When using microbiologically induced calcium carbonate precipitation (MICP) to produce calcium carbonate crystals in the cavities between mineral particles to consolidate them, the inhomogeneous ...distribution of the precipitated calcium carbonate poses a problem for the production of construction materials with consistent parameters. Various approaches have been investigated in the literature to increase the homogeneity of consolidated samples. One approach can be the targeted application of ureolytic organisms by 3D printing. However, to date, this possibility has been little explored in the literature. In this study, the potential to use MICP to print calcium carbonate layers on mineral particles will be investigated. For this purpose, a dispensing unit was modified to apply both a suspension of Sporosarcina pasteurii and a calcination solution containing urea and calcium chloride onto quartz sand. The study showed that after passing through the nozzle, S. pasteurii preserved consistent cell vitality and therefore its potential of MICP. Applying cell suspension and calcination solution through a printing nozzle resulted in a layer of calcium carbonate crystals on quartz sand. This observation demonstrated the proof of concept of printing calcium carbonate by MICP through the nozzle of a dispensing unit. Furthermore, it was shown that cell suspensions of S. pasteurii can be stored at 4°C for a period of 17 days while maintaining its optical density, urease activity and cell vitality and therefore the potential for MICP. This initial concept could be extended in further research to printing three‐dimensional (3D) objects to solve the problem of homogeneity in consolidated mineral particles.
The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of ...diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6μT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.
The cyanobacterium Nostoc sp. BB 92.3. had shown antibacterial activity. A cultivation as biofilm, a self‐forming matrix of cells and extracellular polymeric substances, increased the antibacterial ...effect. A new photobioreactor system was developed that allows a surface‐associated cultivation of Nostoc sp. as biofilm. High‐density polyethylene carriers operated as a moving bed were selected as surface for biomass immobilization. This system, well established in heterotrophic wastewater treatment, was for the first time used for phototrophic biofilms. The aim was a cultivation on a large scale without inhibiting growth while maximizing immobilization. Cultivation in a small photobioreactor (1.5 L) with different volumetric filling degrees of carriers (13.4%–53.8%) in a batch process achieved immobilization rates of 70%–85% and growth was similar to a no‐carrier‐control. In a larger photobioreactor (65 L) essentially all of the biomass was immobilized on the carriers and the space‐time yield of biomass (0.018 gcell dry weight L−1 day−1) was competitive compared to phototrophic biofilm cultivations from literature. The use of carriers increased the gas exchange in the reactor by a factor of 2.5–3 but doubled the mixing time. Enriched gassing with carbon dioxide resulted in a short‐term increase in growth rate, but unexpectedly it also adversely changed the growth morphology.
A new approach for the surface‐associated large‐scale cultivation of phototrophic organisms is presented by Walther et al. The cyanobacterium Nostoc species BB 92.2 was cultivated as a biofilm inside a hollow plastic carrier in a fluidized bed. This system provided the cyanobacteria with a large, well‐defined surface area on which it established a homogeneous self‐formed matrix. A 65‐L photobioreactor was constructed, in which important parameters such as gas transfer rate were studied, which was significantly improved by using carriers.
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•New procedure for separation of EPS into the main components from one sample.•Analysis of the main components of EPS from only one sample.•Characterization of EPS for a deeper ...insight into the state of the cells.•100 % recovery rates of synthetic EPS containing polysaccharides, proteins and lipids.•Successful transfer to real EPS samples from different cultivations.
Phototrophic biofilms produce a matrix of extracellular polymeric substances (EPS), which holds the cells together and functions inter alia as nutrient storage and protection layer. EPS mainly consist of water, polysaccharides, proteins, lipids and nucleic acids as well as lysis and hydrolysis products which makes the composition very complex. Thus, rough simplifications are used and commonly one or at most two components of the EPS are examined. In this work a new procedure for separation and analysis of EPS in the main components (i) polysaccharides, (ii) proteins and (iii) lipids is presented with recovery rates of nearly 100 %. The method was established with synthetic EPS, which based on the composition of real EPS described in literature. Afterwards, the method was transferred to real EPS samples allowing a deeper insight in the composition of EPS from only one sample. The composition of EPS-extracts from Nostoc spec, cultivated under heterotrophic and mixotrophic batch and fed-batch conditions, was analysed during a cultivation period of 14 days. It was observed that mixotrophic cultivation led to higher amounts of carbohydrates, lipids and proteins than heterotrophic cultivation respectively, regardless of batch or fed-batch culture. While the amount of proteins in the EPS increased during the cultivation period, carbohydrates and lipids were dominant in the beginning and decreased afterwards.
In the course of combining the Sustainable Development Goals (SDGs) with the science education curriculum, the relevance of the micro- and macroalgae in education is based on the biotechnological ...future-oriented significance and the ever-growing trend toward plant-based nutrition. So, the micro- and macroalgae are finding their way onto the food shelves and creating biotechnological solutions with regard to climate change (SDG4; SDG13). Their colored photopigments and phycobiliproteins are already established as important natural dyes in the food and textile industries. In addition to being essential in photosynthesis, photopigments have a variety of functions and effects that influence almost all aspects of our lives. The article presents experimental protocols developed based on the established methods for the extraction of photopigments from plants and optimized for the use of phototrophic micro- and macroalgae (Chlorella vulgaris, Arthrospira platensis, and Palmaria palmata). Besides the green chlorophylls and yellow-orange carotenoids in plants, cyanobacteria and red algae developed additional light antenna complexes, so-called phycobilisomes, consisting of different phycobiliproteins. For this purpose, experiments that are simple to execute have been developed to make the colorful world of photopigments visible to the students from the upper secondary level and can be used in both university and school educational settings. Furthermore, a column chromatography was developed, which allows the preparation of the pigment and phycobiliprotein extracts from A. platensis. This procedure is based on the established “supermarket column” and was optimized according to the use of powdered amounts of A. platensis. Additionally, results from a first implementation in a classroom setting will be discussed.
Terrestrial cyanobacteria are phototrophic microorganisms, which grow embedded in a matrix of extracellular polymeric substances (EPS) as biofilms. To perform photosynthesis, cyanobacteria have ...developed, additionally to chlorophyll-a and carotenoids, the so-called phycobilisomes, which consist of differently pigmented phycobiliproteins (PBPs). The production of natural dyes, pharmaceutical substances and their nutrient rich biomass makes cyanobacterial biofilms interesting research targets for industrial development. However, characterization of biofilms during cultivation implies several difficulties, since standard sampling applied in suspended cultures is not possible. The aim of this study was to find new possibilities for documenting biofilm growth. Therefore, two methods for non-invasive examination of phototrophic biofilms were combined. The terrestrial cyanobacteria Trichocoleus sociatus und Coleofasciculus chthonoplastes were cultivated surface-associated in an aerosol. Every three to four days biofilm thickness was measured using optical coherence tomography (OCT) and biofilm area using PAM fluorometry. Additionally, three replicates were harvested to determine biomass formation and pigment composition. According to our results, an increase of biomass, biofilm thickness and biofilm area was detected. The non-linear correlation between these parameters indicates an increase of biofilm area and thickness in the early phase of cultivation with a time-delayed biomass production. Hereby, possibly the proportion of EPS and biomass changed in the beginning in combination with biofilm spreading to increase the utilization of available light and aerosol components. Afterwards, biomass production was increased. Concerning pigment composition in the course of cultivation, for T. sociatus all PBPs were degraded, whereas in C. chthonoplastes an increase of all PBPs was measured. This can be explained with the role of PBPs for photosynthesis but as well as nitrogen storage, which is adjusted strain dependent. Summarizing, the shown methods are suitable for phototrophic biofilm characterization in a small scale. This allows the examination of cyanobacteria and a valuation of their suitability for possible production processes.
•Characterization of cyanobacterial biofilms over a cultivation time of 31 days•Determination of biofilm growth with optical coherence tomography (OCT)•First-time combination of OCT and chlorophyll fluorometry as non-invasive methods•Analysis of biofilm thickness, area, biomass, light adsorption and pigment content•Extensive comparison of biofilm formation of two cyanobacteria strains
The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of ...diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6μT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.
This paper proposes to use a reservoir computation approach to model the non-linear dynamic behaviour of a novel electroactive soft actuator. The soft actuator is fabricated as a unimorph cantilever ...beam, in which the active layer is a mat of electrospun aligned nanofibers of the P(VDF-TrFE-CTFE) electrostrictive polymer integrated into a PDMS silicone matrix. The passive layer consists of kapton, while the soft electrodes are made of conductive carbon powder. The non-linear dynamic response of three specimens of the soft actuator, when stimulated by both DC and complex electric fields of varying frequency, is modeled by means of an echo state network. The proposed architecture is able to achieve a normalized root mean square error of 0.429 for the tip deflections and of 0.265 for the blocking forces, when compared to experimental data.
In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate ...stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.
Abstract
Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. ...The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a
MYC
translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in
MCL1
and
IRF4
. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients.
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