Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin-ergosterol modulated vesicle fusion

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic After fusion with an ergosterol-free artificial bilayer the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid was reduced. Under these conditions, the B-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, the electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems

Lipid-modulated assembly of magnetized iron-filled carbon nanotubes in millimeter-scale structures

Biomolecule-functionalized carbon nanotubes (CNTs) combine the molecular recognition properties of biomaterials with the electrical properties of nanoscale solid state transducers. Application of this hybrid material in bioelectronic devices requires the development of methods for the reproducible self-assembly of CNTs into higher-order structures in an aqueous environment. To this end, we have studied pattern formation of lipid-coated Fe-filled CNTs, with lengths in the 1–5 µm range, by controlled evaporation of aqueous CNT-lipid suspensions. Novel diffusion limited aggregation structures composed of end-to-end oriented nanotubes were observed by optical and atomic force microscopy. Significantly, the lateral dimension of assemblies of magnetized Fe-filled CNTs was in the millimeter range. Control experiments in the absence of lipids and without magnetization indicated that the formation of these long linear nanotube patterns is driven by a subtle interplay between radial flow forces in the evaporating droplet, lipid-modulated van der Waals forces, and magnetic dipole–dipole interactions. Keywords

Circular material flow in the intensive care unit-environmental effects and identification of hotspots

PurposeThe healthcare sector is responsible for 6–7% of CO2 emissions. The intensive care unit (ICU) contributes to these CO2 emissions and a shift from a linear system to a circular system is needed. The aim of our research was to perform a material flow analysis (MFA) in an academic ICU. Secondary aims were to obtain information and numbers on mass, carbon footprint, agricultural land occupation and water usage and to determine so-called “environmental hotspots” in the ICU.MethodsA material flow analysis was performed over the year 2019, followed by an environmental footprint analysis of materials and environmental hotspot identification.Results2839 patients were admitted to our ICU in 2019. The average length of stay was 4.6 days. Our MFA showed a material mass inflow of 247,000 kg in 2019 for intensive care, of which 50,000 kg is incinerated as (hazardous) hospital waste. The environmental impact per patient resulted in 17 kg of mass, 12 kg CO2 eq, 300 L of water usage and 4 m2 of agricultural land occupation per day. Five hotspots were identified: non-sterile gloves, isolation gowns, bed liners, surgical masks and syringes (including packaging).ConclusionThis is the first material flow analysis that identified environmental risks and its magnitude in the intensive care unit

Effect of Phosphate Buffered Saline Solutions on Top-Down Fabricated ZnO Nanowire Field Effect Transistor

ZnO based nanowire FETs have been fabricated by implementing a top-down approach, which uses optical photolithography, atomic layer deposition (ALD) of ZnO thin film, and anisotropic plasma etching. The effects of Phosphate Buffered Saline (PBS) solution on the surface of ZnO nanowire were investigated by measuring the FET characteristics at different PBS dilutions. The drain current, ION, exhibited an increase of 39 times in the highest PBS solution concentration compared to measurement in air. From the measured transfer characteristics and output characteristics in various PBS dilutions, the device was found to maintain n-type behaviour. These results indicate that the device can be effectively used for biomolecules sensing

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex

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A high confidence, manually validated human blood plasma protein reference set

<p>Abstract</p> <p>Background</p> <p>The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.</p> <p>Methods</p> <p>To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.</p> <p>Results</p> <p>Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset.</p> <p>Conclusion</p> <p>Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.</p

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling.

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors

Fatigue in teriflunomide-treated patients with relapsing remitting multiple sclerosis in the real-world Teri-FAST study

BACKGROUND: Fatigue is a frequent and disabling symptom of multiple sclerosis (MS) often associated with impaired quality of life (QoL) in patients. Teriflunomide is a once-daily oral immunomodulator used for the treatment of relapsing remitting forms of MS. However, its effect on fatigue is not well known in real life practice. We evaluated the impact of teriflunomide on fatigue in patients with relapsing remitting MS (RRMS) after 2 years of treatment in the real-world Teri-FAST study. METHODS: Teri-FAST was a 2-year, prospective, observational study conducted in France in RRMS patients treated with teriflunomide 14 mg. Fatigue was assessed using the French version of the modified fatigue impact scale (EMIF-SEP). The primary endpoint was the change from baseline in EMIF-SEP score after 2 years of treatment. Secondary endpoints included evaluation of depression (Beck Depression Inventory [BDI]), health-related QoL (Two-Life Scale TLS-QoL 10), self-reported physical activity, and adverse events. RESULTS: 210 eligible patients were included in the study with a mean age of 45.4 years and a mean ± SD Expanded Disability Status Scale score of 1.76 ± 1.43 at baseline. About half (52.4%) of patients had no previous treatment for MS. In the 163 patients who completed at least 1 follow-up visit, the mean change in EMIF-SEP score at Year 2 was -1.54 (95% CI: -4.02, 0.94) indicating that fatigue remained stable. Similarly, there were no changes in depression level and QoL after 2 years of treatment. Physical activity slightly improved with 57% of patients reporting being physically active after 2 years as compared to 46% at baseline. The safety profile of teriflunomide was consistent with that seen during clinical development, and compliance with treatment was high. CONCLUSION: Fatigue scores remained stable in RRMS patients treated with teriflunomide 14 mg over 2 years in real-life setting. Teriflunomide did not negatively impact depression or QoL