Evaluation of an automated far ultraviolet-C light technology for decontamination of surfaces and aerosolized viruses in bathrooms.

BACKGROUND: Aerosols generated during toilet flushing are a potential source for transmission of viral and bacterial pathogens in bathrooms. However, manual decontamination of bathrooms after each use is not feasible. METHODS: We tested the efficacy of a wall-mounted far ultraviolet-C (UV-C) light technology that only delivers far UV-C when people are not present for decontamination of surfaces and aerosolized viral particles in an unoccupied hospital bathroom. A quantitative disk carrier test method was used to test efficacy against organisms on steel disk carriers placed in 9 sites in the bathroom with an exposure time of 45 min and 2 h; Clostridioides difficile spores were also exposed for 24 h. Efficacy against aerosolized bacteriophage MS2 was tested with a 45-minute exposure. RESULTS: The far UV-C technology reduced methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Candida auris, and bacteriophage MS2 on steel disk carriers by ≥ 1.2 log10 (range, 1.2 to 4.2 log10) at all test sites after 2 h of exposure. The technology reduced C. difficile spores by < 1 log10 after 2 h exposure, but 4 of 9 test locations had ≥ 2 log10 reductions after 24 h exposure. Aerosolized bacteriophage MS2 was reduced by 4 log10 plaque-forming units in 45 min. CONCLUSIONS: The far UV-C light technology could potentially be useful for automated decontamination of air and surfaces in bathrooms in healthcare and community settings.

Bacteria and RNA virus inactivation with a high-irradiance UV-A source.

Disinfection with LED lamps is a promising ecological and economical substitute for mercury lamps. However, the optimal time/dose relationship needs to be established. Pathogen inactivation by UV-A primarily relies on induced reactive oxygen species (ROS) formation and subsequent oxidative damage. While effective against bacteria and enveloped viruses, non-enveloped viruses are less sensitive. In this study, we explored the disinfection properties of 10 W UV-A LED, emitting in the 365-375 nm range. UV-A at high values of irradiance (~ 0.46 W/cm2) can potentially induce ROS formation and direct photochemical damage of the pathogen nucleic acids, thus improving the disinfection. The UV-A inactivation was evaluated for the bacterium Escherichia coli (E. coli), non-enveloped RNA bacteriophage MS2, and enveloped mammalian RNA virus-Semliki Forest virus (SFV). The 4 log10 reduction doses for E. coli and SFV were 268 and 241 J/cm2, respectively. Furthermore, in irradiated E. coli, ROS production positively correlated with the inactivation rate. In the case of MS2 bacteriophage, the 2.5 log10 inactivation was achieved by 679 J/cm2 within 30 min of irradiation. The results demonstrate significant disinfection efficiency of non-enveloped virus MS2 using high-irradiance UV-A. This suggests a potential strategy for improving the inactivation of UV-A-unsusceptible pathogens, particularly non-enveloped viruses. Additionally, the direct UV-A irradiation of self-replicating viral RNA from SFV led to a significant loss of viral gene expression in cells transfected with the irradiated RNA. Therefore, the virus inactivation mechanism of high-irradiance UV-A LED can be partially determined by the direct damage of viral RNA.

Fungicide Metabolite MS2 Spectral Libraries for Comprehensive Human Biomonitoring.

Fungicides undergo rapid metabolism and are excreted in the urine. There are few methods for screening these ubiquitous compounds, which have a high potential for human exposure. High-resolution mass spectrometry (HRMS) is a suitable technique to assess fungicide exposures; however, there is a lack of spectral libraries for fungicide annotation and in particular for downstream metabolites. We created spectral libraries for 32 fungicides for suspect screening. Fungicide standards were administered to mice, and 24-h urine was analyzed using hydrophilic interaction and reversed-phase chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Suspect metabolite MS2 spectra for library creation were selected based on the ratio of exposed-to-control mouse urine. MS2 libraries were applied to urine collected from female university students (n = 73). Several tetraconazole and tebuconazole metabolites were detected in 3% (2/73) of the samples. The creation of comprehensive suspect screening MS2 libraries is a useful tool to detect fungicide exposure for human biomonitoring.

Comparative Removal of Poliovirus, Rotavirus SA11 and MS2 Coliphage by Point-of-Use Devices used to Treat Drinking Water and Water Disinfectants: A Review.

Test protocols have been developed to test water treatment devices/systems for use for treating drinking water that are used at the individual and home level to ensure the removal of waterborne viruses. Current test procedures call for the use of poliovirus type 1 and/or rotavirus SA11. Recently we suggested that selected coliphages could be used as surrogates for poliovirus for testing of point-of-use (POU) water treatment devices, however, rotavirus was not used in those studies. The purpose of this review was to compare studies of POU devices which were tested with poliovirus type 1, simian rotavirus SA11 and coliphage MS2 to determine if the behavior of rotavirus SA11 was significantly different. In addition, an attempt was made to compare the relative resistance of these viruses by various disinfectants used to treat drinking water. In all cases SA11 was removed to an equal or greater degree than poliovirus. SA11 was found to be less resistant to halogens, although one study found it to be more resistance to chloramines than poliovirus and MS2. Based on this review, use of coliphages for testing POU devices appear justified. Additionally, data on chloramines for these viruses would be useful to determine if rotavirus is more resistant than poliovirus and MS2.

Feasibility of magnetomyography with optically pumped magnetometers in a mobile magnetic shield.

While magnetomyography (MMG) using optically pumped magnetometers (OPMs) is a promising method for non-invasive investigation of the neuromuscular system, it has almost exclusively been performed in magnetically shielded rooms (MSRs) to date. MSRs provide extraordinary conditions for biomagnetic measurements but limit the widespread adoption of measurement methods due to high costs and extensive infrastructure. In this work, we address this issue by exploring the feasibility of mobile OPM-MMG in a setup of commercially available components. From field mapping and simulations, we find that the employed zero-field OPM can operate within a large region of the mobile shield, beyond which residual magnetic fields and perturbations become increasingly intolerable. Moreover, with digital filtering and moderate averaging a signal quality comparable to that in a heavily shielded MSR is attained. These findings facilitate practical and cost-effective implementations of OPM-MMG systems in clinical practice and research.

Vicia faba L. Pod Valves: A By-Product with High Potential as an Adjuvant in the Treatment of Parkinson's Disease.

Vicia faba L. is a leguminous plant with seeds rich in nutritional compounds, such as polyphenols and L-dopa, a dopamine precursor and first-line treatment for Parkinson's symptoms. Recently, its by-products have been revalued as a sustainable source of bioactive compounds. In this study, aqueous extracts of Lucan broad bean pod valves (BPs) were characterized to evaluate their potential use as adjuvants in severe Parkinson's disease. L-dopa content, quantified by LC-UV, was much higher in BPs than in seeds (28.65 mg/g dw compared to 0.76 mg/g dw). In addition, vicine and convicine, the metabolites responsible for favism, were not detected in pods. LC-ESI/LTQ-Orbitrap/MS2 allowed the identification of the major polyphenolic compounds, including quercetin and catechin equivalents, that could ensure neuroprotection in Parkinson's disease. ESI(±)-FT-ICR MS was used to build 2D van Krevelen diagrams; polyphenolic compounds and carbohydrates were the most representative classes. The neuroprotective activity of the extracts after MPP+-induced neurotoxicity in SH-SY5Y cells was also investigated. BP extracts were more effective than synthetic L-dopa, even at concentrations up to 100 µg/mL, due to the occurrence of antioxidants able to prevent oxidative stress. The stability and antioxidant component of the extracts were then emphasized by using naturally acidic solutions of Punica granatum L., Ribes rubrum L., and gooseberry (Phyllanthus emblica L.) as extraction solvents.

Determining KRAS4B-Targeting Compound Specificity by Top-Down Mass Spectrometry.

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2.

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.

Multi-labeling Live Imaging to Quantify Gene Expression Dynamics During Drosophila Embryonic Development.

Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.

Deciphering the Thermal Stability of Bacteriophage MS2-Derived Virus-like Particle and Its Engineered Variant.

RNA bacteriophage MS2-derived virus-like particles (VLPs) have been widely used in biomedical research as model systems to study virus assembly, structure-function relationships, vaccine development, and drug delivery. Considering the diverse utility of these VLPs, a systemic engineering approach has been utilized to generate smaller particles with optimal serum stability and tissue penetrance. Additionally, it is crucial to demonstrate the overall stability of these mini MS2 VLPs, ensuring cargo protection until they reach their target cell/organ. However, no detailed analysis of the thermal stability and heat-induced disassembly of MS2 VLPs has yet been attempted. In this work, we investigated the thermal stability of both wild-type (WT) MS2 VLP and its "mini" variant containing S37P mutation (mini MS2 VLP). The mini MS2 VLP exhibits a higher capsid melting temperature (Tm) when compared to its WT MS2 VLP counterpart, possibly attributed to its smaller interdimer angle. Our study presents that the thermal unfolding of MS2 VLPs follows a sequential process involving particle destabilization, nucleic acid exposure/melting, and disassembly of VLP. This observation underscores the disruption of cooperative intersubunit interactions and protein-nucleic acid interactions, shedding light on the mechanism of heat-induced VLP disassembly.

Bioinformatics Pipeline for Processing Single-Cell Data.

Single-cell proteomics can offer valuable insights into dynamic cellular interactions, but identifying proteins at this level is challenging due to their low abundance. In this chapter, we present a state-of-the-art bioinformatics pipeline for single-cell proteomics that combines the search engine Sage (via SearchGUI), identification rescoring with MS2Rescore, quantification through FlashLFQ, and differential expression analysis using MSqRob2. MS2Rescore leverages LC-MS/MS behavior predictors, such as MS2PIP and DeepLC, to recalibrate scores with Percolator or mokapot. Combining these tools into a unified pipeline, this approach improves the detection of low-abundance peptides, resulting in increased identifications while maintaining stringent FDR thresholds.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Phage detection by a bacterial NLR-related protein is mediated by DnaJ.

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link infection detection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we determine the mechanism of RNA phage detection by the bacterial NLR-related protein bNACHT25 in E. coli. bNACHT25 was specifically activated by Emesvirus ssRNA phages and analysis of MS2 phage suppressor mutants that evaded detection revealed Coat Protein (CP) was sufficient for activation. bNACHT25 and CP did not physically interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP. Our data suggest that bNACHT25 detects a wide range of phages by guarding a host cell process rather than binding a specific phage-derived molecule.

Predicting Odor Sensory Attributes of Unidentified Chemicals in Water Using Fragmentation Mass Spectra with Machine Learning Models.

Knowing odor sensory attributes of odorants lies at the core of odor tracking when addressing waterborne odor issues. However, experimental determination covering tens of thousands of odorants in authentic water is not pragmatic due to the complexity of odorant identification and odor evaluation. In this study, we propose the first machine learning (ML) model to predict odor perception/threshold aiming at odorants in water, which can use either molecular structure or MS2 spectra as input features. We demonstrate that model performance using MS2 spectra is nearly as good as that using unequivocal structures, both with outstanding accuracy. We particularly show the model's robustness in predicting odor sensory attributes of unidentified chemicals by using the experimentally obtained MS2 spectra from nontarget analysis on authentic water samples. Interpreting the developed models, we identify the intricate interaction of functional groups as the predominant influence factor on odor sensory attributes. We also highlight the important roles of carbon chain length, molecular weight, etc., in the inherent olfactory mechanisms. These findings streamline the odor sensory attribute prediction and are crucial advancements toward credible tracking and efficient control of off-odors in water.

Studying Plant Specialized Metabolites Using Computational Metabolomics Strategies.

Plant specialized metabolites have diversified vastly over the course of plant evolution, and they are considered key players in complex interactions between plants and their environment. The chemical diversity of these metabolites has been widely explored and utilized in agriculture and crop enhancement, the food industry, and drug development, among other areas. However, the immensity of the plant metabolome can make its exploration challenging. Here we describe a protocol for exploring plant specialized metabolites that combines high-resolution mass spectrometry and computational metabolomics strategies, including molecular networking, identification of structural motifs, as well as prediction of chemical structures and metabolite classes.

Metabolic and elemental profiling as potential discriminating features among the black mahlab seeds (Monechma ciliatum) grown in three different regions.

INTRODUCTION: Black mahlab (Monechma ciliatum) seed is a rich source of metabolites and minerals and is usually believed to have a similar composition between different areas of cultivation. Until now, no studies have assessed changes in black mahlab seeds (BMSs) to determine those constituents that help to discriminate them according to geographical origin. OBJECTIVES: The present study attempted to compare the metabolomics and elemental profiles of BMSs of different geographical origins and identified the potential markers using ultrahigh-performance liquid chromatography quadrupole Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS2), and inductively coupled plasma mass spectrometry (ICP-MS) techniques and established the chemometric model to identify the potential markers and discriminate them according to cultivation sites. MATERIAL AND METHODS: In this work, data from metabolites analysis by UHPLC-Q-Orbitrap-MS2 and multi-elemental data obtained from ICP-MS were combined with chemometrics for tracing the geographical origin of BMSs. Principal component analysis (PCA) was used to evaluate the overall grouping of samples. In contrast, partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed for authentication. RESULTS: PLS-DA and OPLS-DA models were fully validated (R2Y and Q2 values > 0.5). Variable importance of various projections was applied to obtain valuable data about differential elements (seven markers were identified) and metabolites (23 markers were identified) with high discrimination potential. The outcomes presented in this study serve as an appropriate framework for developing novel discrimination approaches in food origin screening.

Polyphenol-rich fermented hempseed ethanol extracts improve obesity, oxidative stress, and neural health in high-glucose diet-induced Caenorhabditis elegans.

Whole hempseed (WHS), fermented whole hempseed (FWHS), dehulled hempseed (DHS), and fermented dehulled hempseed (FDHS) ethanol extracts were tested for their toxicity and physiological benefits in relation to their phenolic profiles. The safety of all samples was confirmed by the absence of toxic effects on HepG2 cells. FWHS exhibited the highest capacity to inhibit lipase activity (70.80%) and acetylcholinesterase (AChE) (78.94%) in vitro. Similarly, in HepG2 cells, FWHS revealed the greatest ability to reduce the accumulation of reactive oxygen species (ROS). Fermented hempseed demonstrated superior antioxidant, neuroprotective and anti-fat potential, counteracting ageing in high glucose diet-induced C. elegans than unfermented. HPLC and UHPLC-Q-TOF-MS/MS2 phenolic identification revealed the presence of diverse flavonoids, phenolic acids, lignanamides, and phenylamides in hempseed extracts. Among these polyphenols, quercetin, gallic acid, and kaempferol exhibited excellent antioxidant potential, whereas N-trans-feruloyl tyramine displayed the highest anti-lipase potential. This study suggests that polyphenol-rich hempseed exhibits potent antioxidant, and anti-obesity effects, and could improve neural health.

BT100, a three-in-one, multipurpose disinfecting, deodorizing, and air-cleaning solution with an effective, gradual, and continuous gaseous chlorine dioxide-releasing substance.

Aerosols carrying viruses that are released from the oral cavity of infected individuals are the primary, if not the only, means of transmission during viral respiratory disease epidemics. This makes crowded rooms and tiny, enclosed public areas like bathrooms prime environments for the transmission of diseases. Volatile organic compounds (VOCs) and formaldehyde are two contaminants that pose serious threats to human health and well-being in indoor environments. The varied disinfectant properties of chlorine dioxide (ClO2) make it a key player in treating a range of air quality issues. To balance effectiveness and safety, however, the careful application of chlorine dioxide is essential to achieving the best results in air quality while preserving human health and well-being. This study explores the many functions of chlorine dioxide, including the prevention of the spread of viruses, the elimination of harmful gases like ammonia and hydrogen sulfide, and its effects on formaldehyde and total volatile organic compounds (TVOCs) in indoor environments using BT100. The results indicate a reduction of 98.5%, 81.01%, 62.22%, 46.5%, and 63.84% in minimizing aerosolized viruses, ammonia, and hydrogen sulfide gas in addition to formaldehyde and total volatile organic compounds.

Purification of Single-Stranded RNA Bacteriophages and Host Receptors for Structural Determination Using Cryo-Electron Microscopy.

Single-stranded RNA bacteriophages (ssRNA phages) are small viruses with a compact genome (~3-4 kb) that infect gram-negative bacteria via retractile pili. These phages have been applied in various fields since their discovery approximately 60 years ago. To understand their biology, it is crucial to analyze the structure of mature virions. Cryo-electron microscopy (cryo-EM) has been employed to determine the structures of two ssRNA phages, MS2 and Qß. This chapter presents a method for purifying these two phages and their receptor, the F-pilus, to allow examination using cryo-EM.

Dissecting quantitative trait nucleotides by saturation genome editing.

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.