Digital teaching and assessment of psychomotor skills of the clinical head and neck examination during COVID-19 pandemic.

PURPOSE: During COVID-19, a fully digital course was established for teaching and assessing the psychomotor skills of clinical head and neck examination. Influence of different digital teaching formats was investigated. METHODS: The students (n = 286) received disposable instruments, a manual, and instructional videos for the examination. 221 students additionally received 45 min of interactive teleteaching. After 5 days of practice, all students were required to submit a video of their examination and report their spent practice time. The assessment was carried out using a checklist which was already established in presence teaching. RESULTS: The average score achieved by digital teaching was 86%. Previously published data show that presence teaching achieved 94%. With a teleteaching unit the total score was significantly better than without (87% vs 83%). Teleteaching leads to a significant positive correlation between practice time and total score. Without teleteaching there is a negative correlation. After the same practice time, presence teaching leads to better total scores than digital teaching. CONCLUSION: Digital teaching and assessing of a complex psychomotor skill is possible. Interactive teaching methods increase learning success. Nevertheless, presence teaching seems to be better at teaching these skills. The results can provide a basis for developing hybrid teaching models.

A framework for P-cycle assessment in wastewater treatment plants.

Phosphorus (P) in wastewater has a variety of negative effects and is usually permanently lost as a non-renewable resource. To mitigate future P shortage, P must be recovered from wastewater, preferably by bio-based technologies to avoid toxic side streams. A standardized procedure for the determination of P types and P concentrations in all liquid and solid process stages was established, which is applicable to all full-scale wastewater treatment plants (WWTPs). Based on this, an equally universal calculation framework for P-cycle assessment based on volume flow and mass load rates was designed to identify the most promising process streams for biological P recovery. As an example, in 16 process streams of a typical WWTP, concentrations of free, bound and total P were calculated and microbial communities were analyzed by flow cytometry over 748 days. The most promising process streams for the recovery of free P were anaerobic digester sludge, centrate and the water-extracts of the biosolids with 0.510 kg P m-3, 0.075 kg P m-3 and 1.023 kg P m-3, while the best process streams for the recovery of bound P were return sludge, excess sludge, anaerobic digester sludge, and the solids of the biosolids with 0.300 kg P m-3, 0.268 kg P m-3, 0.213 kg P m-3 and 1.336 kg P m-3, respectively. Microorganisms capable of P accumulation were active in all process stages and it was observed that chemical P precipitation antagonizes biological P removal. The framework for P-cycle assessment was able to identify process streams that are economically viable to make future in-stream technologies for biological P removal feasible.

Microbiological Control of Cellular Products: The Relevance of the Cellular Matrix, Incubation Temperature, and Atmosphere for the Detection Performance of Automated Culture Systems.

BACKGROUND: The microbiological control of cellular products sometimes causes significant procedural issues for quality control laboratories. According to the European Pharmacopoeia (EP), the microbiological control of cellular products requires a 7- to 14-day incubation period at two different incubation temperatures using aerobic and anaerobic growth media. However, the suitability of these test conditions for efficient quality control can be influenced by many conditions, such as the expected microbial spectrum of contamination or the texture and composition of the cellular product. Because of interference, direct inoculation and membrane filtration as reference methods of pharmacopoeia are largely unsuitable for the microbiological control of cellular products; therefore, alternative and, above all, automated methods are the focus of interest. OBJECTIVE: The aim of our study was to evaluate the method suitability and possible effects of cell matrix, incubation temperature, and oxygen pressure on the detection performance of automated culture systems. METHODS: The BacT/ALERT® 3DTM Dual T system (bioMérieux, Nürtingen, Germany) was used to evaluate the factors influencing automated microbiological control of cellular products. The tests were performed using microbial strains recommended by the EP for microbiological method suitability testing and additional relevant possible contaminants of human-derived stem-cell products under varying culture and cell matrix conditions. RESULTS: All contaminants were detected by the system in the required period of 2-5 days. Low incubation temperatures (22°C) had overall negative effects on the detection kinetics of each type of microbial contamination. The adverse effects of the accompanying cell matrix on the detection properties of the system could be compensated in our study by incubation at 32°C in both the aerobic and the anaerobic culture conditions. CONCLUSION: Automated culture techniques represent a sufficient approach for the microbiological control of cellular products. The negative effects of the cell matrix and microbial contamination on the detection performance can be compensated by the application of variable culture conditions in the automated culture system.

Long-Term Biogas Production from Glycolate by Diverse and Highly Dynamic Communities.

Generating chemical energy carriers and bulk chemicals from solar energy by microbial metabolic capacities is a promising technology. In this long-term study of over 500 days, methane was produced by a microbial community that was fed by the mono-substrate glycolate, which was derived from engineered algae. The microbial community structure was measured on the single cell level using flow cytometry. Abiotic and operational reactor parameters were analyzed in parallel. The R-based tool flowCyBar facilitated visualization of community dynamics and indicated sub-communities involved in glycolate fermentation and methanogenesis. Cell sorting and amplicon sequencing of 16S rRNA and mcrA genes were used to identify the key organisms involved in the anaerobic conversion process. The microbial community allowed a constant fermentation, although it was sensitive to high glycolate concentrations in the feed. A linear correlation between glycolate loading rate and biogas amount was observed (R² = 0.99) for glycolate loading rates up to 1.81 g L-1 day-1 with a maximum in biogas amount of 3635 mL day-1 encompassing 45% methane. The cytometric diversity remained high during the whole cultivation period. The dominating bacterial genera were Syntrophobotulus, Clostridia genus B55_F, Aminobacterium, and Petrimonas. Methanogenesis was almost exclusively performed by the hydrogenotrophic genus Methanobacterium.

Ecological Stability Properties of Microbial Communities Assessed by Flow Cytometry.

Natural microbial communities affect human life in countless ways, ranging from global biogeochemical cycles to the treatment of wastewater and health via the human microbiome. In order to probe, monitor, and eventually control these communities, fast detection and evaluation methods are required. In order to facilitate rapid community analysis and monitor a community's dynamic behavior with high resolution, we here apply community flow cytometry, which provides single-cell-based high-dimensional data characterizing communities with high acuity over time. To interpret time series data, we draw inspiration from macroecology, in which a rich set of concepts has been developed for describing population dynamics. We focus on the stability paradigm as a promising candidate to interpret such data in an intuitive and actionable way and present a rapid workflow to monitor stability properties of complex microbial ecosystems. Based on single-cell data, we compute the stability properties resistance, resilience, displacement speed, and elasticity. For resilience, we also introduce a method which can be implemented for continuous online community monitoring. The proposed workflow was tested in a long-term continuous reactor experiment employing both an artificial and a complex microbial community, which were exposed to identical short-term disturbances. The computed stability properties uncovered the superior stability of the complex community and demonstrated the global applicability of the protocol to any microbiome. The workflow is able to support high temporal sample densities below bacterial generation times. This may provide new opportunities to unravel unknown ecological paradigms of natural microbial communities, with applications to environmental, biotechnological, and health-related microbiomes. IMPORTANCE Microbial communities drive many processes which affect human well-being directly, as in the human microbiome, or indirectly, as in natural environments or in biotechnological applications. Due to their complexity, their dynamics over time is difficult to monitor, and current sequence-based approaches are limited with respect to the temporal resolution. However, in order to eventually control microbial community dynamics, monitoring schemes of high temporal resolution are required. Flow cytometry provides single-cell-based data in the required temporal resolution, and we here use such data to compute stability properties as easy to interpret univariate indicators of microbial community dynamics. Such monitoring tools will allow for a fast, continuous, and cost-effective screening of stability states of microbiomes. Applicable to various environments, including bioreactors, surface water, and the human body, it will contribute to the development of control schemes to manipulate microbial community structures and performances.

Overview of recent advances in phosphorus recovery for fertilizer production.

This Mini Review gives an overview of and respective references for the current situation regarding global phosphorus reserves and the legal situation for P recovery using Germany as the model. Apart from the well-known pilot up to industrial/full scale recovery techniques, emerging chemical and bio-based P recovery techniques are named without claiming to be all-encompassing. Special attention is paid to the biological systems for P recovery that reveal ways for use of renewable resources as raw materials. A few chemically based recovery techniques like AirPrex®, (Ostara)PEARL™, AshDec®, and RecoPhos® have already been used to recover P at a rate and quality which allows for its sale as a fertilizer. Many chemically based processes are at the stage of investigation on a pilot or laboratory scale, e.g. P-RoC, LeachPhos, and Mephrec®. All of the biologically based technologies like P-Bac are still at an early stage of research and show promising results. Of all recovered materials struvite, calcium phosphate and biological bound phosphorous seem to have the best plant availability. Although there is no ultimate "one fits all" technology, potential P-recovery plant operators can choose from a wide range of techniques which will best fit local raw material availability, economic and ecological situation.

Species-sorting and mass-transfer paradigms control managed natural metacommunities.

A complex microbial system consisting of six different interconnected localities was thoroughly investigated at full scale for over a year. The metacommunity concept originating from macro-ecology was used to uncover mechanisms of community assembly by observing microbial interrelationships in and between the different localities via correlation and network analysis. The individual-based observation approach was applied using high-throughput microbial community cytometry in addition to next generation sequencing. We found robust α-diversity values for each of the six localities and high ß-diversity values despite directed connectivity between localities, classifying for endpoint assembly of organisms in each locality. Endpoint characteristics were based on subcommunities with high cell numbers whereas those with lower cell numbers were involved in dispersal. Perturbation caused abiotic parameters to alter local community assembly with especially the rare cells announcing community restructuration processes. The mass-effect paradigm as part of the metacommunity concept was identified by an increase in interlocality biotic correlations under perturbation which, however, did not unbalance the predominant species-sorting paradigm in the studied full scale metacommunity. Data as generated in this study might contribute to the development of individual-based models for controlling managed multispecies natural systems in future.

Non-random distribution of macromolecules as driving forces for phenotypic variation.

Clonal populations employ many strategies of diversification to deal with constraints. All these strategies result in the generation of different phenotypes with diverse functions. Events like cell division are major sources of phenotypic variability due to the unequal partitioning of cellular components. In this review we concentrate on passive and active mechanisms cells employ to distribute macromolecules between their offspring. Different types of segregation are described, addressing both metabolically pertinent molecules such as PHA/PHB or polyphosphates, and components that adversely affect cells by promoting aging, such as damaged protein complexes or extrachromosomal rDNA circles. We also refer to mechanisms generating plasmid copy number (PCN) variation between cells in a population, and how elaborate partitioning systems counteract partitioning errors and ensure equal distribution. Finally, we demonstrate how simple differences in chromosomal copy number determine the fate of a cell, in this case the effect of gene dosage on the onset of sporulation in Bacillus subtilis or on a functional trait in Sinorhizobium meliloti.

Facilitated gate setting by sequential dot plot scanning.

Microbial communities comprising thousands of unknown organisms can be studied flow cytometrically by applying just one fluorescent parameter and using scatter characteristics of cells. Resulting 2D-plots need to represent high numbers of cells to detect the many subcommunities, even rare ones that might be present in the sample. Evaluation of such data can be faulty and subjective due to the low number of parameters available for data discrimination and the high numbers of overlaying events. Here, we describe a procedure that helps to evaluate such data using facilitated gate setting by sequential dot-plot scanning.

Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function.

Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.

Correlation of community dynamics and process parameters as a tool for the prediction of the stability of wastewater treatment.

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a primary clarifier and two activated sludge basins. The stability of microbial community structure was found to be high in the basins and low in the primary clarifier. Despite major abiotic changes certain subcommunities were dominantly present (up to 80% stability), whereas others emerged only sporadically (down to 3% stability, both according to equivalence testing). Additionally, subcommunities of diagnostic value were detected showing positive correlation with substrate influxes. For instance blackwater (r(s) = 0.5) and brewery inflow (both r(s) = 0.6) were mirrored by increases in cell abundances in subclusters 1 and 6 as well as 4 and 8, respectively. Phosphate accumulation was obviously positively correlated with nitrate (r(s) = 0.4) and the presence of denitrifying organisms (Rhodacyclaceae). Various other correlations between community structure and abiotic parameters were apparent. The bacterial composition of certain subcommunities was determined by cell sorting and phylogenetic tools like T-RFLP. In essence, we developed a monitoring tool which is quick, cheap and causal in its interpretation. It will make laborious PCR based technique less obligatory as it allows reliable process monitoring and control in wastewater treatment plants.

Structural basis of enzyme encapsulation into a bacterial nanocompartment.

Compartmentalization is an important organizational feature of life. It occurs at varying levels of complexity ranging from eukaryotic organelles and the bacterial microcompartments, to the molecular reaction chambers formed by enzyme assemblies. The structural basis of enzyme encapsulation in molecular compartments is poorly understood. Here we show, using X-ray crystallographic, biochemical and EM experiments, that a widespread family of conserved bacterial proteins, the linocin-like proteins, form large assemblies that function as a minimal compartment to package enzymes. We refer to this shell-forming protein as 'encapsulin'. The crystal structure of such a particle from Thermotoga maritima determined at 3.1-angstroms resolution reveals that 60 copies of the monomer assemble into a thin, icosahedral shell with a diameter of 240 angstroms. The interior of this nanocompartment is lined with conserved binding sites for short polypeptide tags present as C-terminal extensions of enzymes involved in oxidative-stress response.

Fluorogenic surrogate substrates for toluene-degrading bacteria--are they useful for activity analysis?

Cultivated bacterial toluene degraders use one or several of four described pathways for the aerobic degradation of this priority groundwater contaminant. To be able to identify un-cultivated toluene-degrading bacteria within enriched or natural consortia, we attempted to develop a set of staining techniques that invariably label toluene-degrading bacteria while differentiating between the different degradation pathways. In the literature, we found suggestions for pathway-specific labels of individual cells that rely on the conversion of toluene surrogates into specific colored and fluorescent products. These surrogate substrates were phenylacetylene (PA), cinnamonitrile, 3-hydroxyphenylacetylene (3-HPA), and indole. We were able to confirm that the chromogenic reactions reliably verified the pathway-specific reactions of well-characterized toluene-degrading bacterial species. However, it was most surprising to find out that three (PA, 3-HPA and cinnamonitrile) of the four supplied surrogate substrates did not lead to any product fluorescence above the cultures' autofluorescence, neither inside of cells nor in supernatants. More disturbingly, the original surrogate compound 3-HPA was inherently fluorescent and found to stain cells at intensities that depended on their states in the cell cycle. Indoxyl originating from the surrogate substrate indole was the only fluorescent product that was formed. It was detected intracellularly when the cells were sealed with para-formaldehyde, but its appearance was unrelated to the presence of expressed toluene degradation pathways. These findings were scrutinized by fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. Activity and growth of the test bacteria were determined by analyzing chromosome numbers and membrane integrity. Our results contradict literature reports that propose the surrogate fluorogenic substrates for the identification of toluene degraders and the identification of specific pathways used by them.

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.

Evidence for an indirect transcriptional regulation of glucose-6-phosphatase gene expression by liver X receptors.

Liver X receptor (LXR) paralogues alpha and beta (LXRalpha and LXRbeta) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXRalpha or LXRbeta suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors.

Voice-related quality of life in organic and functional voice disorders.

The voice-related quality of life (V-RQOL) questionnaire was proposed as a disease specific measure of patients' subjective burden elicited by a voice disorder. The purpose of this study was to investigate the influence of gender and aetiology (benign organic versus functional) on V-RQOL results. Fifty-two German-speaking patients completed the questionnaire without prior information about their individual diagnosis. Results allow for the conclusion that women score lower indicating a poorer voice status. Differences between patients diagnosed with an organic or a functional voice disorder revealed no universal difference. This has to be investigated in future, especially against the degree of the voice disorder, the individual personality, health belief and social background.