INTRODUCTION: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. METHODS: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. RESULTS: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. DISCUSSION: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

• Keywords
• Aeromonas hydrophila (A. hydrophila), Cyprinus carpio, bacteria, cytokines, engulfment, inflammation, red blood cell (RBC), teleost fish,
• MeSH
• Aeromonas hydrophila * immunology   MeSH
• Cytokines * metabolism   immunology   MeSH
• Erythrocytes * immunology   metabolism   MeSH
• Phagocytosis immunology   MeSH
• Gram-Negative Bacterial Infections * immunology   MeSH
• Carps * immunology   microbiology   MeSH
• Fish Diseases * immunology   microbiology   MeSH
• Pathogen-Associated Molecular Pattern Molecules immunology   MeSH
• Immunity, Innate MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokines * MeSH
• Pathogen-Associated Molecular Pattern Molecules MeSH

Objective.This work presents a method for enhanced detection, imaging, and measurement of the thermal neutron flux.Approach. Measurements were performed in a water tank, while the detector is positioned out-of-field of a 20 MeV ultra-high pulse dose rate electron beam. A semiconductor pixel detector Timepix3 with a silicon sensor partially covered by a6LiF neutron converter was used to measure the flux, spatial, and time characteristics of the neutron field. To provide absolute measurements of thermal neutron flux, the detection efficiency calibration of the detectors was performed in a reference thermal neutron field. Neutron signals are recognized and discriminated against other particles such as gamma rays and x-rays. This is achieved by the resolving power of the pixel detector using machine learning algorithms and high-resolution pattern recognition analysis of the high-energy tracks created by thermal neutron interactions in the converter.Main results. The resulting thermal neutrons equivalent dose was obtained using conversion factor (2.13(10) pSv·cm2) from thermal neutron fluence to thermal neutron equivalent dose obtained by Monte Carlo simulations. The calibrated detectors were used to characterize scattered radiation created by electron beams. The results at 12.0 cm depth in the beam axis inside of the water for a delivered dose per pulse of 1.85 Gy (pulse length of 2.4μs) at the reference depth, showed a contribution of flux of 4.07(8) × 103particles·cm-2·s-1and equivalent dose of 1.73(3) nSv per pulse, which is lower by ∼9 orders of magnitude than the delivered dose.Significance. The presented methodology for in-water measurements and identification of characteristic thermal neutrons tracks serves for the selective quantification of equivalent dose made by thermal neutrons in out-of-field particle therapy.

• Keywords
• 6LiF converter, FLASH electron radiotherapy, Timepix3 pixel detector, equivalent dose, out-of-field dose from neutrons, particle type discrimination, thermal neutrons,
• MeSH
• Algorithms * MeSH
• Electrons * MeSH
• Calibration MeSH
• Neutrons MeSH
• Gamma Rays MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA-ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings.

• Keywords
• endoplasmic reticulum, mRNA, proteosynthesis, ribosome stalling, signal peptide,
• Publication type
• Journal Article MeSH

Detection of enantiomers is a challenging problem in drug development as well as environmental and food quality monitoring where traditional optical detection methods suffer from low signals and sensitivity. Application of surface enhanced Raman scattering (SERS) for enantiomeric discrimination is a powerful approach for the analysis of optically active small organic or large biomolecules. In this work, we proposed the coupling of disposable chiral plasmonic shurikens supporting the chiral near-field distribution with SERS active silver nanoclusters for enantio-selective sensing. As a result of the plasmonic coupling, significant difference in SERS response of optically active analytes is observed. The observations are studied by numerical simulations and it is hypothesized that the silver particles are being excited by superchiral fields generated at the surface inducing additional polarizations in the probe molecules. The plasmon coupling phenomena was found to be extremely sensitive to slight variations in shuriken geometry, silver nanostructured layer parameters, and SERS excitation wavelength(s). Designed structures were able to discriminate cysteine enantiomers at concentrations in the nanomolar range and probe biomolecular chirality, using a common Raman spectrometer within several minutes. The combination of disposable plasmonic substrates with specific near-field polarization can make the SERS enantiomer discrimination a commonly available technique using standard Raman spectrometers.

• Publication type
• Journal Article MeSH

Reliable tools for artefact rejection and signal classification are a must for cosmic ray detection experiments based on CMOS technology. In this paper, we analyse the fitness of several feature-based statistical classifiers for the classification of particle candidate hits in four categories: spots, tracks, worms and artefacts. We use Zernike moments of the image function as feature carriers and propose a preprocessing and denoising scheme to make the feature extraction more efficient. As opposed to convolution neural network classifiers, the feature-based classifiers allow for establishing a connection between features and geometrical properties of candidate hits. Apart from basic classifiers we also consider their ensemble extensions and find these extensions generally better performing than basic versions, with an average recognition accuracy of 88%.

• Keywords
• CMOS sensors, Zernike moments, computer vision, feature-based classification, machine learning,
• MeSH
• Artifacts * MeSH
• Neural Networks, Computer * MeSH
• Publication type
• Journal Article MeSH

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.

• Keywords
• Ffh, FtsY, LECA, evolution, mitochondrion, protein targeting, protists, signal recognition particle,
• MeSH
• Bacterial Proteins genetics   MeSH
• Biological Evolution * MeSH
• Genome, Mitochondrial * MeSH
• Naegleria genetics   MeSH
• Escherichia coli Proteins genetics   MeSH
• Receptors, Cytoplasmic and Nuclear genetics   MeSH
• Sequence Homology, Nucleic Acid MeSH
• Signal Recognition Particle genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Ffh protein, E coli MeSH Browser
• FtsY protein, Bacteria MeSH Browser
• Escherichia coli Proteins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Signal Recognition Particle MeSH

Gamification is known to enhance users' participation in education and research projects that follow the citizen science paradigm. The Cosmic Ray Extremely Distributed Observatory (CREDO) experiment is designed for the large-scale study of various radiation forms that continuously reach the Earth from space, collectively known as cosmic rays. The CREDO Detector app relies on a network of involved users and is now working worldwide across phones and other CMOS sensor-equipped devices. To broaden the user base and activate current users, CREDO extensively uses the gamification solutions like the periodical Particle Hunters Competition. However, the adverse effect of gamification is that the number of artefacts, i.e., signals unrelated to cosmic ray detection or openly related to cheating, substantially increases. To tag the artefacts appearing in the CREDO database we propose the method based on machine learning. The approach involves training the Convolutional Neural Network (CNN) to recognise the morphological difference between signals and artefacts. As a result we obtain the CNN-based trigger which is able to mimic the signal vs. artefact assignments of human annotators as closely as possible. To enhance the method, the input image signal is adaptively thresholded and then transformed using Daubechies wavelets. In this exploratory study, we use wavelet transforms to amplify distinctive image features. As a result, we obtain a very good recognition ratio of almost 99% for both signal and artefacts. The proposed solution allows eliminating the manual supervision of the competition process.

• Keywords
• CREDO, citizen science, convolutional neural networks, deep learning, gamification, global sensor network, image classification, image sensors,
• MeSH
• Artifacts MeSH
• Humans MeSH
• Neural Networks, Computer * MeSH
• Image Processing, Computer-Assisted * MeSH
• Machine Learning MeSH
• Wavelet Analysis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

We report that the immunogenicity of colloidal gold nanoparticles coated with polyvinylpyrrolidone (PVP-AuNPs) in a model organism, the sea urchin Paracentrotus lividus, can function as a proxy for humans for in vitro immunological studies. To profile the immune recognition and interaction from exposure to PVP-AuNPs (1 and 10 μg mL-1), we applied an extensive nano-scale approach, including particle physicochemical characterisation involving immunology, cellular biology, and metabolomics. The interaction between PVP-AuNPs and soluble proteins of the sea urchin physiological coelomic fluid (blood equivalent) results in the formation of a protein "corona" surrounding the NPs from three major proteins that influence the hydrodynamic size and colloidal stability of the particle. At the lower concentration of PVP-AuNPs, the P. lividus phagocytes show a broad metabolic plasticity based on the biosynthesis of metabolites mediating inflammation and phagocytosis. At the higher concentration of PVP-AuNPs, phagocytes activate an immunological response involving Toll-like receptor 4 (TLR4) signalling pathway at 24 hours of exposure. These results emphasise that exposure to PVP-AuNPs drives inflammatory signalling by the phagocytes and the resolution at both the low and high concentrations of the PVP-AuNPs and provides more details regarding the immunogenicity of these NPs.

• Keywords
• Immune metabolic rewiring, Immunoreactivity, Innate defence response, Nano-recognition, Sea urchin immune cells,
• MeSH
• Phagocytes MeSH
• Metal Nanoparticles * toxicity   MeSH
• Humans MeSH
• Paracentrotus * MeSH
• Povidone MeSH
• Gold MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Povidone MeSH
• Gold MeSH

The pyrimidine-pyrimidone (6-4) photoproduct (64-PP) is an important photoinduced DNA lesion constituting a mutational signature for melanoma. The structural impact of 64-PP on DNA complexed with histones affects the lesion mutagenicity and repair but remains poorly understood. Here we investigate the conformational dynamics of DNA-containing 64-PP within the nucleosome core particle by atomic-resolution molecular dynamics simulations and multiscale data analysis. We demonstrate that the histone core exerts important mechanical restraints that largely decrease global DNA structural fluctuations. However, the local DNA flexibility at the damaged site is enhanced due to imperfect structural adaptation to restraints imposed by the histone core. If 64-PP faces the histone core and is therefore not directly accessible by the repair protein, the complementary strand facing the solvent is deformed and exhibits higher flexibility than the corresponding strand in a naked, undamaged DNA. This may serve as an initial recognition signal for repair. Our simulations also pinpoint the structural role of proximal residues from the truncated histone tails.

• MeSH
• DNA chemistry   MeSH
• Histones chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Pyrimidine Dimers chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Ultraviolet Rays MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Histones MeSH
• pyrimidine-pyrimidone dimer MeSH Browser
• Pyrimidine Dimers MeSH

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

• MeSH
• Cell Nucleus ultrastructure   MeSH
• Cytological Techniques MeSH
• Fluorescent Dyes MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Molecular Imaging methods   MeSH
• Cell Line, Tumor MeSH
• Nanodiamonds * MeSH
• Spectrum Analysis, Raman MeSH
• Dental Pulp cytology   diagnostic imaging   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Nanodiamonds * MeSH