scholarly journals Flow Cytometric Analysis of Bacterial Protein Synthesis: Monitoring Vitality After Water Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Mathilde Lindivat ◽  
Gunnar Bratbak ◽  
Aud Larsen ◽  
Ole-Kristian Hess-Erga ◽  
Ingunn Alne Hoell
Keyword(s):  
Heat Treatment ◽  
Protein Synthesis ◽  
Uv Irradiation ◽  
Single Cells ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Residual Activity ◽  
Water Disinfection ◽  
Dna Integrity ◽  
Bacterial Strains

Bacterial vitality after water disinfection treatment was investigated using bio-orthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM). Protein synthesis activity and DNA integrity (BONCAT–SYBR Green) was monitored in Escherichia coli monocultures and in natural marine samples after UV irradiation (from 25 to 200 mJ/cm2) and heat treatment (from 15 to 45 min at 55°C). UV irradiation of E. coli caused DNA degradation followed by the decrease in protein synthesis within a period of 24 h. Heat treatment affected both DNA integrity and protein synthesis immediately, with an increased effect over time. Results from the BONCAT method were compared with results from well-known methods such as plate counts (focusing on growth) and LIVE/DEAD™ BacLight™ (focusing on membrane permeability). The methods differed somewhat with respect to vitality levels detected in bacteria after the treatments, but the results were complementary and revealed that cells maintained metabolic activity and membrane integrity despite loss of cell division. Similarly, analysis of protein synthesis in marine bacteria with BONCAT displayed residual activity despite inability to grow or reproduce. Background controls (time zero blanks) prepared using different fixatives (formaldehyde, isopropanol, and acetic acid) and several different bacterial strains revealed that the BONCAT protocol still resulted in labeled, i.e., apparently active, cells. The reason for this is unclear and needs further investigation to be understood. Our results show that BONCAT and FCM can detect, enumerate, and differentiate bacterial cells after physical water treatments such as UV irradiation and heating. The method is reliable to enumerate and explore vitality of single cells, and a great advantage with BONCAT is that all proteins synthesized within cells are analyzed, compared to assays targeting specific elements such as enzyme activity.

scholarly journals Deep-Ultraviolet Transparent Conductive MWCNT/SiO2 Composite Thin Film Fabricated by UV Irradiation at Ambient Temperature onto Spin-Coated Molecular Precursor Film

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1348
Author(s):  
Hiroki Nagai ◽  
Naoki Ogawa ◽  
Mitsunobu Sato
Keyword(s):  
Thin Film ◽  
Thin Films ◽  
Heat Treatment ◽  
Uv Irradiation ◽  
Composite Thin Film ◽  
Precursor Film ◽  
Mixed Solution ◽  
Pencil Hardness ◽  
Strong Base ◽  
Deep Ultraviolet

Deep-ultraviolet (DUV) light-transparent conductive composite thin films, consisting of dispersed multiwalled carbon nanotubes (MWCNTs) and SiO2 matrix composites, were fabricated on a quartz glass substrate. Transparent and well-adhered amorphous thin films, with a thickness of 220 nm, were obtained by weak ultraviolet (UV) irradiation (4 mW cm−2 at 254 nm) for more than 6 h at 20−40 °C onto the precursor films, which were obtained by spin coating with a mixed solution of MWCNT in water and Si(IV) complex in ethanol. The electrical resistivity of MWCNT/SiO2 composite thin film is 0.7 Ω·cm, and transmittance in the wavelength region from DUV to visible light is higher than 80%. The MWCNT/SiO2 composite thin film showed scratch resistance at pencil hardness of 8H. Importantly, the resistivity of the MWCNT/SiO2 composite thin film was maintained at the original level even after heat treatment at 500 °C for 1 h. It was observed that the heat treatment of the composite thin film improved durability against both aqueous solutions involving a strong acid (HCl) and a strong base (NaOH).

scholarly journals Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells

2007 ◽  
Vol 97 (2) ◽  
pp. 321-328 ◽  
Author(s):  
S. Aisling Aherne ◽  
Joseph P. Kerry ◽  
Nora M. O'Brien
Keyword(s):  
Dna Damage ◽  
Plant Extract ◽  
Plant Extracts ◽  
Antioxidant Status ◽  
Cell Injury ◽  
Membrane Integrity ◽  
Neutral Red ◽  
Dna Integrity ◽  
Cell Membrane Integrity ◽  
Wide Range

Experimental evidence suggests that most herbs and spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage. The objectives of the present study were: first, to determine the effects of plant extracts on the viability, membrane integrity, antioxidant status and DNA integrity of Caco-2 cells and second, to investigate the cytoprotective and genoprotective effects of these plant extracts against oxidative stress in Caco-2 cells. The plant extracts examined were rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), sage (Salvia officinalis L.) and echinacea (Echinacea purpurea L.). Cell membrane integrity was assessed by the lactate dehydrogenase release assay. Viability was determined by the neutral red uptake assay (NRUA) and the concentration of compound that resulted in 50 % cell death (IC50) was calculated. Antioxidant status of the cells was assessed by measuring GSH content, catalase activity and superoxide dismutase activity. To examine their cytoprotective and genoprotective effects, Caco-2 cells were pre-treated with each plant extract for 24 h followed by exposure to H2O2. DNA damage was assessed by the comet assay and cell injury was determined by the NRUA. Rosemary was the most toxic (IC50 123 μg/ml) and echinacea the least toxic (IC50 1421 μg/ml). Sage was the only plant extract to affect the antioxidant status of the cells by increasing GSH content. Sage, oregano and rosemary protected against H2O2-induced DNA damage (olive tail moment and percentage tail DNA), whereas protection against H2O2-induced cytotoxicity was afforded by sage only.

scholarly journals Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

2017 ◽  
Vol 29 (8) ◽  
pp. 1556 ◽  
Author(s):  
S. Morrow ◽  
J. Gosálvez ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
W. V. Holt ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Subsequent Development ◽  
Xenopus Tropicalis ◽  
Model Organisms ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Cryopreservation ◽  
Sperm Plasma Membrane

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P < 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

scholarly journals Cytotoxic Potential of the Novel Horseshoe Crab Peptide Polyphemusin III

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 466 ◽  
Author(s):  
Mariana Marggraf ◽  
Pavel Panteleev ◽  
Anna Emelianova ◽  
Maxim Sorokin ◽  
Ilia Bolosov ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Horseshoe Crab ◽  
Inhibitory Concentration ◽  
Human Cancer ◽  
Membrane Integrity ◽  
Antimicrobial Effect ◽  
Human Leukemia ◽  
Bacterial Strains ◽  
Trypan Blue Exclusion

Biological activity of the new antimicrobial peptide polyphemusin III from the horseshoe crab Limulus polyphemus was examined against bacterial strains and human cancer, transformed, and normal cell cultures. Polyphemusin III has the amino acid sequence RRGCFRVCYRGFCFQRCR and is homologous to other β-hairpin peptides from the horseshoe crab. Antimicrobial activity of the peptide was evaluated and MIC (minimal inhibitory concentration) values were determined. IC50 (half-maximal inhibitory concentration) values measured toward human cells revealed that polyphemusin III showed a potent cytotoxic activity at concentrations of <10 μM. Polyphemusin III caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60, which was measured with trypan blue exclusion assay and lactate dehydrogenase-release assay. Flow cytometry experiments for annexin V-FITC/ propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antimicrobial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.

scholarly journals The Effect of Glutathione on The Quality of Aceh Local Catfish (Clarias gariepinus) Spermatozoa After Cryopreservation

2020 ◽  
Vol 12 (1) ◽  
pp. 125-131
Author(s):  
Raudhah Mahfudhah ◽  
Kartini Eriani ◽  
Zainal Abidin Muchlisin ◽  
Cut Ruhul Muthmainnah
Keyword(s):  
Plasma Membrane ◽  
Clarias Gariepinus ◽  
Membrane Integrity ◽  
Dna Integrity ◽  
Freezing And Thawing ◽  
Randomized Design ◽  
Fetal Bovine ◽  
Fresh Semen ◽  
Different Doses

The cryopreservation process might reduce the quality of spermatozoa due to an increase in the production of reactive oxygen species (ROS) compounds during cooling, freezing, and thawing. The quality of spermatozoa can be maintained by adding glutathione as an exogenous antioxidant into cryo-diluent. This study aimed to examine the effect of the addition of different doses of glutathione in cryopreservation of Aceh Local catfish (Clarias gariepinus) spermatozoa after freezing. The method used was a completely randomized design (CRD) with four treatments and four replications. Fresh semen was diluted in Ringer, 15% DMSO, and 20% Fetal Bovine Serum (FBS) and then added with glutathione antioxidants of 0.0 mgL-1, 0.5 mgL-1, 1.0 mgL-1, and 2.0 mgL-1. The parameters observed in this study were motility, integrity of the plasma membrane, fertility, and DNA integrity. The results showed that the concentration of glutathione had no effect on motility, integrity ofthe plasma membrane, or fertility, but had an effect on DNA integrity. The highest percentage of motility and plasma membrane integrity respectively was 40.50% (P3) and 70.87% (P2). Furthermore, the assessment of DNA integrity showed that there was no DNA fragmentation both treatments and fresh spermatozoa. This research is the first study regarding glutathione supplementation in cryo-diluent of Aceh Local catfish spermatozoa. Finally, the results obtained can provide information about the exact concentration of glutathione in the extender on the quality of spermatozoa of Aceh Local catfish (C. gariepinus) after the cryopreservation process. These results can also increase the success of fertility be used by the seed hall unit and the aquaculture industry to increase the productivity and supply high quality seeds.

scholarly journals A tunable multicellular timer in bacterial consortia

2020 ◽  
Author(s):  
Carlos Toscano-Ochoa ◽  
Jordi Garcia-Ojalvo
Keyword(s):  
Collective Behavior ◽  
Bacterial Population ◽  
Single Cells ◽  
Bacterial Strains ◽  
Intracellular Space ◽  
Bacterial Consortia ◽  
Robust Implementation ◽  
Time Encoding ◽  
Stationary Conditions ◽  
Molecular Components

Processing time-dependent information requires cells to quantify the durations of past regulatory events and program the time span of future signals. Such timer mechanisms are difficult to implement at the level of single cells, however, due to saturation in molecular components and stochasticity in the limited intracellular space. Multicellular implementations, on the other hand, outsource some of the components of information-processing circuits to the extracellular space, and thereby might escape those constraints. Here we develop a theoretical framework, based on a trilinear coordinate representation, to study the collective behavior of a three-strain bacterial population under stationary conditions. This framework reveals that distributing different processes (in our case the production, detection and degradation of a time-encoding signal) across distinct bacterial strains enables the robust implementation of a multicellular timer. Our analysis also shows the circuit to be easily tunable by varying the relative frequencies of the bacterial strains composing the consortium.

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