Systematic comparison of commercial devices for temporal characterization of few-cycle laser pulses in the 500-1000†nm spectral range.

We compare multiple temporal pulse characterization techniques in three different pulse duration regimes from 15 fs to sub-5 fs, as there are no available standards yet for measuring such ultrashort pulses. To accomplish this, a versatile post-compression platform was developed, where the 100 fs near infrared pulses were post-compressed to the sub-two-cycle regime in a hybrid, three-stage configuration. After each stage, the duration of the compressed pulse was measured with the d-scan, TIPTOE and SRSI techniques and the retrieved temporal intensity profiles, spectrum and spectral phases were compared. Spectral homogeneity was also measured with an imaging spectrometer to understand the input coupling conditions of the temporal measurements. Our findings suggest that the different devices give similar results in terms of temporal intensity profile, however they are extremely sensitive to alignment and to beam quality, especially in the case of the shortest pulses. We address specific steps of measurement procedures, which paves the way towards the standardization of pulse characterization in the near future.

Effect of hypercholesterolemia on circulating and cardiomyocyte-derived extracellular vesicles.

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements.

Few-cycle laser pulse characterization on-target using high-harmonic generation from nano-scale solids.

We demonstrate high-harmonic generation for the time-domain observation of the electric field (HHG-TOE) and use it to measure the waveform of ultrashort mid-infrared (MIR) laser pulses interacting with ZnO thin-films or WS2 monolayers. The working principle relies on perturbing HHG in solids with a weak replica of the pump pulse. We measure the duration of few-cycle pulses at 3200 nm, in reasonable agreement with the results of established pulse characterization techniques. Our method provides a straightforward approach to accurately characterize femtosecond laser pulses used for HHG experiments right at the point of interaction.

Packing up the genome.

Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.

Correction: Synthesis and preclinical application of a Prussian blue-based dual fluorescent and magnetic contrast agent (CA).

[This corrects the article DOI: 10.1371/journal.pone.0264554.].

Inferring mechanical properties of the SARS-CoV-2 virus particle with nano-indentation tests and numerical simulations.

The pandemic caused by the SARS-CoV-2 virus has claimed more than 6.5 million lives worldwide. This global challenge has led to accelerated development of highly effective vaccines tied to their ability to elicit a sustained immune response. While numerous studies have focused primarily on the spike (S) protein, less is known about the interior of the virus. Here we propose a methodology that combines several experimental and simulation techniques to elucidate the internal structure and mechanical properties of the SARS-CoV-2 virus. The mechanical response of the virus was analyzed by nanoindentation tests using a novel flat indenter and evaluated in comparison to a conventional sharp tip indentation. The elastic properties of the viral membrane were estimated by analytical solutions, molecular dynamics (MD) simulations on a membrane patch and by a 3D Finite Element (FE)-beam model of the virion's spike protein and membrane molecular structure. The FE-based inverse engineering approach provided a reasonable reproduction of the mechanical response of the virus from the sharp tip indentation and was successfully verified against the flat tip indentation results. The elastic modulus of the viral membrane was estimated in the range of 7-20 MPa. MD simulations showed that the presence of proteins significantly reduces the fracture strength of the membrane patch. However, FE simulations revealed an overall high fracture strength of the virus, with a mechanical behavior similar to the highly ductile behavior of engineering metallic materials. The failure mechanics of the membrane during sharp tip indentation includes progressive damage combined with localized collapse of the membrane due to severe bending. Furthermore, the results support the hypothesis of a close association of the long membrane proteins (M) with membrane-bound hexagonally packed ribonucleoproteins (RNPs). Beyond improved understanding of coronavirus structure, the present findings offer a knowledge base for the development of novel prevention and treatment methods that are independent of the immune system.

Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA-Lipid Complexes.

Despite the worldwide success of mRNA-LNP Covid-19 vaccines, the nanoscale structures of these formulations are still poorly understood. To fill this gap, we used a combination of atomic force microscopy (AFM), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and the determination of the intra-LNP pH gradient to analyze the nanoparticles (NPs) in BNT162b2 (Comirnaty), comparing it with the well-characterized PEGylated liposomal doxorubicin (Doxil). Comirnaty NPs had similar size and envelope lipid composition to Doxil; however, unlike Doxil liposomes, wherein the stable ammonium and pH gradient enables accumulation of 14C-methylamine in the intraliposomal aqueous phase, Comirnaty LNPs lack such pH gradient in spite of the fact that the pH 4, at which LNPs are prepared, is raised to pH 7.2 after loading of the mRNA. Mechanical manipulation of Comirnaty NPs with AFM revealed soft, compliant structures. The sawtooth-like force transitions seen during cantilever retraction imply that molecular strands, corresponding to mRNA, can be pulled out of NPs, and the process is accompanied by stepwise rupture of mRNA-lipid bonds. Unlike Doxil, cryo-TEM of Comirnaty NPs revealed a granular, solid core enclosed by mono- and bilipid layers. Negative staining TEM shows 2-5 nm electron-dense spots in the LNP's interior that are aligned into strings, semicircles, or labyrinth-like networks, which may imply cross-link-stabilized RNA fragments. The neutral intra-LNP core questions the dominance of ionic interactions holding together this scaffold, raising the possibility of hydrogen bonding between mRNA and the lipids. Such interaction, described previously for another mRNA/lipid complex, is consistent with the steric structure of the ionizable lipid in Comirnaty, ALC-0315, displaying free ═O and -OH groups. It is hypothesized that the latter groups can get into steric positions that enable hydrogen bonding with the nitrogenous bases in the mRNA. These structural features of mRNA-LNP may be important for the vaccine's activities in vivo.

Selective and optimal dynamic pricing strategy for residential electricity consumers based on genetic algorithms.

The continuous increase in residential population implies an increase in electricity demand. The demand prediction performed by the utility company (UC) to schedule and plan the next energy purchase from the market may not be efficient since the actual demand consistently exceeds the supply. To use the energy production infrastructure efficiently and decrease the grid system's overload, the UC seeks to match the demand with the supply. Dynamic pricing is a simple yet effective way to influence consumption as price-sensitive consumers may reschedule the operation of some of the appliances. This paper presents a novel, consumption data-driven, selective and dynamic Time-of-Use (ToU) electricity pricing strategy applicable by the UC for residential electricity consumers. The method consists of clustering real consumption data using the k-means clustering technique and heuristically categorizing clusters based only on their consumption data to simplify the design of the ToU tariffs and limit the number of different tariffs in the same population. The method includes a heuristic determination of the time period for the ToU tariff changes for each category. The ToU tariff parameters are determined by minimizing a single cost function using genetic algorithms and considering all consumer clusters such that the consumer behavior model is based on price elasticity. The optimal ToU tariffs result in decreasing the demand in power peaks by targeting the overconsumer categories in the latter. Implementing the ToU tariff results in a profit margin distributed among the UC and the consumer. Moreover, the optimal prices guarantee positive gains for both of them with the presence of a wide range of parameter uncertainties. The proposed pricing strategy necessitates the presence of consumption data only, which implies the conservation of the consumers' privacy. Real consumption data obtained for two towns in Hungary over three years is used to show the efficiency of the proposed pricing strategy.

Imaging the Infection Cycle of T7 at the Single Virion Level.

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage-displayed isotropic spatial orientation. The viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle.

Synthesis and preclinical application of a Prussian blue-based dual fluorescent and magnetic contrast agent (CA).

The aim of this study was to develop and characterize a Prussian Blue based biocompatible and chemically stable T1 magnetic resonance imaging (MRI) contrast agent with near infrared (NIR) optical contrast for preclinical application. The physical properties of the Prussian blue nanoparticles (PBNPs) (iron (II); iron (III);octadecacyanide) were characterized with dynamic light scattering (DLS), zeta potential measurement, atomic force microscopy (AFM), and transmission electron microscopy (TEM). In vitro contrast enhancement properties of PBNPs were determined by MRI. In vivo T1-weighted contrast of the prepared PBNPs was investigated by MRI and optical imaging modality after intravenous administration into NMRI-Foxn1 nu/nu mice. The biodistribution studies showed the presence of PBNPs predominantly in the cardiovascular system. Briefly, in this paper we show a novel approach for the synthesis of PBNPs with enhanced iron content for T1 MRI contrast. This newly synthetized PBNP platform could lead to a new diagnostic agent, replacing the currently used Gadolinium based substances.

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the ß-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Topography, Spike Dynamics, and Nanomechanics of Individual Native SARS-CoV-2 Virions.

SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, displays a corona-shaped layer of spikes which play a fundamental role in the infection process. Recent structural data suggest that the spikes possess orientational freedom and the ribonucleoproteins segregate into basketlike structures. How these structural features regulate the dynamic and mechanical behavior of the native virion are yet unknown. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, here, we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. The dynamics and the mechanics of SARS-CoV-2 are likely to affect its stability and interactions.

Development, structure and mechanics of a synthetic E. coli outer membrane model.

The outer membrane (OM) of Gram-negative bacteria is a complex asymmetric bilayer containing lipids, lipopolysaccharides (LPS) and proteins. While it is a mechanical and chemical barrier, it is also the primary surface of bacterial recognition processes that involve infection by and of the bacterium. Uncovering the mechanisms of these biological functions has been hampered by the lack of suitable model systems. Here we report the step-by-step assembly of a synthetic OM model from its fundamental components. To enable the efficient formation of a supported lipid bilayer at room temperature, dimyristoyl-phosphocholine (DMPC) was used as the lipid component to which we progressively added LPS and OM proteins. The assembled system enabled us to explore the contribution of the molecular components to the topographical structure and stability of the OM. We found that LPS prefers solid-state membrane regions and forms stable vesicles in the presence of divalent cations. LPS can gradually separate from DMPC membranes to form independent vesicles, pointing at the dynamic nature of the lipid-LPS system. The addition of OM proteins from E. coli and saturating levels of LPS to DMPC liposomes resulted in a thicker and more stable bilayer the surface of which displayed a nanoscale texture formed of parallel, curved, long (>500 nm) stripes spaced apart with a 15 nm periodicity. The synthetic membrane may facilitate the investigation of binding and recognition processes on the surface of Gram-negative bacteria.

Fluorescent, Prussian Blue-Based Biocompatible Nanoparticle System for Multimodal Imaging Contrast.

(1) Background. The main goal of this work was to develop a fluorescent dye-labelling technique for our previously described nanosized platform, citrate-coated Prussian blue (PB) nanoparticles (PBNPs). In addition, characteristics and stability of the PB nanoparticles labelled with fluorescent dyes were determined. (2) Methods. We adsorbed the fluorescent dyes Eosin Y and Rhodamine B and methylene blue (MB) to PB-nanoparticle systems. The physicochemical properties of these fluorescent dye-labeled PBNPs (iron(II);iron(III);octadecacyanide) were determined using atomic force microscopy, dynamic light scattering, zeta potential measurements, scanning- and transmission electron microscopy, X-ray diffraction, and Fourier-transformation infrared spectroscopy. A methylene-blue (MB) labelled, polyethylene-glycol stabilized PBNP platform was selected for further assessment of in vivo distribution and fluorescent imaging after intravenous administration in mice. (3) Results. The MB-labelled particles emitted a strong fluorescent signal at 662 nm. We found that the fluorescent light emission and steric stabilization made this PBNP-MB particle platform applicable for in vivo optical imaging. (4) Conclusion. We successfully produced a fluorescent and stable, Prussian blue-based nanosystem. The particles can be used as a platform for imaging contrast enhancement. In vivo stability and biodistribution studies revealed new aspects of the use of PBNPs.

Single-particle virology.

The development of advanced experimental methodologies, such as optical tweezers, scanning-probe and super-resolved optical microscopies, has led to the evolution of single-molecule biophysics, a field of science that allows direct access to the mechanistic detail of biomolecular structure and function. The extension of single-molecule methods to the investigation of particles such as viruses permits unprecedented insights into the behavior of supramolecular assemblies. Here we address the scope of viral exploration at the level of individual particles. In an era of increased awareness towards virology, single-particle approaches are expected to facilitate the in-depth understanding, and hence combating, of viral diseases.

Laser-induced inner-shell excitations through direct electron re-collision versus indirect collision.

The dynamics and the decay processes of inner-shell excited atoms are of great interest in physics, chemistry, biology, and technology. The highly excited state decays very quickly through different channels, both radiative and non-radiative. It is therefore a long-standing goal to study such dynamics directly in the time domain. Using few-cycle infrared laser pulses, we investigated the excitation and ionization of inner-shell electrons through laser-induced electron re-collision with the original parent ions and measured the dependence of the emitted x-ray spectra on the intensity and ellipticity of the driving laser. These directly re-colliding electrons can be used as the initiating pump step in pump/probe experiments for studying core-hole dynamics at their natural temporal scale. In our experiment we found that the dependence of the x-ray emission spectrum on the laser intensity and polarization state varies distinctly for the two kinds of atomic systems. Relying on our data and numerical simulations, we explain this behavior by the presence of different excitation mechanisms that are contributing in different ratios to the respective overall x-ray emission yields. Direct re-collision excitation competes with indirect collisions with neighboring atoms by electrons having "drifted away" from the original parent ion.

Carrier-envelope-phase measurement of few-cycle mid-infrared laser pulses using high harmonic generation in ZnO.

High-harmonic generation (HHG) in crystals offers a simple, affordable and easily accessible route to carrier-envelope phase (CEP) measurements, which scales favorably towards longer wavelengths. We present measurements of HHG in ZnO using few-cycle pulses at 3.1µm. Thanks to the broad bandwidth of the driving laser pulses, spectral overlap between adjacent harmonic orders is achieved. The resulting spectral interference pattern provides access to the relative harmonic phase, and hence, the CEP.

Single-Molecule Mechanics in Ligand Concentration Gradient.

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

Single-shot CEP drift measurement at arbitrary repetition rate based on dispersive Fourier transform.

This paper presents a single-shot technique for measuring CEP. The Temporal dispersion based One-shot Ultrafast Carrier envelope phase Analysis method (TOUCAN) is an arbitrary repetition rate single-shot CEP drift measurement technique based on dispersive Fourier transformations and has been experimentally tested at 100 kHz. TOUCAN was validated by a direct comparison of decimated data with an independent traditional CEP drift measurement technique. The impact of a temporal jitter on the CEP drift measurement is investigated and a new mitigation technique is shown to produce high accuracy jitter-free CEP drift extraction.