Development of a Fluorescent Multiwell Assay for Evaluating the Capacity of the Ciliated Protozoan Tetrahymena for Bacterivory in Water Samples

2006 ◽  
Vol 41 (3) ◽  
pp. 307-315 ◽  
Author(s):  
Mary E. Power ◽  
Dana Sotornik ◽  
Marcel D.O. Pinheiro ◽  
Vivian R. Dayeh ◽  
Barbara J. Butler ◽  
...  
Keyword(s):  
Water Samples ◽  
Ecosystem Health ◽  
Cytochalasin B ◽  
Microbial Population ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Microtitre Plate ◽  
Population Composition ◽  
Different Types ◽  
Water Ecosystems

Abstract Bacterivory by ciliates in various water ecosystems, both natural and artificial, plays a significant role on the microbial population composition and consequently affects water quality. A convenient, rapid and inexpensive methodology to evaluate the capacity of the ciliate protozoan Tetrahymena thermophila for bacterivory was developed utilizing fluorescent protein expressing bacteria (FPEB) in a microtitre plate fluorimeter. Bacterivory was correlated with a loss in fluorescence measured in the fluorimeter and confirmed by fluorescence microscopy showing that the FPEB were engulfed during the assay and subsequently lost their fluorescence, whereas Cytochalasin B, a known inhibitor of phagocytosis, prevented a decrease in relative fluorescence units (RFUs). The ciliate bacterivory (CB) assay has a great dynamic range allowing the assay to be performed with a variety of predator:prey concentrations. A model toxicant, CuCl2, known to have a toxicological impact on protozoa and often present in different types of wastewater, resulted in measurable decreases in bacterivory. As well, starvation of Tetrahymena for 24 h resulted in reduced bacterivory. In the future, the CB assay could be developed for water monitoring purposes to rapidly assess water samples for the capacity to support bacterivory as an indicator of ecosystem health.

scholarly journals A split ribozyme that links detection of a native RNA to orthogonal protein outputs

2022 ◽  
Author(s):  
Lauren Gambill ◽  
August Staubus ◽  
Andrea Ameruoso ◽  
James Chappell
Keyword(s):  
Antibiotic Resistance ◽  
Synthetic Biology ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Tetrahymena Thermophila ◽  
Laboratory Evolution ◽  
Input Signals ◽  
Different Types

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we used laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivered a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We resolved a thermodynamic model to guide RENDR design, showed how input signals can be transduced into diverse visual, chemical, and regulatory outputs, and used RENDR to detect an antibiotic resistance phenotype in bacteria. This work shows how transcriptional signals can be monitored in situ using RNA synthetic biology and converted into different types of biochemical information.

Assessment of viral assemblages in different types of wetland water in northeast China using RAPD-PCR

2020 ◽  
Vol 85 ◽  
pp. 183-196
Author(s):  
Y Sun ◽  
J Liu ◽  
Q Yao ◽  
J Jin ◽  
X Liu ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Water Samples ◽  
Northeast China ◽  
Bacterial Community Composition ◽  
Freshwater Wetlands ◽  
Viral Community ◽  
Rapd Pcr ◽  
Wetland Water ◽  
Different Types ◽  
Viral Communities

Viruses are the most abundant and ubiquitous biological entities in various ecosystems, yet few investigations of viral communities in wetlands have been performed. To address this data gap, water samples from 6 wetlands were randomly collected across northeast China; viruses in the water were concentrated by sequential tangential flow filtration, and viral communities were assessed through randomly amplified polymorphic DNA-PCR (RAPD-PCR) with 4 decamer oligonucleotide primers. Principal coordinate analysis and hierarchical clustering analysis of the DNA fingerprints showed that viral community compositions differed among the water samples: communities in the 2 coastal wetlands were more similar to each other than to those in the 4 freshwater wetlands. The Shannon-Weaver index (H) and evenness index (E) of the RAPD-PCR fingerprint also differed among the 6 wetlands. Mantel test revealed that the changes in viral communities in wetland water were most closely related to the water NH4+-N and inorganic C content, followed by total K, P, C and NO3--N. DNA sequence analysis of the excised bands revealed that viruses accounted for ~40% of all sequences. Among the hit viral homologs, the majority belonged to the Microviridae. Moreover, variance partitioning analysis showed that the viral community contributed 24.58% while environmental factors explained 30.56% of the bacterial community variation, indicating that the bacterial community composition was strongly affected by both viral community and water variables. This work provides an initial outline of the viral communities from different types of wetlands in northeast China and improves our understanding of the viral diversity in these ecosystems.

Enumeration of Cryptosporidium oocysts from surface water concentrates by laser-scanning cytometry

2000 ◽  
Vol 41 (7) ◽  
pp. 197-202 ◽  
Author(s):  
F. Zanelli ◽  
B. Compagnon ◽  
J. C. Joret ◽  
M. R. de Roubin
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Laser Scanning ◽  
Immunomagnetic Separation ◽  
Entire Surface ◽  
Cryptosporidium Oocysts ◽  
Different Types ◽  
Laser Scanning Cytometry ◽  
Direct Microscopic Observation

The utilization of the ChemScan® RDI was tested for different types of water concentrates. Concentrates were prepared by cartridge filtration or flocculation, and analysed either without purification, or after Immunomagnetic separation (IMS) or flotation on percoll-sucrose gradients. Theenumeration of the oocysts was subsequently performed using the ChemScan® RDI Cryptosporidium application. Enumeration by direct microscopic observation of the entire surface of the membrane was carried out as a control, and recoveries were calculated as a ratio between the ChemScan® RDI result and the result obtained with direct microscopic enumeration. The Chemscan enumeration technique proved reliable, with recoveries yielding close to 100% in most cases (average 125%, range from 86 to 467%) for all the concentration/purification techniques tested. The quality of the antibodies was shown to be critical, with antibodies from some suppliers yielding recoveries a low as 10% in some cases. This difficulty could, however, be overcome by the utilization of the antibody provided by Chemunex. These data conclusively prove that laser scanning cytometry, which greatly facilitates the microscopic enumeration of Cryptosporidium oocysts from water samples and decreases the time of observation by four to six times, can be successfully applied to water concentrates prepared from a variety of concentration/purification techniques.

scholarly journals Chromosome segregation fidelity is controlled by small changes in phospho-occupancy at the kinetochore-microtubule interface

2021 ◽  
Author(s):  
Thomas J. Kucharski ◽  
Rufus Hards ◽  
Kristina M. Godek ◽  
Scott A. Gerber ◽  
Duane A. Compton
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Cyclin B1 ◽  
Quantitative Mass Spectrometry ◽  
Kinetochore Protein ◽  
Microtubule Attachment ◽  
Different Types ◽  
Narrow Dynamic Range

SummaryKinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.

Calculating Irradiance Penetration into Water Bodies from the Measured Beam Attenuation Coefficient, II: Application of the Improved Model to Different Types of Lakes

2002 ◽  
Vol 33 (2-3) ◽  
pp. 227-240 ◽  
Author(s):  
Helgi Arst ◽  
Ants Erm ◽  
Anu Reinart ◽  
Liis Sipelgas ◽  
Antti Herlevi
Keyword(s):  
Attenuation Coefficient ◽  
Water Samples ◽  
Water Bodies ◽  
Diffuse Attenuation Coefficient ◽  
Beam Attenuation ◽  
Underwater Light Climate ◽  
Different Types ◽  
Underwater Irradiance ◽  
Beam Attenuation Coefficient

The method suggested earlier for estimating the spectra of diffuse attenuation coefficient of light in the water bodies relying on the beam attenuation coefficient measured from water samples, was improved and applied to different types of lakes. Measurement data obtained in 1994-95 and 1997-98 for 18 Estonian and Finnish lakes were used. The spectra of two characteristics were available for our investigations: 1) beam attenuation coefficient estimated from water samples in the laboratory with a spectrophotometer Hitachi U1000; 2) vertical irradiance (diffuse) attenuation coefficient measured in situ with an underwater spectroradiometer LI 1800UW. A total of 70 spectra were considered. Relying on these data the parameters of our earlier model were changed. The criterion of the efficiency of the new version of our model is the coincidence of the spectra of diffuse attenuation coefficient derived from Hitachi U1000 data (Kdc) with those obtained by underwater irradiance measurements (Kdm). Correlation analysis of the model's results gave the relationship Kdm=1.0023Kdc with correlation coefficient 0.961. The respective values of mean relative difference and standard deviation were 5.4% and 0.55 m−1. This method may be useful in conditions where in situ measuring of underwater irradiance spectra cannot be performed because of weather conditions. As the measurement of the underwater radiation field is often a complicated and expensive procedure, our numerical method may be useful for estimating the underwater light climate.

scholarly journals Simultaneous Determination of Glycyrrhizin and 15 Flavonoids in Licorice and Blood by High Performance Liquid Chromatography with Ultraviolet Detector

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Yongqian Zhang ◽  
Jin Cao ◽  
Yingping Wang ◽  
Shengyuan Xiao
Keyword(s):  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Dynamic Range ◽  
High Dynamic Range ◽  
Rat Plasma ◽  
Simple Method ◽  
Ultraviolet Detector ◽  
Different Types ◽  
High Dynamic

Licorice is the most frequently used herb in traditional Chinese medicine (TCM) with versatile functions. It is also a popular natural dietary supplement. While the dosages is very important for there are some side effects caused by licorice. The composition of licorice, its products should be well determined thereof. A simple method for simultaneous determining sixteen compounds in vary high dynamic range of content has been established. This method based on the detection at the characteristic ultraviolet spectra of different types of compounds in licorice. Glycyrrhizin and fifteen flavonoids were well measured. All of these compounds can be precisely quantified at their characteristic wavelengths. This method has been successfully applied to the analyses of different licorices, Sini Tang decoction, and rat plasma after oral administration of Sini Tang decoction. These compounds were found to be over 3000 times in content (from 0.01 μg/g to 34.5 μg/g) in some samples.

scholarly journals Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness

Sensors ◽  
2019 ◽  
Vol 19 (13) ◽  
pp. 2982 ◽  
Author(s):  
Liubov A. Kost ◽  
Violetta O. Ivanova ◽  
Pavel M. Balaban ◽  
Konstantin A. Lukyanov ◽  
Evgeny S. Nikitin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Response Speed ◽  
Red Fluorescent Protein ◽  
Positive Slope ◽  
Reporter Protein ◽  
Fluorescent Indicators ◽  
Linker Length ◽  
Final Performance ◽  
Voltage Indicator

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188—a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn’t show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

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