A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K+ efflux assays confirm that high cholesterol concentration reduces K+ conductance. We then determine the structure of the cholesterol-KirBac1.1 complex using Xplor-NIH simulated annealing calculations driven by solid-state NMR distance measurements. These calculations identified an α-α cholesterol dimer docked to a cleft formed by adjacent subunits of the homotetrameric protein. We compare these results to coarse grain molecular dynamics simulations. This is one of the first examples of a cholesterol oligomer performing a distinct biological function and structural characterization of a conserved promiscuous lipid binding region.

Magnetic Susceptibility Modeling of Magic-Angle Spinning Modules for Part Per Billion Scale Field Homogeneity.

Magic-angle spinning (MAS) solid-state NMR methods are crucial in many areas of biology and materials science. Conventional probe designs have often been specified with 0.1 part per million (ppm) or 100 part per billion (ppb) magnetic field resolution, which is a limitation for many modern scientific applications. Here we describe a novel 5-mm MAS module design that significantly improves the linewidth and line shape for solid samples by an improved understanding of the magnetic susceptibility of probe materials and geometrical symmetry considerations, optimized to minimize the overall perturbation to the applied magnetic field (B0). The improved spinning module requires only first and second order shimming adjustments to achieve a sub-Hz resolution of 13C resonances of adamantane at 150 MHz Larmor frequency (14.1Tesla magnetic field). Minimal use of third and higher order shims improves experimental reproducibility upon sample changes and the exact placement within the magnet. Furthermore, the shimming procedure is faster, and the required gradients smaller, thus minimizing thermal drift of the room temperature (RT) shims. We demonstrate these results with direct polarization (Bloch decay) and cross polarization experiments on adamantane over a range of sample geometries and with multiple superconducting magnet systems. For a direct polarization experiment utilizing the entire active sample volume of a 5-mm rotor (90 µl), we achieved full width at half maximum (FWHM) of 0.76 Hz (5 ppb) and baseline resolved the 13C satellite peaks for adamantane as a consequent of the 7.31 Hz (59 ppb) width at 2% intensity. We expect these approaches to be increasingly pivotal for high-resolution solid-state NMR spectroscopy at and above 1 GHz 1H frequencies.

Tetrakis(trimethylsilyl)silane as a standard compound for fast spinning Solid-State NMR experiments.

The development of magic angle spinning (MAS) at rates ranging from 30 kHz to greater than 100 kHz has substantially advanced solid-state nuclear magnetic resonance (SSNMR) spectroscopy 1H-detection methods. The small rotors required for such MAS rates have a limited sample volume and low 13C-detection sensitivity, rendering the traditional set of standard compounds for SSNMR insufficient or highly inconvenient for shimming and magic-angle calibration. Additionally, the reproducibility of magic angle setting, chemical shift referencing, and probe position can be especially critical for SSNMR experiments at high fields. These conditions suggest the need for a high signal-to-noise ratio (SNR) 1H-detection standard compound, which is preferably multi-purpose, to simplify instrument set up for ultra-fast MAS SSNMR instruments at high magnetic fields. In this study, we present the results for setting magic angle and shimming using tetrakis(trimethylsilyl)silane (TTMSS, or TKS), a tetramethylsilane (TMS) analogue, at near 40 kHz and demonstrate that we can achieve favorable results in less time but with equal or superior precision as traditional KBr and adamantane standards. The high SNR and TMS-like chemical shift of TKS also opens the possibilities for using TKS as an internal standard with biological samples. A single rotor containing a four-component mixture of TKS, adamantane, uniformly 13C, 15N-labeled N-acetyl valine and KBr was used to perform a complete configuration and calibration of a SSNMR probe without sample changes. We anticipate TKS as a standard compound to be especially effective at very high MAS conditions and to greatly simplify the instrument set up for high and ultra-high field SSNMR instruments.

Solid-State NMR 13C sensitivity at high magnetic field.

Sensitivity is the foundation of every NMR experiment, and the signal-to-noise ratio (SNR) should increase with static (B0) magnetic field, by a proportionality that primarily depends on the design of the NMR probe and receiver. In the low B0 field limit, where the coil geometry is much smaller than the wavelength of the NMR frequency, SNR can increase in proportion to B0 to the power 7/4. For modern magic-angle spinning (MAS) probes, this approximation holds for rotor sizes up to 3.2 mm at 14.1 Tesla (T), corresponding to 600 MHz 1H and 151 MHz 13C Larmor frequencies. To obtain the anticipated benefit of larger coils and/or higher B0 fields requires a quantitative understanding of the contributions to SNR, utilizing standard samples and protocols that reproduce SNR measurements with high accuracy and precision. Here, we present such a systematic and comprehensive study of 13C SNR under MAS over the range of 14.1 to 21.1 T. We evaluate a range of probe designs utilizing 1.6, 2.5 and 3.2 mm rotors, including 24 different sets of measurements on 17 probe configurations using five spectrometers. We utilize N-acetyl valine as the primary standard and compare and contrast with other commonly used standard samples (adamantane, glycine, hexamethylbenzene, and 3-methylglutaric acid). These robust approaches and standard operating procedures provide an improved understanding of the contributions from probe efficiency, receiver noise figure, and B0 dependence in a range of custom-designed and commercially available probes. We find that the optimal raw SNR is obtained with balanced 3.2 mm design at 17.6 T, that the best mass-limited SNR is achieved with a balanced 1.6 mm design at 21.1 T, and that the raw SNR at 21.1 T reaches diminishing returns with rotors larger than 2.5 mm.

Cross polarization stability in multidimensional NMR spectroscopy of biological solids.

Magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful and versatile technique for probing structure and dynamics in large, insoluble biological systems at atomic resolution. With many recent advances in instrumentation and polarization methods, technology development in SSNMR remains an active area of research and presents opportunities to further improve data collection, processing, and analysis of samples with low sensitivity and complex tertiary and quaternary structures. SSNMR spectra are often collected as multidimensional data, requiring stable experimental conditions to minimize signal fluctuations (t1 noise). In this work, we examine the factors adversely affecting signal stability as well as strategies used to mitigate them, considering laboratory environmental requirements, configuration of amplifiers, and pulse sequence parameter selection. We show that Thermopad® temperature variable attenuators (TVAs) can partially compensate for the changes in amplifier output power as a function of temperature and thereby ameliorate one significant source of instability for some spectrometers and pulse sequences. We also consider the selection of tangent ramped cross polarization (CP) waveform shapes, to balance the requirements of sensitivity and instrumental stability. These findings collectively enable improved stability and overall performance for CP-based multidimensional spectra of microcrystalline, membrane, and fibrous proteins performed at multiple magnetic field strengths.

A complete 3D-printed tool kit for Solid-State NMR sample and rotor handling.

Solid state NMR (SSNMR) is a highly versatile and broadly applicable method for studying the structure and dynamics of biomolecules and materials. For scientists entering the field of SSNMR, the many quotidian activities required in the workflow to prepare samples for data collection can present a significant barrier to adoption. These steps include transfer of samples into rotors, marking the reflective surfaces for high sensitivity tachometer signal detection, inserting rotors into the magic-angle spinning (MAS) stator, achieving stable spinning, and removing and storing rotors to ensure reproducibility of data collection conditions. Even experienced spectroscopists experience occasional problems with these operations, and the cumulative probability of a delay to successful data collection is high enough to cause frequent disruptions to instrument schedules, particularly in the context of large facilities serving a diverse community of users. These problems are all amplified when utilizing rotors smaller than about 4 mm in diameter. Therefore, to improve the reliability and robustness of SSNMR sample preparation workflows, here we describe a set of tools for rotor packing, unpacking, tachometer marking, extraction and storage. Stereolithography 3D printing was employed as a cost-effective and convenient method for prototyping and manufacturing a full range of designs suitable for several types of probes and rotor geometries.

Comparison of alcalase- and pepsin-treated oilseed protein hydrolysates - Experimental validation of predicted antioxidant, antihypertensive and antidiabetic properties.

There is emerging evidence on the importance of food-derived bioactive peptides to promote human health. Compared with animal derived proteins, plant proteins, in particular oilseed proteins, are considered as affordable and sustainable sources of bioactive peptides. Based on our previous bioinformatic analysis, five oilseed proteins (flaxseed, rapeseed, sunflower, sesame and soybean) were enzymatically hydrolysed using alcalase and pepsin (pH 1.3 and pH 2.1). Further, low molecular weight (Mw â€‹< â€‹3 â€‹kDa) fractions were generated using ultrafiltration. The protein hydrolysates and their low Mw fractions were evaluated for their in vitro antioxidant, antihypertensive and antidiabetic capabilities, in comparison with samples obtained from two dairy proteins (whey and casein). Apart from dipeptidyl-peptidase IV inhibition, significantly stronger bioactivities were detected for the low Mw fractions. In partial agreement with in silico predictions, most oilseed hydrolysates exerted comparable angiotensin-converting enzyme inhibitory capability to dairy proteins, whilst whey protein was the most promising source of dipeptidyl-peptidase IV inhibitors. Apart from alcalase-treated soybean, dairy proteins were more efficient in releasing antioxidant peptides as compared to oilseed proteins. On the other hand, soybean protein hydrolysates showed the highest α-glucosidase inhibitory activity amongst all protein sources. Overall, there was limited correlation between in silico predictions and in vitro experimental results. Nevertheless, our results indicate that oilseed proteins have potential as bioactive peptide sources, and they might therefore be suitable replacers for dairy proteins as well as good sources for development of functional foods.

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State.

NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1-300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

Identification of angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory peptides derived from oilseed proteins using two integrated bioinformatic approaches.

Angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) play critical roles in the development of hypertension and type 2 diabetes, respectively. Inhibiting ACE and DPP-IV activity using peptides has become part of new therapeutic strategies for supporting medicinal treatment of both diseases. In this study, oilseed proteins, including soybean, flaxseed, rapeseed, sunflower and sesame are evaluated for the possibility of generating ACE and DPP-IV inhibitory peptides using different integrated bioinformatic approaches (UniProt knowledgebase, ProtParam, BLAST, BIOPEP, PeptideRanker, Pepsite2 and ToxinPred), and three bovine proteins (ß-lactoglobulin, ß-casein and κ-casein) as comparisons. Compared with bovine proteins, the potency indices of ACE and DPP-IV inhibitory peptides, calculated using the BIOPEP database, suggest that oilseed proteins may be considered as good precursors of ACE inhibitory peptides but generate a relative lower yield of DPP-IV inhibitory peptides following subtilisin, pepsin (pH = 1.3) or pepsin (pH > 2) hydrolysis. Average scores aligned using PeptideRanker confirmed oilseed proteins as significant potential sources of bioactive peptides: over 105 peptides scored over 0.8. Pepsite2 predicted that these peptides would largely bind via Gln281, His353, Lys511, His513, Tyr520 and Tyr523 of ACE to inhibit the enzyme, while Trp629 would be the predominant binding site of peptides in reducing DPP-IV activity. All peptides were capable of inhibiting ACE and DPP-IV whilst 65 of these 105 peptides are not currently recorded in BIOPEP database. In conclusion, our in silico study demonstrates that oilseed proteins could be considered as good precursors of ACE and DPP-IV inhibitory peptides as well as so far unexplored peptides that potentially have roles in ACE and DPP-IV inhibition and beyond.

Effectiveness and safety of chemotherapy combined with dendritic cells co-cultured with cytokine-induced killer cells in the treatment of advanced non-small-cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: Lung cancer, particularly non-small-cell lung cancer (NSCLC) is the leading cause of cancer mortality. Chemotherapy combined dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) immunotherapy has been applied in advanced NSCLC patients' treatment, but couldn't provide consistent beneficial results. Therefore, it is necessary to evaluate the efficiency and safety of combination therapy to promote the application. METHODS: A literature search for randomized controlled trials of NSCLC was conducted in PubMed database. Before meta-analysis was performed, studies were evaluated heterogeneity. Pooled risk ratios (RRs) were estimated and 95% confidence intervals (CIs) were calculated using a fixed-effect model. Sensitivity analysis was also performed. RESULTS: Six eligible trials were enrolled. Efficiency and safety of chemotherapy followed by DC-CIK immunotherapy (experimental group) and chemotherapy alone (control group) were compared. 1-year overall survival (OS) (P = 0.02) and progression free survival (PFS) (P = 0.005) in the experimental group were significantly increased compared with the control. Disease control rate (DCR) (P = 0.006) rose significantly in experimental group. However, no significant differences between the two groups were observed in 2-year OS (P = 0.21), 2-year PFS (P = 0.10), overall response rate (ORR) (P = 0.76) and partial response (PR) (P = 0.22). Temporary fever, anemia, leukopenia and nausea were the four major adverse events (AEs) treated by chemotherapy. The incidence of anemia, leukopenia and nausea in the experimental group was obviously lower than the control group. Temporary fever rate was higher in experimental group than that in the control, but could be alleviated by taking sufficient rest. CONCLUSIONS: Chemotherapy combined with DC-CIK immunotherapy showed superiority in DCR, 1-year OS and PFS, and no more AEs appeared, however, there was no significant improvement in ORR, PR, 2-year OS and PFS. As a whole, the combination therapy is safer but modest in efficacy for advanced NSCLC patients.