Assessment of viability of microorganisms employing fluorescence techniques

Viability assessment of microorganisms is relevant for a wide variety of applications in industry, including evaluation of inactivation treatments and quality assessment of starter cultures for beer, wine, and yoghurt production.Usually, the ability of microbial cells to reproduce is considered as the benchmark method for determination of cell viability, and this is most commonly determined by the plate count method. The time needed to form visible colonies, however, is relatively long. Therefore, there is an increasing interest in rapid methods which exploit criteria other than reproduction. The advantages of fluorescent methods are a high sensitivity, a high time resolution and the potential to analyze individual cells.Fluorescent probes such as fluorescein, carboxyfluorescein (cF), and BCECF may be incorporated in microorganisms as (non-fluorescent) acetoxymethyl or diacetyl esters. These esters are membrane permeable and are cleaved in the cytoplasm by esterases, which results in accumulation of the fluorescent form. In Saccharomycescerevisiae it was found that the carrier- mediated efflux of cF was coupled to the energy metabolism. Subsequently, a twostep procedure was developed, consisting of loading the cells with cF, followed by incubation in the presence of glucose. The efflux experiments showed an excellent correlation between the viability of S.cerevisiae cells (determined by plate count) and the ability to translocate cF.The intracellular pH (pH in ) is critical for the control of many cellular processes, such as DNA transcription, protein synthesis, and enzyme activities. To determine the intracellular pH of bacteria a novel method was developed based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE). This cFSE method significantly reduced problems due to efflux of fluorescent probe from the cells. Moreover, the method was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.The viability of R. oligosporus sporangiospores was determined microscopically and with flow cytometry. Swelling of the sporangiospores was accompanied by an increase of the pH in . In the presence of nonanoic acid, a self-inhibitor produced by various fungi, an increase of the pH in , was prevented and swelling inhibited, suggesting a crucial role of the pHin in the germination of the spores.The effects of detergents on the viability of Lactobacillus lactis and Bacillus subtilis were investigated. Triton X-100 and Lauryl sulfobetaine efficiently inhibited growth at concentrations below the critical micelle concentration. Surprisingly, the application of fluorescence techniques showed that Triton X-100 did not permeabilize the cytoplasmic membrane of L. lactis .In conclusion, fluorescence techniques offer the possibility to study viability of a cell population employing microscopy, flow cytometry and image analysis. This may contribute to a better understanding of the mechanisms involved in selective survival of microorganisms under different stress conditions

Parthenogenesis and sex-ratio distorting bacteria in Empoasca (Hemiptera: Cicadellidae) Leafhoppers

Endosymbiotic bacteria can induce parthenogenesis in many haplo-diploid species, but little is known about their role in asexual reproduction in diplodiploids. Bacteria are essential for reproduction in the asexual lin eages of springtails and booklice, and possibly some weevils, but whether they are the causative agent of par thenogenesis remains to be proven. This would require comparing unisexual forms and their closely related bisexual species, but such model species are very rare. Empoasca leafhoppers from the Madeira Archipelago provide an excellent case to test whether bacterial infections can cause parthenogenesis. Here we examined the diversity of the sex-distorting bacteria, Wolbachia, Cardinium, Rickettsia, and Arsenophonus, in three apo mictic morphotypes (A, B, C) and three bisexual relatives of Empoasca. Wolbachia of group B was present in all unisexual and bisexual species. Rickettsia were the only bacteria restricted to the three unisexual forms, suggesting an association between infection and asexual reproduction. In addition, we found Asaia for the third time in the Cicadellidae, although this may have little to do with the reproductive mode of its host. Adult females of morphotype C reared on plants watered with tetracycline solution did not result in the production of male offspring. To our knowledge, this is the first study to suggest a possible association between bacterial infection and parthenogenesis in apomictic XO/XX diplodiploid bisexual species based on a range of sex ratio distorting bacteria.info:eu-repo/semantics/publishedVersio

rt-me-fMRI: a task and resting state dataset for real-time, multi-echo fMRI methods development and validation

Latest published: 04 Feb 2021A multi-echo fMRI dataset (N=28 healthy participants) with four task-based and two resting state runs was collected, curated and made available to the community. Its main purpose is to advance the development of methods for real-time multi-echo functional magnetic resonance imaging (rt-me-fMRI) analysis with applications in neurofeedback, real-time quality control, and adaptive paradigms, although the variety of experimental task paradigms supports a multitude of use cases. Tasks include finger tapping, emotional face and shape matching, imagined finger tapping and imagined emotion processing. This work provides a detailed description of the full dataset; methods to collect, prepare, standardize and preprocess it; quality control measures; and data validation measures. A web-based application is provided as a supplementary tool with which to interactively explore, visualize and understand the data and its derivative measures: https://rt-me-fmri.herokuapp.com/. The dataset itself can be accessed via a data use agreement on DataverseNL at https://dataverse.nl/dataverse/rt-me-fmri. Supporting information and code for reproducibility can be accessed at https://github.com/jsheunis/rt-me-fMR

A Deep Learning Approach Utilizing Covariance Matrix Analysis for the ISBI Edited MRS Reconstruction Challenge

This work proposes a method to accelerate the acquisition of high-quality edited magnetic resonance spectroscopy (MRS) scans using machine learning models taking the sample covariance matrix as input. The method is invariant to the number of transients and robust to noisy input data for both synthetic as well as in-vivo scenarios

A review of machine learning applications for the proton MR spectroscopy workflow

This literature review presents a comprehensive overview of machine learning (ML) applications in proton MR spectroscopy (MRS). As the use of ML techniques in MRS continues to grow, this review aims to provide the MRS community with a structured overview of the state-of-the-art methods. Specifically, we examine and summarize studies published between 2017 and 2023 from major journals in the MR field. We categorize these studies based on a typical MRS workflow, including data acquisition, processing, analysis, and artificial data generation. Our review reveals that ML in MRS is still in its early stages, with a primary focus on processing and analysis techniques, and less attention given to data acquisition. We also found that many studies use similar model architectures, with little comparison to alternative architectures. Additionally, the generation of artificial data is a crucial topic, with no consistent method for its generation. Furthermore, many studies demonstrate that artificial data suffers from generalization issues when tested on in vivo data. We also conclude that risks related to ML models should be addressed, particularly for clinical applications. Therefore, output uncertainty measures and model biases are critical to investigate. Nonetheless, the rapid development of ML in MRS and the promising results from the reviewed studies justify further research in this field.</p

Verified and potential pathogens of predatory mites (Acari: Phytoseiidae)

Several species of phytoseiid mites (Acari: Phytoseiidae), including species of the genera Amblyseius, Galendromus, Metaseiulus, Neoseiulus, Phytoseiulus and Typhlodromus, are currently reared for biological control of various crop pests and/or as model organisms for the study of predator¿prey interactions. Pathogen-free phytoseiid mites are important to obtain high efficacy in biological pest control and to get reliable data in mite research, as pathogens may affect the performance of their host or alter their reproduction and behaviour. Potential and verified pathogens have been reported for phytoseiid mites during the past 25 years. The present review provides an overview, including potential pathogens with unknown host effects (17 reports), endosymbiotic Wolbachia (seven reports), other bacteria (including Cardinium and Spiroplasma) (four reports), cases of unidentified diseases (three reports) and cases of verified pathogens (six reports). From the latter group four reports refer to Microsporidia, one to a fungus and one to a bacterium. Only five entities have been studied in detail, including Wolbachia infecting seven predatory mite species, other endosymbiotic bacteria infecting Metaseiulus (Galendromus, Typhlodromus) occidentalis (Nesbitt), the bacterium Acaricomes phytoseiuli infecting Phytoseiulus persimilis Athias-Henriot, the microsporidium Microsporidium phytoseiuli infecting P. persimilis and the microsporidium Oligosproridium occidentalis infecting M. occidentalis. In four cases (Wolbachia, A. phytoseiuli, M. phytoseiuli and O. occidentalis) an infection may be connected with fitness costs of the host. Moreover, infection is not always readily visible as no obvious gross symptoms are present. Monitoring of these entities on a routine and continuous basis should therefore get more attention, especially in commercial mass-production. Special attention should be paid to field-collected mites before introduction into the laboratory or mass rearing, and to mites that are exchanged among rearing facilities. However, at present general pathogen monitoring is not yet practical as effects of many entities are unknown. More research effort is needed concerning verified and potential pathogens of commercially reared arthropods and those used as model organisms in research

Advancing GABA-edited MRS Research through a Reconstruction Challenge

Purpose To create a benchmark for the comparison of machine learning-based Gamma-Aminobutyric Acid (GABA)-edited Magnetic Resonance Spectroscopy (MRS) reconstruction models using one quarter of the transients typically acquired during a complete scan.Methods The Edited-MRS reconstruction challenge had three tracks with the purpose of evaluating machine learning models trained to reconstruct simulated (Track 1), homogeneous in vivo (Track 2), and heterogeneous in vivo (Track 3) GABA-edited MRS data. Four quantitative metrics were used to evaluate the results: mean squared error (MSE), signal-to-noise ratio (SNR), linewidth, and a shape score metric that we proposed. Challenge participants were given three months to create, train and submit their models. Challenge organizers provided open access to a baseline U-NET model for initial comparison, as well as simulated data, in vivo data, and tutorials and guides for adding synthetic noise to the simulations.Results The most successful approach for Track 1 simulated data was a covariance matrix convolutional neural network model, while for Track 2 and Track 3 in vivo data, a vision transformer model operating on a spectrogram representation of the data achieved the most success. Deep learning (DL) based reconstructions with reduced transients achieved equivalent or better SNR, linewidth and fit error as conventional reconstructions with the full amount of transients. However, some DL models also showed the ability to optimize the linewidth and SNR values without actually improving overall spectral quality, pointing to the need for more robust metrics.Conclusion The edited-MRS reconstruction challenge showed that the top performing DL based edited-MRS reconstruction pipelines can obtain with a reduced number of transients equivalent metrics to conventional reconstruction pipelines using the full amount of transients. The proposed metric shape score was positively correlated with challenge track outcome indicating that it is well-suited to evaluate spectral quality.Competing Interest StatementThe authors have declared no competing interest

Life and Death of an Influential Passenger: Wolbachia and the Evolution of CI-Modifiers by Their Hosts

Wolbachia are intracellular bacteria widely distributed among arthropods and nematodes. In many insect species these bacteria induce a cytoplasmic incompatibility (CI) between sperm of infected males and eggs of uninfected females. From an evolutionary point of view, CI is puzzling: In order to induce this modification-rescue system, Wolbachia affect sperm of infected males even though Wolbachia are only transmitted maternally. Phylogenetic studies of Wolbachia and hosts show that the bacteria rarely cospeciate with their hosts, indicating that infections are lost in host species. However, the mechanisms leading to Wolbachia loss are not well understood.Using a population genetic model, we investigate the spread of host mutants that enhance or repress Wolbachia action by affecting either bacterial transmission or the level of CI. We show that host mutants that decrease CI-levels in males (e.g. by reducing Wolbachia-density during spermatogenesis) spread, even at cost to mutant males. Increase of these mutants can lead to loss of Wolbachia infections, either as a direct consequence of their increase or in a step-wise manner, and we derive analytically a threshold penetrance above which a mutation's spread leads to extinction of Wolbachia. Selection on host modifiers is sexually antagonistic in that, conversely, host mutants that enhance Wolbachia in females are favoured whereas suppressors are not.Our results indicate that Wolbachia is likely to be lost from host populations on long evolutionary time scales due to reduction of CI levels in males. This can occur either by evolution of single host modifiers with large effects or through accumulation of several modifier alleles with small effects on Wolbachia action, even at cost to mutant males and even if infected hosts do not incur fecundity costs. This possibility is consistent with recent findings and may help to explain the apparent short evolutionary persistence times of Wolbachia in many host systems

Intracellular Vesicles as Reproduction Elements in Cell Wall-Deficient L-Form Bacteria

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms