function of the molar ratio Q of DMPC to DHPC. At Q = 0 (pure DHPC lipids), oblate core-shell micelles are present. At Q = 0.5, the bicelles have a tablet-shaped core-shell cylindrical form with an ellipsoidal cross section. For Q = 1, 2, 3.2, and 4, the bicelles have a rectangular cuboidal structure with a core and a shell, for which the overall length and width increase with Q. At Q = ∞ (pure DMPC), there is coexistence between multilamellar structures and free bilayers. For Q = 1-4, the hydrocarbon core is relatively narrow and the headgroup thickness on the flat areas is larger than that of, respectively, pure DHPC and DMPC, suggesting some mixing of DHPC into these areas and staggering of the molecules. This is further supported by comparisons of the ratio of the areas of rim and flat parts and estimates of the composition of the flat areas.

Fast Wide-Line Solid-State NMR on a Low-Cost Benchtop Spectrometer.

Solid-state NMR may provide access to a wealth of information on molecular structure and dynamics. However, for many applications, the acquisition is challenged by broad resonances implying large spectral linewidths and low sensitivity. Conventionally, this is tackled by using costly and laboratory-fixed spectrometers based on large high-field superconducting magnets. In this Communication, we demonstrate that a range of challenging wide-line solid-state NMR spectra can be acquired on a robust, maintenance-free, low-cost benchtop/mobile NMR spectrometer with a sensitivity comparable to common high-field instruments. The performance and versatility for recording sensitive wide-line spectra is demonstrated through acquisition of 31 P NMR of paramagnetic FePO4 and full quadupolar lineshapes of Al2 O3 (27 Al) and KNO3 (14 N). Also, we introduce interleaved acquisition of frequency-stepped slices providing a dramatic reduction of the required experiment time.

Quantification of Ammonium Phosphatide Emulsifiers in Chocolate Using 31P NMR Spectroscopy.

31P NMR is a valuable tool to study phosphorus-containing biomolecules from complex mixtures. One important group of such molecules are phosphorus-containing emulsifiers, including lecithins and ammonium phosphatides (AMPs), which are used in chocolate production. By developing extraction protocols and applying high resolution 31P nuclear magnetic resonance (NMR), we enable identification of the type of emulsifier used in chocolate. We furthermore demonstrate that this method allows quantification of AMPs in chocolate. To our knowledge, this is the first method that allows verification of the type and amount of emulsifier present in chocolate samples.

Fast simulations of multidimensional NMR spectra of proteins and peptides.

To simulate full multidimensional nuclear magnetic resonance spectra of peptides and proteins in a reasonable time frame, a strategy for separating the time-consuming full-density matrix calculations from the chemical shift prediction and calculation of coupling patterns is presented. The simulation setup uses SIMulation Program for SOlid-state NMR (SIMPSON) to calculate total correlation spectroscopy transfer amplitudes and average distances as a source for nuclear Overhauser effect spectroscopy transfer amplitudes. Simulated 1 H 1D, 2D total correlation spectroscopy, and 2D nuclear Overhauser effect spectroscopy nuclear magnetic resonance spectra of peptides with sequence Pro─Ala─Gly─Tyr─Asn and Asn─Phe─Gly─Ala─Ile─Leu and of ubiquitin are presented. In all cases, the simulations lasted from a few seconds to tens of seconds on a normal laptop computer.

Myoglobin and α-Lactalbumin Form Smaller Complexes with the Biosurfactant Rhamnolipid Than with SDS.

Biosurfactants (BSs) attract increasing attention as sustainable alternatives to petroleum-derived surfactants. This necessitates structural insight into how BSs interact with proteins encountered by current chemical surfactants. Thus, small-angle x-ray scattering (SAXS) has been used for studying the structures of complexes made of the proteins α-Lactalbumin (αLA) and myoglobin (Mb) with the biosurfactant rhamnolipid (RL). For comparison, complexes between αLA and the chemical surfactant sodium dodecyl sulfate (SDS) were also investigated. The SAXS data for pure RL micelles can be described by prolate core-shell structures with a core radius of 7.7 Å and a shell thickness of 12 Å, giving an aggregation number of 11. The small core radius is attributed to RL's complex hydrophobic tail. Data for the αLA-RL complex agree with a 12-molecule micelle with a single protein molecule in the shell. For Mb-RL, the analysis gives complexes of two connected micelles, each containing 10 RL and one protein in the shells. αLA-RL and Mb-RL form surfactant-saturated complexes above 5.6 and 4.7 mM RL, respectively, leaving the remaining RL in free micelles. The SAXS data for SDS agree with oblate-shaped micelles with a core of 20 Å, core eccentricity 0.7, and shell thickness of 5.45 Å, with an aggregation number of 74. The αLA-SDS complexes contain a prolate micelle with a core radius of 11-14 Å and a shell of 8-12 Å with up to 3 αLA per particle and up to 43 SDS per αLA, both considerably larger than for RL. Unlike the RL-protein complexes, the number of surfactant molecules in αLA-SDS complexes increases with surfactant concentration, and saturate at higher surfactant concentrations than αLA-RL complexes. The results highlight how RL and SDS follow similar overall rules of self-assembly and interactions with proteins, but that differences in the strength of protein-surfactant interactions affect the formed structures.

Applications of nuclear magnetic resonance in lipid analyses: An emerging powerful tool for lipidomics studies.

The role of lipids in cell, tissue, and organ physiology is crucial; as many diseases, including cancer, diabetes, neurodegenerative, and infectious diseases, are closely related to absorption and metabolism of lipids. Mass spectrometry (MS) based methods are the most developed powerful tools to study the synthetic pathways and metabolic networks of cellular lipids in biological systems; leading to the birth of an emerging subject lipidomics, which has been extensively reviewed. Nuclear magnetic resonance (NMR), another powerful analytical tool, which allows the visualization of single atoms and molecules, is receiving increasing attention in lipidomics analyses. However, very little work focusing on lipidomic studies using NMR has been critically reviewed. This paper presents a first comprehensive summary of application of 1H, 13C &31P NMR in lipids and lipidomics analyses. The scientific basis, principles and characteristic diagnostic peaks assigned to specific atoms/molecular structures of lipids are presented. Applications of 2D NMR in mapping and monitoring of the components and their changes in complex lipids systems, as well as alteration of lipid profiling over disease development are also reviewed. The applications of NMR lipidomics in diseases diagnosis and food adulteration are exemplified.

Epoxy Matrices Modified by Green Additives for Recyclable Materials.

Epoxy-based thermosets are one of the most popular matrix materials in many industries, and significant environmental benefits can be obtained by developing a recyclable variant of this widely utilized material. Incorporation of a bio-based disulfide additive within a commercial epoxy system leads to a cross-linked material that can be fractionated under mild and environmentally benign conditions. The material has been analyzed by FTIR and solid-state NMR. Furthermore, modified epoxy matrices with low additive concentrations are demonstrated to have similar mechanical and thermal properties compared to commercially available benchmarks. Thus, additive formulation and fractionation based on green chemistry principles have been demonstrated, and a recyclable epoxy matrix has been developed.

How a short pore forming peptide spans the lipid membrane.

Many antimicrobial peptides function by forming pores in the plasma membrane of the target cells. Intriguingly, some of these peptides are very short, and thus, it is not known how they can span the membrane, or whether other mechanisms of cell disruption are dominant. Here, the conformation and orientation of the 14-residue peptaibol SPF-5506-A4 (SPF) are investigated in lipid environments by atomistic and coarse grained molecular dynamics (MD) simulations, circular dichroism, and nuclear magnetic resonance (NMR) experiments. The MD simulations show that SPF is inserted spontaneously in a transmembrane orientation in both 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers resulting in thinning of the bilayers near the peptides, which drives the peptide aggregation. Furthermore, the backbone conformation of the peptide in the bilayer bound state is different from that of the NMR model solved in small bicelles. These results demonstrate that mutual adaption between the peptides and the membrane is likely to be important for pore formation.

A general theoretical description of the influence of isotropic chemical shift in dipolar recoupling experiments for solid-state NMR.

We present a general theoretical description that allows us to describe the influence of isotropic chemical shift in homonuclear and heteronuclear dipolar recoupling experiments in magic-angle-spinning solid-state NMR. Through a transformation of the Hamiltonian into an interaction frame with the combined radio-frequency irradiation and the isotropic chemical shift, we determine an effective Hamiltonian to first order with respect to the relevant internal nuclear spin interactions. This unravels the essential resonance conditions for efficient dipolar recoupling. Furthermore, we propose how to handle situations where the resonance conditions are not exactly fulfilled. To verify the general theoretical description, we compare numerical simulations using a time-sliced time-dependent Hamiltonian with simulations using the calculated effective Hamiltonian for propagation. The comparisons are exemplified for the homonuclear dipolar recoupling experiments C721 and POST-C721.

Synthesis of ent-BE-43547A1 reveals a potent hypoxia-selective anticancer agent and uncovers the biosynthetic origin of the APD-CLD natural products.

Tumour hypoxia is speculated to be a key driver of therapeutic resistance and metastatic dissemination. Consequently, the discovery of new potent agents that selectively target the hypoxic cell population may reveal new and untapped antitumour mechanisms. Here we demonstrate that the BE-43547 subclass of the APD-CLD (amidopentadienoate-containing cyclolipodepsipeptides) natural products possesses highly hypoxia-selective growth-inhibitory activity against pancreatic cancer cells. To enable this discovery, we have developed the first synthesis of the BE-43547-macrocyclic scaffold in 16 steps (longest linear sequence), which also allowed access to the full panel of relative stereoisomers and ultimately to the assignment of stereochemical configuration. Discrepancies between the spectroscopic signatures of the synthetic compounds with that originally reported for the BE-43547 members stimulated us to re-isolate the natural product from a BE-43547-producing microorganism during which we elucidated the biosynthetic gene clusters for the BE-43547 family as well as for all other known APD-CLDs. Our studies underline the exciting possibilities for the further development of the anticancer activities of these natural products.

Local SAR, global SAR, and power-constrained large-flip-angle pulses with optimal control and virtual observation points.

PURPOSE: To present a constrained optimal-control (OC) framework for designing large-flip-angle parallel-transmit (pTx) pulses satisfying hardware peak-power as well as regulatory local and global specific-absorption-rate (SAR) limits. The application is 2D and 3D spatial-selective 90° and 180° pulses. THEORY AND METHODS: The OC gradient-ascent-pulse-engineering method with exact gradients and the limited-memory Broyden-Fletcher-Goldfarb-Shanno method is proposed. Local SAR is constrained by the virtual-observation-points method. Two numerical models facilitated the optimizations, a torso at 3 T and a head at 7 T, both in eight-channel pTx coils and acceleration-factors up to 4. RESULTS: The proposed approach yielded excellent flip-angle distributions. Enforcing the local-SAR constraint, as opposed to peak power alone, reduced the local SAR 7 and 5-fold with the 2D torso excitation and inversion pulse, respectively. The root-mean-square errors of the magnetization profiles increased less than 5% with the acceleration factor of 4. CONCLUSION: A local and global SAR, and peak-power constrained OC large-flip-angle pTx pulse design was presented, and numerically validated for 2D and 3D spatial-selective 90° and 180° pulses at 3 T and 7 T. Magn Reson Med 77:374-384, 2017. © 2015 Wiley Periodicals, Inc.