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The enticing aroma of truffles is a key factor for their culinary value. Although all truffle species tend to be pricy, the most intensely aromatic species are the most sought after. Research into the aroma of truffles encompasses various disciplines including chemistry, biology, and sensory science. This study focusses on the chemical composition of the aroma of black truffles (Tuber melanosporum) and the changes occurring under different storage conditions. For this, truffle samples were stored under different treatments, at different temperatures, and measured over a total storage time of 12 days. Measurements of the truffle aroma profiles were taken with SPME/GC-MS at regular intervals. To handle the ample data collected, a systematic approach utilizing multivariate data analysis techniques was taken. This approach led to a vast amount of data which we made publicly available for future exploration. Results reveal the complexity of aroma changes, with 695 compounds identified, highlighting the need for a comprehensive understanding. Principal component analyses offer initial insights into truffle composition, while individual compounds may serve as markers for age (formic acid, 1-methylpropyl ester), freshness (2-Methyl-1-propanal; 1-(methylthio)-propane), freezing (tetrahydrofuran), salt treatment (1-chloropentane), or heat exposure (4-hydroxy-3-methyl-2-butanone). This research suggests that heat treatment or salt contact significantly affects truffle aroma, while freezing and cutting have less pronounced effects in comparison. The enrichment of compounds showing significant changes during storage was investigated with a metabolomic pathway analysis. The involvement of some of the enriched compounds on the pyruvate/glycolysis and sulfur pathways was shown.
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Antimicrobial resistance is an emerging global threat to humanity. As resistance outpaces development, new perspectives are required. For decades, scientists have prioritized chemical optimization, while largely ignoring the physical process of delivery. Here, we used biophysical simulations and microfluidic experiments to explore how fluid flow delivers antimicrobials into communities of the highly resistant pathogen Pseudomonas aeruginosa . We discover that increasing flow overcomes bacterial resistance towards three chemically distinct antimicrobials: hydrogen peroxide, gentamicin, and carbenicillin. Without flow, resistant P. aeruginosa cells generate local zones of depletion by neutralizing all three antimicrobials through degradation or chemical modification. As flow increases, delivery overwhelms neutralization, allowing antimicrobials to regain effectiveness against resistant bacteria. Additionally, we discover that cells on the edge of a community shield internal cells, and cell-cell shielding is abolished in higher flow regimes. Collectively, our quantitative experiments reveal the unexpected result that physical flow and chemical dosage are equally important to antimicrobial effectiveness. Thus, our results should inspire the incorporation of flow into the discovery, development, and implementation of antimicrobials, and could represent a new strategy to combat antimicrobial resistance.
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The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.
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Fimbrias Bacterianas , Fagos Pseudomonas , Pseudomonas aeruginosa , Virus ARN , Internalización del Virus , Humanos , Microscopía por Crioelectrón , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/virología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/virología , Virus ARN/química , Virus ARN/fisiología , Fagos Pseudomonas/química , Fagos Pseudomonas/fisiología , Proteínas Virales/metabolismoRESUMEN
Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.
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Ergotamina , Ergotamina/síntesis química , Ergotamina/análisis , Isótopos de Carbono , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Marcaje IsotópicoRESUMEN
The statistical tool eCerto was developed for the evaluation of measurement data to assign property values and associated uncertainties of reference materials. The analysis is based on collaborative studies of expert laboratories and was implemented using the R software environment. Emphasis was put on comparability of eCerto with SoftCRM, a statistical tool based on the certification strategy of the former Community Bureau of Reference. Additionally, special attention was directed towards easy usability from data collection through processing, archiving, and reporting. While the effects of outlier removal can be flexibly explored, eCerto always retains the original data set and any manipulation such as outlier removal is (graphically and tabularly) documented adequately in the report. As a major reference materials producer, the Bundesanstalt für Materialforschung und -prüfung (BAM) developed and will maintain a tool to meet the needs of modern data processing, documentation requirements, and emerging fields of RM activity. The main features of eCerto are discussed using previously certified reference materials.
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Inhibition of γ-secretase, an intramembrane protease, to reduce secretion of Amyloid-ß (Aß) peptides has been considered for treating Alzheimer's disease. However, γ-secretase inhibitors suffer from severe side effects. As an alternative, γ-secretase modulators (GSM) reduce the generation of toxic peptides by enhancing the cleavage processivity without diminishing the enzyme activity. Starting from a known γ-secretase structure without substrate but in complex with an E2012 GSM, we generated a structural model that included a bound Aß43 peptide and studied interactions among enzyme, substrate, GSM, and lipids. Our result suggests that E2012 binding at the enzyme-substrate-membrane interface attenuates the membrane distortion by shielding the substrate-membrane interaction. The model predicts that the E2012 modulation is charge-dependent and explains the preserved hydrogen acceptor and the aromatic ring observed in many imidazole-based GSM. Predicted effects of γ-secretase mutations on E2012 modulation were confirmed experimentally. We anticipate that the study will facilitate the future development of effective GSMs.
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Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Mutación , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.
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Bacteriófagos , Infecciones por Pseudomonas , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Pseudomonas aeruginosa/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Virulencia , Antibacterianos/farmacología , Infecciones por Pseudomonas/microbiologíaRESUMEN
Fluid flow is thought to prevent bacterial adhesion, but some bacteria use adhesins with catch bond properties to enhance adhesion under high shear forces. However, many studies on bacterial adhesion either neglect the influence of shear force or use shear forces that are not typically found in natural systems. In this study, we use microfluidics and single-cell imaging to examine how the human pathogen Pseudomonas aeruginosa interacts with surfaces when exposed to shear forces typically found in the human body (0.1 pN to 10 pN). Through cell tracking, we demonstrate that the angle between the cell and the surface predicts if a cell will depart the surface. We discover that at lower shear forces, type IV pilus retraction tilts cells away from the surface, promoting surface departure. Conversely, we show that higher shear forces counterintuitively enhance adhesion by counteracting type IV pilus retraction-dependent cell tilting. Thus, our results reveal that P. aeruginosa exhibits behavior reminiscent of a catch bond, without having a specific adhesin that is enhanced by force. Instead, P. aeruginosa couples type IV pilus dynamics and cell geometry to tune adhesion to its mechanical environment, which likely provides a benefit in dynamic host environments.
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Fimbrias Bacterianas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/metabolismo , Fimbrias Bacterianas/metabolismo , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Fenómenos Físicos , Proteínas Fimbrias/metabolismoRESUMEN
Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-ß (Aß) peptides and defines the proportion of short-to-long Aß peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aß peptide length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APPC99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aß length. In addition, our study reveals that APPC99 -ECD facilitates the paradoxical production of longer Aßs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , ProteolisisRESUMEN
Colonies of the social bacterium Myxococcus xanthus go through a morphological transition from a thin colony of cells to three-dimensional droplet-like fruiting bodies as a strategy to survive starvation. The biological pathways that control the decision to form a fruiting body have been studied extensively. However, the mechanical events that trigger the creation of multiple cell layers and give rise to droplet formation remain poorly understood. By measuring cell orientation, velocity, polarity, and force with cell-scale resolution, we reveal a stochastic local polar order in addition to the more obvious nematic order. Average cell velocity and active force at topological defects agree with predictions from active nematic theory, but their fluctuations are anomalously large due to polar active forces generated by the self-propelled rod-shaped cells. We find that M. xanthus cells adjust their reversal frequency to tune the magnitude of this local polar order, which in turn controls the mechanical stresses and triggers layer formation in the colonies.
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Bacterial populations are highly adaptive. They can respond to stress and survive in shifting environments. How the behaviours of individual bacteria vary during stress, however, is poorly understood. To identify and characterize rare bacterial subpopulations, technologies for single-cell transcriptional profiling have been developed. Existing approaches show some degree of limitation, for example, in terms of number of cells or transcripts that can be profiled. Due in part to these limitations, few conditions have been studied with these tools. Here we develop massively-parallel, multiplexed, microbial sequencing (M3-seq)-a single-cell RNA-sequencing platform for bacteria that pairs combinatorial cell indexing with post hoc rRNA depletion. We show that M3-seq can profile bacterial cells from different species under a range of conditions in single experiments. We then apply M3-seq to hundreds of thousands of cells, revealing rare populations and insights into bet-hedging associated with stress responses and characterizing phage infection.
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Bacteriófagos , Bacteriófagos/genética , Bacterias/genética , ARN Ribosómico/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Being able to predict molecular properties and interactions is of utmost interest for academia as well as industry. But the vast complexity of strongly correlated molecular systems limits the performance of classical algorithms. In contrast, quantum computation has the potential to be a game changer in the field of molecular simulations. Despite the hope in quantum computation, the capabilities of current quantum computers are still insufficient for handling molecular systems of interest. In this paper, we propose a variational ansatz for today's noisy quantum computers to calculate the ground state with the help of imaginary time evolution. Although the imaginary time evolution operator is not unitary, it can be implemented on a quantum computer by a linear decomposition and subsequent Taylor series expansion. This has the advantage that only a set of shallow circuits needs to be computed on a quantum computer. The parallel nature of this algorithm can be exploited to speed-up simulations even further, if a privileged access to quantum computers is granted.
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Bacterial pathogenicity relies on both firm surface adhesion and cell dissemination. How twitching bacteria resolve the fundamental contradiction between adhesion and migration is unknown. To address this question, we employ live-cell imaging of type-IV pili (T4P) and therewith construct a comprehensive mathematical model of Pseudomonas aeruginosa migration. The data show that only 10% to 50% of T4P bind to substrates and contribute to migration through random extension and retraction. Individual T4P do not display a measurable sensory response to surfaces, but their number increases on cellular surface contact. Attachment to surfaces is mediated, besides T4P, by passive adhesive forces acting on the cell body. Passive adhesions slow down cell migration and result in local random motion on short time scales, which is followed by directionally persistent, superdiffusive motion on longer time scales. Moreover, passive adhesions strongly enhance surface attachment under shear flow. Δ pilA mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating Δ pilH cells, which produce an excessive number of T4P, are easily detached by shear. Wild-type cells sacrifice migration speed for robust surface attachment by maintaining a low number of active pili. The different cell strains pertain to disjunct regimes in a generic adhesion-migration trait space. Depending on the nature of the adhesion structures, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization under different conditions.
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Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.
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Alcaloides de Claviceps , Ácido Lisérgico , Espectrometría de Masas en Tándem , Ergolinas , Harina/análisisRESUMEN
Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.
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Bacterias , Peróxido de Hidrógeno , Humanos , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Bacterias/metabolismo , Microfluídica , Técnicas de Cultivo Celular por Lotes , Pseudomonas aeruginosa/genéticaRESUMEN
Volatile organic compounds (VOCs) are of interest in many different fields. Among them are food and fragrance analysis, environmental and atmospheric research, industrial applications, security or medical and life science. In the past, the characterization of these compounds was mostly performed via sample collection and off-site analysis with gas chromatography coupled to mass spectrometry (GC-MS) as the gold standard. While powerful, this method also has several drawbacks such as being slow, expensive, and demanding on the user. For decades, intense research has been dedicated to find methods for fast VOC analysis on-site with time and spatial resolution. We present the working principles of the most important, utilized, and researched technologies for this purpose and highlight important publications from the last five years. In this overview, non-selective gas sensors, electronic noses, spectroscopic methods, miniaturized gas chromatography, ion mobility spectrometry and direct injection mass spectrometry are covered. The advantages and limitations of the different methods are compared. Finally, we give our outlook into the future progression of this field of research.
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Compuestos Orgánicos Volátiles , Cromatografía de Gases y Espectrometría de Masas/métodos , Compuestos Orgánicos Volátiles/análisis , Espectrometría de MasasRESUMEN
PURPOSE: Anterior cruciate ligament (ACL) injuries are a common severe type of football injury. Injury prevention measures should be adapted to the respective type of sports and be based on sports-specific strategies. A football-specific ACL registry including prospective ACL injury data of both sexes and at different skill levels is lacking in the literature. METHODS: In Germany, a prospective 'ACL registry for German Football' was implemented in the 2014-15 football season. Professional football leagues (1st to 3rd national league), semi-professional football leagues (4th until 6th leagues) and amateur football leagues (7th league and below) were monitored regarding the incidence of ACL injuries, risk factors, general treatment and rehabilitation. After the registration of an injury, injured players were sent a standardised questionnaire. RESULTS: Overall, 1,206 ACL ruptures were registered in the investigated population of more than 56,000 players, resulting in a prevalence of 2.1%. The highest prevalence was found in men's amateur football (2.7%) as well as in men's and women's professional football (2.1% each). CONCLUSION: This football-specific ACL registry provides for the first-time longitudinal data about ACL injury patterns and treatment at all football levels. This report describes the considerations for data collection and presents first epidemiological results of 6 years of ACL injury registration.
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Lesiones del Ligamento Cruzado Anterior , Fútbol Americano , Masculino , Femenino , Humanos , Lesiones del Ligamento Cruzado Anterior/epidemiología , Fútbol Americano/lesiones , Estudios Prospectivos , Incidencia , Sistema de RegistrosRESUMEN
Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.
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Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Inhibidores y Moduladores de Gamma Secretasa , Enfermedad de Alzheimer/metabolismo , Imidazoles/uso terapéuticoRESUMEN
A brief history of optical forces, the invention of optical tweezers, and their application to biological problems.
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Pinzas ÓpticasRESUMEN
(1) Background: Isolated posterior cruciate ligament (PCL) tears represent a severe type of injury. In hospitals, PCL reconstruction (PCL-R) is less frequently performed than other types of knee surgery. It is unclear whether there is consensus among surgeons on how to perform rehabilitation after PCL-R or if there are different, more individual approaches in daily routines. (2) Methods: Rehabilitation protocols and their main criteria (the progression of weight bearing and range of motion, the use of knee braces, rehabilitation training, and sports-specific training) were retrospectively analyzed after PCL-R. (3) Results: Only 33 of 120 (27.5%) analyzed institutes use rehabilitation protocols after PCL-R. The applied protocols showed vast differences between the individual rehabilitation criteria, especially with regard to the progression of weight bearing and the range of motion. The only standardized recommendations were the obligatory use of knee braces and the general restriction of weight bearing and range of motion immediately post-surgery. Therefore, because of the lack of a consensus about a standardized rehabilitation protocol after PCL-R, no recommendation can be made on one particular protocol. (4) Conclusion: There is no acknowledged standardized rehabilitation protocol after PCL-R. In clinical practice, recommendations are influenced by, i.a., surgeons' opinions and experience. The lack of scientific evidence on a particular standardized rehabilitation protocol after PCL-R suggests that rehabilitation protocols need to be tailored to the individual patient.