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Dissemination of food-borne L. monocytogenes in the host relies on internalin-mediated invasion, but the underlying invasion strategies remain elusive. Here we use live-cell microscopy to follow single cell interactions between individual human cells and L. monocytogenes and elucidate mechanisms associated with internalin B (InlB)-mediated invasion. We demonstrate that whilst a replicative invasion of nonphagocytic cells is a rare event even at high multiplicities of invasion, L. monocytogenes overcomes this by utilising a strategy relaying on PrfA-mediated ActA-based aggregation. We show that L. monocytogenes forms aggregates in extracellular host cell environment, which promote approximately 5-fold more host cell adhesions than the non-aggregating actA-ΔC mutant (which lacks the C-terminus coding region), with the adhering bacteria inducing 3-fold more intracellular invasions. Aggregation is associated with robust MET tyrosine kinase receptor clustering in the host cells, a hallmark of InlB-mediated invasion, something not observed with the actA-ΔC mutant. Finally, we show via RNA-seq analyses that aggregation involves a global adaptive response to host cell environment (including iron depletion), resulting in metabolic changes in L. monocytogenes and upregulation of the PrfA virulence regulon. Overall, our analyses provide new mechanistic insights into internalin-mediated host-pathogen interactions of L. monocytogenes.
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Aderência Bacteriana , Proteínas de Bactérias , Listeria monocytogenes , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , Listeria monocytogenes/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Interações Hospedeiro-Patógeno , Listeriose/microbiologia , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/genética , Regulação Bacteriana da Expressão Gênica , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Microbiomes are ecosystems, and their stability can impact the health of their hosts. Theory predicts that predators influence ecosystem stability. Phages are key predators of bacteria in microbiomes, but phages are unusual predators because many have lysogenic life cycles. It has been hypothesized that lysogeny can destabilize microbiomes, but lysogeny has no direct analog in classical ecological theory, and no formal theory exists. We studied the stability of computationally simulated microbiomes with different numbers of temperate (lysogenic) and virulent (obligate lytic) phage species. Bacterial populations were more likely to fluctuate over time when there were more temperate phages species. After disturbances, bacterial populations returned to their pre-disturbance densities more slowly when there were more temperate phage species, but cycles engendered by disturbances dampened more slowly when there were more virulent phage species. Our work offers the first formal theory linking lysogeny to microbiome stability.
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Bacteriófagos , Lisogenia , Microbiota , Bacteriófagos/fisiologia , Simulação por Computador , Bactérias/virologia , Modelos BiológicosRESUMO
The liquid structure of three common ionic liquids (ILs) was investigated by neutron scattering for the first time. The ILs were based on the bis(trifluoromethanesulfonyl)imide anion, abbreviated in the literature as [NTf2]- or [TFSI]-, and on the following cations: 1-ethyl-3-methylimidazolium, [C2mim]+; 1-decyl-3-methylimidazolium, [C10mim]+; and trihexyl(tetradecyl)phosphonium, [P666,14]+. Comparative analysis of the three ILs confirmed increased size of nonpolar nanodomains with increasing bulk of alkyl chains. It also sheds light on the cation-anion interactions, providing experimental insight into strength, directionality, and angle of hydrogen bonds between protons on the imidazolium ring, as well as H-C-P protons in [P666,14]+, to oxygen and nitrogen atoms in the [NTf2]-. The new Dissolve data analysis package enabled, for the first time, the analysis of neutron scattering data of ILs with long alkyl chains, in particular, of [P666,14][NTf2]. Results generated with Dissolve were validated by comparing outputs from three different models, starting from three different sets of cation charges, for each of the three ILs, which gave convergent outcomes. Finally, a modified method for the synthesis of perdeuterated [P666,14][NTf2] has been reported, with the aim of reporting a complete set of synthetic and data processing approaches, laying robust foundations that enable the study of the phosphonium ILs family by neutron scattering.
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We report the development of a versatile Ru-porphyrin catalyst system which performs the aerobic epoxidation of aromatic and aliphatic (internal) alkenes under mild conditions, with product yields of up to 95% and turnover numbers (TON) up to 300. Water is shown to play a crucial role in the reaction, significantly increasing catalyst efficiency and substrate scope. Detailed mechanistic investigations employing both computational studies and a range of experimental techniques revealed that water activates the RuVI di-oxo complex for alkene epoxidation via hydrogen bonding, stabilises the RuIV mono-oxo intermediate, and is involved in the regeneration of the RuVI di-oxo complex leading to oxygen atom exchange. Distinct kinetics are obtained in the presence of water, and side reactions involved in catalyst deactivation have been identified.
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Wacker oxidations are ubiquitous in the direct synthesis of carbonyl compounds from alkenes. While the reaction mechanism has been widely studied under aerobic conditions, much less is known about such processes promoted with peroxides. Here, we report an exhaustive mechanistic investigation of the Wacker oxidation of styrene using hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (TBHP) as oxidants by combining density functional theory and microkinetic modeling. Our results with H2O2 uncover a previously unreported reaction pathway that involves an intermolecular proton transfer assisted by the counterion [OTf]- present in the reaction media. Furthermore, we show that when TBHP is used as an oxidant instead of H2O2, the reaction mechanism switches to an intramolecular protonation sourced by the HOtBu moiety generated in situ. Importantly, these two mechanisms are predicted to outcompete the 1,2-hydride shift pathway previously proposed in the literature and account for the level of D incorporation in the product observed in labeling experiments with α-d-styrene and D2O2. We envision that these insights will pave the way for the rational design of more efficient catalysts for the industrial production of chemical feedstocks and fine chemicals.
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Transcription of almost all mammalian genes occurs in stochastic bursts, however the fundamental control mechanisms that allow appropriate single-cell responses remain unresolved. Here we utilise single cell genomics data and stochastic models of transcription to perform global analysis of the toll-like receptor (TLR)-induced gene expression variability. Based on analysis of more than 2000 TLR-response genes across multiple experimental conditions we demonstrate that the single-cell, gene-by-gene expression variability can be empirically described by a linear function of the population mean. We show that response heterogeneity of individual genes can be characterised by the slope of the mean-variance line, which captures how cells respond to stimulus and provides insight into evolutionary differences between species. We further demonstrate that linear relationships theoretically determine the underlying transcriptional bursting kinetics, revealing different regulatory modes of TLR response heterogeneity. Stochastic modelling of temporal scRNA-seq count distributions demonstrates that increased response variability is associated with larger and more frequent transcriptional bursts, which emerge via increased complexity of transcriptional regulatory networks between genes and different species. Overall, we provide a methodology relying on inference of empirical mean-variance relationships from single cell data and new insights into control of innate immune response variability.
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It is well-known that mood and pain interact with each other, however individual-level variability in this relationship has been less well quantified than overall associations between low mood and pain. Here, we leverage the possibilities presented by mobile health data, in particular the "Cloudy with a Chance of Pain" study, which collected longitudinal data from the residents of the UK with chronic pain conditions. Participants used an App to record self-reported measures of factors including mood, pain and sleep quality. The richness of these data allows us to perform model-based clustering of the data as a mixture of Markov processes. Through this analysis we discover four endotypes with distinct patterns of co-evolution of mood and pain over time. The differences between endotypes are sufficiently large to play a role in clinical hypothesis generation for personalised treatments of comorbid pain and low mood.
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Epoxidation of alkenes is a valuable transformation in the synthesis of fine chemicals. Described herein are the design and development of a continuous flow process for carrying out the epoxidation of alkenes with a homogeneous manganese catalyst at metal loadings as low as 0.05 mol%. In this process, peracetic acid is generated in situ and telescoped directly into the epoxidation reaction, thus reducing the risks associated with its handling and storage, which often limit its use at scale. This flow process lessens the safety hazards associated with both the exothermicity of this epoxidation reaction and the use of the highly reactive peracetic acid. Controlling the speciation of manganese/2-picolinic acid mixtures by varying the ligand:manganese ratio was key to the success of the reaction. This continuous flow process offers an inexpensive, sustainable, and scalable route to epoxides.
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BACKGROUND: Campylobacter is one of the leading causes of human bacterial gastroenteritis worldwide. Campylobacter infections are most often associated with the consumption of raw milk, undercooked poultry, and contaminated water. OBJECTIVE: The RapidChek®Campylobacter test system (PTM number 052201) was validated for the detection of Campylobacter jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. METHODS: The method uses a proprietary enrichment medium. Following aerobic enrichment, an immunochromatographic test strip is inserted into the tube containing the enrichment, developed for 20 min, and interpreted. Campylobacter-inoculated food samples were tested by the method, as well as the USDA/FSIS cultural reference method; Isolation and Identification of Campylobacter jejuni/coli/lari from Poultry Rinse, Sponge and Raw Product Samples MLG 41.04. The candidate method was also confirmed by an alternative cultural method. The RapidChek method was tested with 50 Campylobacter strains comprised of C. jejuni, C. coli, and C. lari, and 30 non-target strains. RESULTS: A total of 80 low-level spiked samples were tested by both methods in the study. The candidate method yielded 49 presumptive positives: all presumptive results were confirmed culturally. The reference method produced a total of 41 confirmed positive results. No difference between the alternate confirmation method and reference confirmation method was observed. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the candidate method and cultural reference method. The RapidChek method detected all 50 Campylobacter strains and none of the 30 non-target strains, including Campylobacter spp. other than C. jejuni, C. coli, and C. lari. CONCLUSION: The candidate method performed as well as the reference method in the detection of C. jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. HIGHLIGHTS: Aerobic enrichment of selected matrixes for 48 h yielded reliable presumptive results for Campylobacter.
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Campylobacter coli , Campylobacter jejuni , Campylobacter lari , Campylobacter , Animais , Humanos , Aves Domésticas , Galinhas/microbiologia , Perus , Água , Microbiologia de AlimentosRESUMO
Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1ß involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.
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RNA Mensageiro/genética , Receptores Toll-Like/metabolismo , Humanos , Modelos Teóricos , Transdução de SinaisRESUMO
Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.
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Inflamação/imunologia , Ativação de Macrófagos , Macrófagos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Inflamação/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Células RAW 264.7 , Análise de Célula Única , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The Romer Labs RapidChek® Listeria monocytogenes test system (Performance Tested Method 011805) was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (USDA-FSIS/MLG), U.S. Food and Drug Association Bacteriological Analytical Manual (FDA/BAM), and AOAC Official Methods of Analysis (AOAC/OMA) cultural reference methods for the detection of L. monocytogenes on selected foods including hot dogs, frozen cooked breaded chicken, frozen cooked shrimp, cured ham, and ice cream, and environmental surfaces including stainless steel and plastic in an unpaired study design. The RapidChek method uses a proprietary enrichment media system, a 44-48 h enrichment at 30 ± 1°C, and detects L. monocytogenes on an immunochromatographic lateral flow device within 10 min. Different L. monocytogenes strains were used to spike each of the matrixes. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. A total of 140 low-level spiked samples were tested by the RapidChek method after enrichment for 44-48 h in parallel with the cultural reference method. There were 88 RapidChek presumptive positives. One of the presumptive positives was not confirmed culturally. Additionally, one of the culturally confirmed samples did not exhibit a presumptive positive. No difference between the alternate confirmation method and reference confirmation method was observed. The respective cultural reference methods (USDA-FSIS/MLG, FDA/BAM, and AOAC/OMA) produced a total of 63 confirmed positive results. Nonspiked samples from all foods were reported as negative for L. monocytogenes by all methods. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the RapidChek method and the respective cultural reference method.
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Análise de Alimentos/métodos , Imunoensaio/métodos , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia , Animais , Monitoramento Ambiental/economia , Monitoramento Ambiental/métodos , Microbiologia de Alimentos , Humanos , Imunoensaio/economia , Limite de Detecção , Fatores de TempoRESUMO
Kinetic studies, isotope labeling, and in situ high-resolution mass spectrometry are used to elucidate the mechanism for the catalytic oxidation of styrenes using aqueous hydrogen peroxide (H2O2) and the cationic palladium(II) compound, [(PBO)Pd(NCMe)2][OTf]2 (PBO = 2-(pyridin-2-yl)benzoxazole). Previous studies have shown that this reaction yields acetophenones with high selectivity. We find that H2O2 binds to Pd(II) followed by styrene binding to generate a Pd-alkylperoxide that liberates acetophenone by at least two competitive processes, one of which involves a palladium enolate intermediate that has not been previously observed in olefin oxidation reactions. We suggest that acetophenone is formed from the palladium enolate intermediate by protonation from H2O2. We replaced hydrogen peroxide with t-butyl hydroperoxide and found that, although the palladium enolate intermediate was observed, it was not on the major product-generating pathway, indicating that the form of the oxidant plays a key role in the reaction mechanism.
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2-Ynamides can be synthesised through PdII catalysed oxidative carbonylation, utilising low catalyst loadings. A variety of alkynes and amines can be used to afford 2-ynamides in high yields, whilst overcoming the drawbacks associated with previous oxidative methods, which rely on dangerous solvents and gas mixtures. The use of [NBu4 ]I allows the utilisation of the industrially recommended solvent ethyl acetate. O2 can be used as the terminal oxidant, and the catalyst can operate under safer conditions with low O2 concentrations.
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Alcinos/química , Amidas/síntese química , Paládio/química , Aminas/química , Catálise , Gases , Oxirredução , Oxigênio/química , SolventesRESUMO
A homogeneous Pd(II) catalyst, utilizing a simple and inexpensive amine ligand (TMEDA), allows 2-alkynoates to be prepared in high yields by an oxidative carbonylation of terminal alkynes and alcohols. The catalyst system overcomes many of the limitations of previous palladium carbonylation catalysts. It has an increased substrate scope, avoids large excesses of alcohol substrate and uses a desirable solvent. The catalyst employs oxygen as the terminal oxidant and can be operated under safer gas mixtures.
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Prostate cancer (PCa) cells use matrix metalloproteinases (MMPs) to degrade tissue during invasion. Perlecan/HSPG2 is degraded at basement membranes, in reactive stroma and in bone marrow during metastasis. We previously showed MMP-7 efficiently degrades perlecan. We now analyzed PCa tissue and serum from 288 prostatectomy patients of various Gleason grades to decipher the relationship between perlecan and MMP-7 in invasive PCa. In 157 prostatectomy specimens examined by tissue microarray, perlecan levels were 18% higher than their normal counterparts. In Gleason grade 4 tissues, MMP-7 and perlecan immunostaining levels were highly correlated with each other (average correlation coefficient of 0.52) in PCa tissue, regardless of grade. Serial sections showed intense, but non-overlapping, immunostaining for MMP-7 and perlecan at adjacent borders, reflecting the protease-substrate relationship. Using a capture assay, analysis of 288 PCa sera collected at prostatectomy showed elevated levels of perlecan fragments, with most derived from domain IV. Perlecan fragments in PCa sera were associated with overall MMP-7 staining levels in PCa tissues. Domain IV perlecan fragments were present in stage IV, but absent in normal, sera, suggesting perlecan degradation during metastasis. Together, perlecan fragments in sera and MMP-7 in tissues of PCa patients are measures of invasive PCa.
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Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Proteoglicanas de Heparan Sulfato/sangue , Metaloproteinase 7 da Matriz/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica/patologia , Prostatectomia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/cirurgia , Microglobulina beta-2/sangue , Microglobulina beta-2/metabolismoRESUMO
A palladium-catalyzed oxidative reaction is reported which converts dihydropyrans to their corresponding ortholactone. The products are formed in good to excellent yields with a very high level of chemoselectivity and functional group tolerance. Mechanistic studies confirm that the reaction proceeds by a Wacker-type mechanism.
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Traditionally, serotyping of Escherichia coli has been performed via slide agglutination methods using antisera. More recently, multiplex immunoassays and "molecular serotyping" via polymerase chain reaction (PCR) have been validated for this purpose. In this study, the serogroups of 161 Shiga toxin-producing Escherichia coli (STEC) strains isolated from fecal samples of California cattle were typed by conventional methods using antisera as well as two newly developed multiplex PCR- and antibody-based microbead assays using the Luminex technology. Using the Luminex assays, we were capable of serotyping 11 STEC isolates that were previously determined untypeable for the O antigen by conventional methods using antisera. Except for 14 isolates, results from the 2 Luminex assays agreed.
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Escherichia coli Shiga Toxigênica/classificação , Animais , California , Bovinos , Proteínas de Escherichia coli/análise , Fezes/microbiologia , Imunoensaio de Fluorescência por Polarização , Microesferas , Tipagem Molecular , Reação em Cadeia da Polimerase Multiplex , Antígenos O/análise , Sorotipagem , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
We describe a new approach for the real-time detection and identification of pathogens in food and environmental samples undergoing culture. Surface Enhanced Raman Scattering (SERS) nanoparticles are combined with a novel homogeneous immunoassay to allow sensitive detection of pathogens in complex samples such as stomached food without the need for wash steps or extensive sample preparation. SERS-labeled immunoassay reagents are present in the cultural enrichment vessel, and the signal is monitored real-time through the wall of the vessel while culture is ongoing. This continuous monitoring of pathogen load throughout the enrichment process enables rapid, hands-free detection of food pathogens. Furthermore, the integration of the food pathogen immunoassay directly into the enrichment vessel enables fully biocontained food safety testing, thereby significantly reducing the risk of contaminating the surrounding environment with enriched pathogens. Here, we present experimental results showing the detection of E. coli, Salmonella, or Listeria in several matrices (raw ground beef, raw ground poultry, chocolate milk, tuna salad, spinach, brie cheese, hot dogs, deli turkey, orange juice, cola, and swabs and sponges used to sample a stainless steel surface) using the SERS system and demonstrate the accuracy of the approach compared to plating results.