Influence of analytical procedures on miRNA expression analyses in saliva samples.

Aim of this study was to demonstrate the influence of different analytical procedures and techniques on the resulting miRNA expression profile in healthy control subjects and tumor patients using the oral squamous cell carcinoma (OSCC) model and to demonstrate the technical and biological reproducibility. Body fluids such as saliva are suitable for non-invasive miRNA analysis because ubiquitously circulating miRNA can be found in them. It was technically possible to distinguish between healthy and diseased samples based on the miRNA expression profile found. Regardless of the methodology used, good technical reproducibility of the results seems to be achievable. On the other hand, biological reproducibility was inadequate, which is why prompt sampling and sequencing is recommended. The data indicate that malignant lesions can be detected using miRNA signatures extracted from saliva. This could stimulate further research to establish standardized protocols and kits for sample collection, miRNA extraction, sequencing and interpretation of results.

A Conjoined Rectilinear Collision Cell and Pulsed Extraction Ion Trap with Auxiliary DC Electrodes.

Ion traps are routinely directly coupled to mass analyzers, where they serve to suitably cool and shape an ion population prior to pulsed extraction into the analyzer proper. Such devices benefit from high duty cycle and transmission but suffer slow ion processing times caused by a compromise in the buffer gas pressure range that suitably dampens the ion kinetic energy without causing excessive scatter during extraction or within the analyzer. A rectilinear RF quadrupole ion trap has been characterized, conjoining a pressurized collision region with a pumped extraction region, and an unbroken RF interface for seamless ion transfer between them. Auxiliary electrodes mounted between the RF electrodes provide DC voltage gradients that serve to both guide ions through the device and position them at the extraction slot. The influence of the auxiliary DC upon the trapping RF field was measured, and suitable parameters were defined. A mode of operation was developed that allowed parallel processing of ions in both regions, enabling a repetition rate of 200 Hz when the device was coupled to a high-resolution accurate-mass analyzer.

Focused laser differential interferometry post-processing methodology for flowfields with circular symmetry.

An analytic methodology is presented to reconstruct the pressure waveform of flowfields with circular symmetry from the phase shift detected with Focused Laser Differential Interferometry (FLDI). A weak blast wave generated by an electric spark in ambient air is investigated with the proposed approach. Values of separation distance between the differentiating foci of the FLDI Δx of 76, 120, 175, and 252 µm are employed to probe the flowfield at locations between 3 and 50 mm from the spark source. In a subset of these distances, reference measurements of peak pressure obtained with a surface pressure sensor indicate good agreement with the reconstructed data when small separation distances are used. Further analysis of FLDI reconstructed data is conducted using theoretical correlations for N-waves in terms of the distribution of pressure peak amplitude and compression phase as the wave front propagates. Agreement with theory is verified for all differentiation separation distances except the largest, for which peak pressure comparison shows a 10% loss of measured vs predicted value. A computational FLDI is employed to scrutinize the simplifying hypotheses supporting the waveform reconstruction approach. The direct comparison between experimental and computational FLDI output reveals additional discrepancies for intermediate Δx values but very good agreement for the smallest Δx. The proposed methodology is thus verified to be reasonable, upon appropriate minimization of the FLDI differentiation distance. A parametric analysis using computational FLDI indicates the adequate value of FLDI Δx to be 20% or less of the flowfield characteristic length in terms of density gradient.

Application of the endogenous CRISPR-Cas type I-D system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are widely distributed among bacteria and archaea. In this study, we demonstrate the successful utilization of the type I-D CRISPR-Cas system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Given its extreme growth conditions characterized by a temperature of 75°C and pH 3, an uracil auxotrophic selection system was previously established, providing a basis for our investigations. We developed a novel plasmid specifically designed for genome editing, which incorporates a mini-CRISPR array that can be induced using xylose, resulting in targeted DNA cleavage. Additionally, we integrated a gene encoding the ß-galactosidase of Saccharolobus solfataricus into the plasmid, enabling blue-white screening and facilitating the mutant screening process. Through the introduction of donor DNA containing genomic modifications into the plasmid, we successfully generated deletion mutants and point mutations in the genome of S. acidocaldarius. Exploiting the PAM (protospacer adjacent motif) dependence of type I systems, we experimentally confirmed the functionality of three different PAMs (CCA, GTA, and TCA) through a self-targeting assessment assay and the gene deletion of upsE. Our findings elucidate the application of the endogenous Type I-D CRISPR-Cas system for genetic engineering in S. acidocaldarius, thus expanding its genetic toolbox.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Design principles and applications of synthetic self-replicating RNAs.

With the advent of ever more sophisticated methods for the in vitro synthesis and the in vivo delivery of RNAs, synthetic mRNAs have gained substantial interest both for medical applications, as well as for biotechnology. However, in most biological systems exogeneous mRNAs possess only a limited half-life, especially in fast dividing cells. In contrast, viral RNAs can extend their lifetime by actively replicating inside their host. As such they may serve as scaffolds for the design of synthetic self-replicating RNAs (srRNA), which can be used to increase both the half-life and intracellular concentration of coding RNAs. Synthetic srRNAs may be used to enhance recombinant protein expression or induce the reprogramming of differentiated cells into pluripotent stem cells but also to create cell-free systems for research based on experimental evolution. In this article, we discuss the applications and design principles of srRNAs used for cellular reprogramming, mRNA-based vaccines and tools for synthetic biology. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Bartonella taylorii: A Model Organism for Studying Bartonella Infection in vitro and in vivo.

Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species have hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain Bartonella taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro was correlating to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.

Overrelaxation in a diffusive integer lattice gas.

One of the most striking drawbacks of standard lattice gas methods over lattice Boltzmann methods is a much more limited range of transport parameters that can be achieved. It is common for lattice Boltzmann methods to use over-relaxation to achieve arbitrarily small transport parameters in the hydrodynamic equations. Here, we show that it is possible to implement over-relaxation for integer lattice gases. For simplicity, we focus here on lattice gases for the diffusion equation. We demonstrate that adding a flipping operation to lattice gases results in a multirelaxation time lattice Boltzmann scheme with over-relaxation in the Boltzmann limit.

Integer lattice gas with a sampling collision operator for the fluctuating diffusion equation.

We developed an integer lattice gas method for the fluctuating diffusion equation. Such a method is unconditionally stable and able to recover the Poisson distribution for the microscopic densities. A key advance for integer lattice gases introduced in this paper is a sampling collision operator that replaces particle collisions with sampling from an equilibrium distribution. This can increase the efficiency of our integer lattice gas by several orders of magnitude.

In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity.

The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.

The extent of ionization in simulations of radio-loud AGNs impacting kpc gas discs.

We use the results of relativistic hydrodynamic simulations of jet-interstellar medium (ISM) interactions in a galaxy with a radio-loud AGN to quantify the extent of ionization in the central few kpcs of the gaseous galactic disc. We perform post-process radiative transfer of AGN radiation through the simulated gaseous jet-perturbed disc to estimate the extent of photo-ionization by the AGN with an incident luminosity of 1045 erg s-1. We also map the gas that is collisionally ionized due to shocks driven by the jet. The analysis was carried out for simulations with similar jet power (1045 erg s-1) but different jet orientations with respect to the gas disc. We find that the shocks from the jets can ionize a significant fraction (up to 33 [Formula: see text]) of dense gas ([Formula: see text]) in the disc, and that the jets clear out the central regions of gas for AGN radiation to penetrate to larger distances in the disc. Jets inclined towards the disc plane couple more strongly with the ISM and ionize a larger fraction of gas in the disc as compared to the vertical jet. However, similar to previous studies, we find that the AGN radiation is quickly absorbed by the outer layers of dense clouds in the disc, and is not able to substantially ionize the disc on a global scale. Thus, compared to jet-ISM interactions, we expect that photo-ionization by the AGN radiation only weakly affects the star-formation activity in the central regions of the galactic disc (≲ 1 kpc), although the jet-induced shocks can spread farther out.

Shaping the equation of state to improve numerical accuracy and stability of the pseudopotential lattice Boltzmann method.

It has recently been shown that altering the shape of the metastable and unstable branches of an equation of state (EOS) can substantially improve the numerical accuracy of liquid and vapor densities in the pseudopotential lattice Boltzmann method [Peng et al., Phys. Rev. E 101, 063309 (2020)2470-004510.1103/PhysRevE.101.063309]. We found that this approach reduces stability of the method in nonequilibrium conditions and is unstable for bubbles at low reduced temperatures. Here we present an improved method for altering the shape of the metastable and unstable branches of the EOS which remains stable for both equilibrium and nonequilibrium situations and has no issues with bubbles. We also performed a detailed study of the stability of the methods for a droplet impact on a liquid film for reduced temperatures down to 0.35 with Reynolds number of 300. Our approach remained stable for a density ratio of up to 3.38×10^{4}.

Molecular dynamics lattice gas equilibrium distribution function for Lennard-Jones particles.

The molecular dynamics lattice gas (MDLG) method maps a molecular dynamics (MD) simulation onto a lattice gas using a coarse-graining procedure. This is a novel fundamental approach to derive the lattice Boltzmann method (LBM) by taking a Boltzmann average over the MDLG. A key property of the LBM is the equilibrium distribution function, which was originally derived by assuming that the particle displacements in the MD simulation are Boltzmann distributed. However, we recently discovered that a single Gaussian distribution function is not sufficient to describe the particle displacements in a broad transition regime between free particles and particles undergoing many collisions in one time step. In a recent publication, we proposed a Poisson weighted sum of Gaussians which shows better agreement with the MD data. We derive a lattice Boltzmann equilibrium distribution function from the Poisson weighted sum of Gaussians model and compare it to a measured equilibrium distribution function from MD data and to an analytical approximation of the equilibrium distribution function from a single Gaussian probability distribution function. This article is part of the theme issue 'Progress in mesoscale methods for fluid dynamics simulation'.

Nonuniqueness of fluctuating momentum in coarse-grained systems.

Coarse-grained descriptions of microscopic systems often require a mesoscopic definition of momentum. The question arises as to the uniqueness of such a momentum definition at a particular coarse-graining scale. We show here that particularly the fluctuating properties of common definitions of momentum in coarse-grained methods like lattice gas and lattice Boltzmann do not agree with a fundamental definition of momentum. In the case of lattice gases, the definition of momentum will even disagree in the limit of large wavelength. For short times we derive analytical representations for the distribution of different momentum measures and thereby give a full account of these differences.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

Non-Gaussian distribution of displacements for Lennard-Jones particles in equilibrium.

Most mesoscale simulation methods assume Gaussian distributions of velocity-like quantities. These quantities are not true velocities, however, but rather time-averaged velocities or displacements of particles. We show that there is a large range of coarse-graining scales where the assumption of a Gaussian distribution of these displacements fails, and a more complex distribution is required to adequately express these distribution functions of displacements.

Force approach for the pseudopotential lattice Boltzmann method.

One attractive feature of the original pseudopotential method consists on its simplicity of adding a force dependent on a nearest-neighbor potential function. In order to improve the method, regarding thermodynamic consistency and control of surface tension, different approaches were developed in the literature, such as multirange interactions potential and modified forcing schemes. In this work, a strategy to combine these enhancements with an appropriate interaction force field using only nearest-neighbor interactions is devised, starting from the desired pressure tensor. The final step of our procedure is implementing this external force by using the classical Guo forcing scheme. Numerical tests regarding static and dynamic flow conditions were performed. Static tests showed that current procedure is suitable to control the surface tension and phase densities. Based on thermodynamic principles, it is devised a solution for phase densities in a droplet, which states explicitly dependence on the surface tension and interface curvature. A comparison with numerical results suggest a physical inconsistency in the pseudopotential method. This fact is not commonly discussed in the literature, since most of studies are limited to the Maxwell equal area rule. However, this inconsistency is shown to be dependent on the equation of state (EOS), and its effects can be mitigated by an appropriate choice of Carnahan-Starling EOS parameters. Also, a droplet oscillation test was performed, and the most divergent solution under certain flow conditions deviated 7.5% from the expected analytical result. At the end, a droplet impact test against a solid wall was performed to verify the method stability, and it was possible to reach stable simulation results with density ratio of almost 2400 and Reynolds number of Re=373. The observed results corroborate that the proposed method is able to replicate the desired macroscopic multiphase behavior.

Large Fluctuations in Nonideal Coarse-Grained Systems.

Using the recently introduced molecular dynamics lattice gas approach, we test fluctuations of coarse-grained quantities. We show that as soon as the system can no longer be considered an ideal gas fluctuations fail to diminish upon coarse graining as is usually expected. These results suggest that current approaches to simulating fluctuating hydrodynamics may have to be augmented to achieve quantitative results for systems with a nonideal equation of state. The molecular dynamics lattice gas method gives a guidance to the exact nature of the fluctuation in such systems.

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression.

The thermophilic archaeon Sulfolobus acidocaldarius can use different carbon sources for growth, including the pentoses D-xylose and L-arabinose. In this study, we identified the activator XylR (saci_2116) responsible for the transcriptional regulation of the pentose transporter and pentose metabolizing genes in S. acidocaldarius. A xylR deletion mutant showed growth retardation on D-xylose/L-arabinose containing media and the lack of transcription of the respective ABC transporter. In contrast to so far used promoters for expression in S. acidocaldarius, the xylR responsive promoters have a very low background activity. Finally, two XylR dependent promoters next to the long-established maltose inducible promotor were used to construct a high-throughput expression vector system for S. acidocaldarius to efficiently clone and express proteins in S. acidocaldarius.

How Monitor Characteristics Affect Human Perception in Visual Computer Experiments: CRT vs. LCD Monitors in Millisecond Precise Timing Research.

Liquid crystal display (LCD) monitors are nowadays standard in computerized visual presentation. However, when millisecond precise presentation is concerned, they have often yielded imprecise and unreliable presentation times, with substantial variation across specific models, making it difficult to know whether they can be used for precise vision experiments or not. The present paper intends to act as hands-on guide to set up an experiment requiring millisecond precise visual presentation with LCD monitors. It summarizes important characteristics relating to precise visual stimulus presentation, enabling researchers to transfer parameters reported for cathode ray tube (CRT) monitors to LCD monitors. More importantly, we provide empirical evidence from a preregistered study showing the suitability of LCD monitors for millisecond precise timing research. Using sequential testing, we conducted a masked number priming experiment using CRT and LCD monitors. Both monitor types yielded comparable results as indicated by Bayes factor favoring the null hypothesis of no difference between display types. More specifically, we found masked number priming under conditions of zero awareness with both types of monitor. Thus, the present study highlights the importance of hardware settings for empirical psychological research; inadequate settings might lead to more "noise" in results thereby concealing potentially existing effects.