Structure and hydration of membranes embedded with voltage-sensing domains.

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field

Status of Underground Radioactivity Measurements in HADES

The IRMM (Institute for Reference materials and Measurements) performs ultra low-level gamma-ray spectrometry at a depth of 225 m in the underground laboratory HADES. The facility currently houses 7 HPGe-detectors that are built and shielded using specially selected radiopure materials. The sandclay overburden of about 500 m water equivalent assures a muon flux reduction factor of about 5000, with subsequent reduction of the background of the detectors, which makes it possible to obtain detection limits close to 100 µBq for certain radionuclides. This paper describes the aim of the IRMM activities in the HADES laboratory, the equipment and the measurement program and gives examples of radiopurity measurements carried out in order to develop better low-level measurements.JRC.DG.D.5-Nuclear physic

INTERACTIONS OF THE SARS-COV-2 VIRAL GENOME 3’-UNTRANSLATED REGION WITH VIRAL AND HOST RNAS

This dissertation focuses on the characterization of RNA-RNA interactions within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and with host microRNAs. As the causative agent of coronavirus disease 2019 (COVID-19), SARS-CoV-2 has evolved rapidly since its appearance. This has warranted prompt characterization of the virus particularly of its single stranded RNA (ssRNA) genome. By using a combination of bioinformatics, biophysics, and/or biological assays, we analyzed the SARS-CoV-2 viral genomic RNA and uncovered interactions of genomic RNA with host RNAs, highlighting an underutilized method of targeting RNA viruses. We showed here that the conserved elements in the viral genomic RNA, particularly within the 3’-untranslated region (3’-UTR), are involved in intramolecular and intermolecular RNA-RNA interactions that can have key roles in the viral life cycle. The stem-loop II-like motif (s2m) was one of these conserved elements. We show here that s2m, which is known for its role in genome dimerization, underwent a stepwise mutation that eliminates its interactions with host miR-1307-3p alongside its ability to dimerize. We showed that this interaction can inhibit viral translation, suggesting the loss of binding to be a mechanism of immune evasion. Additionally, we identified other microRNA interactions which could mediate ACE2 expression in infected and nearby cells, allowing hijacking of the immune system through JAK/STAT3 manipulation. Our work also demonstrated that these interactions can inhibit viral translation, similar to the miR-1307-3p interactions. We then implemented 2’-fluoro-D-arabinonucleic acids as effective competitive inhibitors for these microRNA binding interactions. Finally, we also characterized a second dimerization site, the stem-loop III-like motif (s3m), which utilizes a kissing dimer intermediate like s2m and can bind miR-1236-3p with the conserved octanucleotide motif. Taken together, this work elucidated a set of RNA-RNA interactions with SARS-CoV-2 viral RNAs that allow for specific interactions with immune-related regulatory microRNAs. While we suggest here that these microRNAs could be hijacked for benefit of the virus, we also suggest that these interactions could be an antiviral response targeting SARS-CoV-2. Through the identification of these interactions, and the implementation of antisense oligonucleotide competitors, we establish a foundation for therapeutic intervention against SARS-CoV-2 aimed at the virus-host interface

Hydration of POPC bilayers studied by 1H-PFG-MAS-NOESY and neutron diffraction

The stability of lipid bilayers is ultimately linked to the hydrophobic effect and the properties of water of hydration. Magic angle spinning (MAS) nuclear Overhauser enhancement spectroscopy (NOESY) with application of pulsed magnetic field gradients (PFG) was used to study the interaction of water with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers in the fluid phase. NOESY cross-relaxation between water and polar groups of lipids, but also with methylene resonances of hydrophobic hydrocarbon chains, has been observed previously. This observation led to speculations that substantial amounts of water may reside in the hydrophobic core of bilayers. Here, the results of a quantitative analysis of cross-relaxation in a lipid 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC)/water mixture are reported. Coherences were selected via application of pulsed magnetic field gradients. This technique shortens acquisition times of NOESY spectra to 20 min and reduces t (1)-spectral noise, enabling detection of weak crosspeaks, like those between water and lipids, with higher precision than with non-gradient NOESY methods. The analysis showed that water molecules interact almost exclusively with sites of the lipid-water interface, including choline, phosphate, glycerol, and carbonyl groups. The lifetime of lipid-water associations is rather short, on the order of 100 ps, at least one order of magnitude shorter than the lifetime of lipid-lipid associations. The distribution of water molecules over the lipid bilayer was measured at identical water content by neutron diffraction. Water molecules penetrate deep into the interfacial region of bilayers but water concentration in the hydrophobic core is below the detection limit of one water molecule per lipid, in excellent agreement with the cross-relaxation data

AND/R: Advanced neutron diffractometer/reflectometer for investigation of thin films and multilayers for the life sciences

An elastic neutron scattering instrument, the advanced neutron diffractometer/reflectometer (AND/R), has recently been commissioned at the National Institute of Standards and Technology Center for Neutron Research. The AND/R is the centerpiece of the Cold Neutrons for Biology and Technology partnership, which is dedicated to the structural characterization of thin films and multilayers of biological interest. The instrument is capable of measuring both specular and nonspecular reflectivity, as well as crystalline or semicrystalline diffraction at wave-vector transfers up to approximately 2.20 Å(-1). A detailed description of this flexible instrument and its performance characteristics in various operating modes are given.D. J. M. is supported through a NSF NIRT grant Contract No. 0304062

Clinical and Experimental Biomechanical Studies Regarding Innovative Implants in Traumatology

Fracture treatment has experienced a fascinating evolution in the last years. The aim of this chapter is to reveal some clinical and biomechanical studies regarding innovative implants. After a short introduction (1), we intend to present our results regarding (2) dynamic condylar screw versus condylar blade plate in complex supracondylar femoral fractures; (3) biomechanical analysis of four types of implants in humeral fractures; (4) clinical and experimental studies for optimal stabilization of trochanteric fractures: the gliding nail; (5) intramedullary XS nail for pilon and ankle fractures: design, biomechanics, and clinical results; (6) the XS nail for the treatment of patella and olecranon fractures; and (7) plates with polyaxial stability for fractures of distal radius and proximal humerus. In conclusion, the authors highlight the advantages of these innovative implants in difficult trauma cases

Uncontrolled Type 2 Diabetes Mellitus - a Rare Cause of Hemiballismus-Hemichorea. A Case Report and Literature Review

Introduction: Chorea, hyperglycemia, basal ganglia syndrome (CHBG) is a rare neurological complication of nonketotic hyperglycemia which occurs more often in elderly female patients with undiagnosed or poorly controlled diabetes mellitus. Case report: We present the case of a 82-year old female, diagnosed with type 2 diabetes mellitus, but untreated, admitted with non-ketotic hyperglycemia and hemiballistic movements in her left limbs, with acute onset, that changed to choreic movements and then disappeared. Brain magnetic resonance imaging (MRI) showed characteristic hyper intensity in the right (contralateral) putamen on the T1-weighted images. Other secondary causes for ballismus and chorea were excluded. Hemiballismus/hemichorea non-ketotic hyperglycemic basal ganglia (CHBG) syndrome was considered and blood glucose was lowered using insulin. As symptomatic treatment, Haloperidol was started but, due to adverse effects, it was stopped and Clonazepam was associated. The movement disorders disappeared in two weeks after glycemic control. Signifi cance: Movement disorders, like chorea and/or ballismus (hemichorea/hemiballismus) can be a marker of uncontrolled known diabetes mellitus or a presenting sign for an undiagnosed diabetes mellitus. The chorea hyperglycemia basal ganglia (CHBG) syndrome is rare and likely undiagnosed but, being aware of it’s existence is of high importance, as normalising blood sugar values severe neurological complications can be avoided

Structure and Dynamics of Cholesterol-Containing Polyunsaturated Lipid Membranes Studied by Neutron Diffraction and NMR

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, 2H-NMR and 13C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 Å away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid–water interface. Addition of cholesterol partially redistributes DHA density from near the lipid–water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C8–C18 is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin

Antiviral activity of the host defense peptide piscidin 1: investigating a membrane-mediated mode of action

Outbreaks of viral diseases are on the rise, fueling the search for antiviral therapeutics that act on a broad range of viruses while remaining safe to human host cells. In this research, we leverage the finding that the plasma membranes of host cells and the lipid bilayers surrounding enveloped viruses differ in lipid composition. We feature Piscidin 1 (P1), a cationic host defense peptide (HDP) that has antimicrobial effects and membrane activity associated with its N-terminal region where a cluster of aromatic residues and copper-binding motif reside. While few HDPs have demonstrated antiviral activity, P1 acts in the micromolar range against several enveloped viruses that vary in envelope lipid composition. Notably, it inhibits HIV-1, a virus that has an envelope enriched in cholesterol, a lipid associated with higher membrane order and stability. Here, we first document through plaque assays that P1 boasts strong activity against SARS-CoV-2, which has an envelope low in cholesterol. Second, we extend previous studies done with homogeneous bilayers and devise cholesterol-containing zwitterionic membranes that contain the liquid disordered (Ld; low in cholesterol) and ordered (Lo, rich in cholesterol) phases. Using dye leakage assays and cryo-electron microscopy on vesicles, we show that P1 has dramatic permeabilizing capability on the Lo/Ld, an effect matched by a strong ability to aggregate, fuse, and thin the membranes. Differential scanning calorimetry and NMR experiments demonstrate that P1 mixes the lipid content of vesicles and alters the stability of the Lo. Structural studies by NMR indicate that P1 interacts with the Lo/Ld by folding into an α-helix that lies parallel to the membrane surface. Altogether, these results show that P1 is more disruptive to phase-separated than homogenous cholesterol-containing bilayers, suggesting an ability to target domain boundaries. Overall, this multi-faceted research highlights how a peptide that interacts strongly with membranes through an aromatic-rich N-terminal motif disrupt viral envelope mimics. This represents an important step towards the development of novel peptides with broad-spectrum antiviral activity