Fatty Acyl Availability Modulates Cardiolipin Composition and Alters Mitochondrial Function in HeLa Cells

The molecular assembly of cells depends not only on their balance between anabolism and catabolism, but to a large degree also on the building blocks available in the environment. For cultivated mammalian cells, this is largely determined by the composition of the growth medium used. Here we study the impact of medium lipids on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum- and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins (CL) strongly depends on the lipid environment in cultured cells and prefers the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This suggests a link between membrane composition and respiratory capacity. In summary, we find a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. Thus, this underlines the importance of considering these factors when using and establishing cell culture models in biomedical research.

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Cell Culture in the Biological Evaluation of Dental Materials: A Review

Alternatives to Laboratory Animals ◽

10.1177/026119298501300305 ◽

1985 ◽

Vol 13 (3) ◽

pp. 194-202

Author(s):

Alf Wennberg

Keyword(s):

Cell Culture ◽

World War I ◽

Mammalian Cells ◽

Cultured Cells ◽

Dental Materials ◽

Biological Evaluation ◽

Culture Technique ◽

Early Years ◽

Systematic Research

Systematic research on dental materials began after World War I. For a long time the research was focused on the physical properties of the materials, and papers dealing with biological aspects were scarce. By the late 1950s a growing interest in biological responses to dental materials developed, and from the 1970s biological and physical evaluations were deemed equally important (1). Mammalian cells have been maintained in vitro since the early years of this century, but the use of cultured cells to evaluate the effects of chemicals and drugs is a more recent occurrence. The first practical application of this technique was in pharmacological investigations (2), but applications in other fields soon followed, and in 1955 the first studies were reported where a cell culture technique had been applied to the biological evaluation of dental materials (3,4). Since then the use of cell culture systems in dental materials research has grown rapidly. The main application has been for the assessment of cytotoxic effects, and the purpose of this paper is to review different test methods and discuss some facets of the problems posed by the cytotoxicity testing of dental materials.

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Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle Threshold Value

Frontiers in Medicine ◽

10.3389/fmed.2021.822633 ◽

2022 ◽

Vol 8 ◽

Author(s):

Li-Teh Liu ◽

Jih-Jin Tsai ◽

Chun-Hong Chen ◽

Ping-Chang Lin ◽

Ching-Yi Tsai ◽

...

Keyword(s):

Cell Culture ◽

Cultured Cells ◽

Nucleic Acid Amplification ◽

Post Inoculation ◽

Rt Pcr ◽

Isolation And Identification ◽

Spike Gene ◽

Double Deletion ◽

Blind Passage ◽

The Impact

Coronavirus disease 2019 (COVID-19) is an emerging life-threatening pulmonary disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, Hubei Province, China, in December 2019. COVID-19 develops after close contact via inhalation of respiratory droplets containing SARS-CoV-2 during talking, coughing, or sneezing by asymptomatic, presymptomatic, and symptomatic carriers. This virus evolved over time, and numerous genetic variants have been reported to have increased disease severity, mortality, and transmissibility. Variants have also developed resistance to antivirals and vaccination and can escape the immune response of humans. Reverse transcription polymerase chain reaction (RT–PCR) is the method of choice among diagnostic techniques, including nucleic acid amplification tests (NAATs), serological tests, and diagnostic imaging, such as computed tomography (CT). The limitation of RT–PCR is that it cannot distinguish fragmented RNA genomes from live transmissible viruses. Thus, SARS-CoV-2 isolation by using cell culture has been developed and makes important contributions in the field of diagnosis, development of antivirals, vaccines, and SARS-CoV-2 virology research. In this research, two SARS-CoV-2 strains were isolated from four RT–PCR-positive nasopharyngeal swabs using VERO E6 cell culture. One isolate was cultured successfully with a blind passage on day 3 post inoculation from a swab with a Ct > 35, while the cells did not develop cytopathic effects without a blind passage until day 14 post inoculation. Our results indicated that infectious SARS-CoV-2 virus particles existed, even with a Ct > 35. Cultivable viruses could provide additional consideration for releasing the patient from quarantine. The results of the whole genome sequencing and bioinformatic analysis suggested that these two isolates contain a spike 68-76del+spike 675-679del double-deletion variation. The double deletion was confirmed by amplification of the regions spanning the spike gene deletion using Sanger sequencing. Phylogenetic analysis revealed that this double-deletion variant was rare (one per million in public databases, including GenBank and GISAID). The impact of this double deletion in the spike gene on the SARS-CoV-2 virus itself as well as on cultured cells and/or humans remains to be further elucidated.

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The Use of Artemia Salina in Toxicity Testing

Alternatives to Laboratory Animals ◽

10.1177/026119299202000222 ◽

1992 ◽

Vol 20 (2) ◽

pp. 297-301 ◽

Cited By ~ 10

Author(s):

Lillemor Lewan ◽

Marianne Andersson ◽

Paloma Morales-Gomez

Keyword(s):

Cell Culture ◽

Mammalian Cells ◽

Culture Medium ◽

Cultured Cells ◽

Sea Water ◽

Toxicity Testing ◽

Artemia Salina ◽

Good Survival ◽

Cell Culture Medium ◽

A Cell

This study shows that the Artemia assay, which is usually performed by incubation for a 24-hour period in artificial sea water, can also be performed in phosphate buffered saline (PBS) at pH 7.2, or in a cell culture medium, both of which are used in toxicity assays employing mammalian cells. Thus, by using the equivalent media for incubation, toxic effects in the Artemia assay and toxic mechanisms can more accurately be compared with results obtained in mammalian cell toxicity. Survival of control animals is good for 72 hours, provided that infection can be avoided. A pH greater than 6 is essential for good survival of Artemia Salina, and a pH greater than 10.5 should be avoided. Because of the risk of infection at low saline concentrations, a decreased incubation time of 16 or 12 hours is recommended. The lethal concentrations (LC10 and LC50) in the 24-hour Artemia assay of the first ten chemicals in the MEIC programme were measured in PBS, and the results compared with those from a previous study of the effects in a 10-minute acute ATP leakage assay, specifically indicating lesions in the cell membrane. The EC10 and EC50 values for the alcohols in the Artemia assay were 35–75% of the corresponding values in the ATP leakage assay. For paracetamol and amitriptyline, the EC10 and EC50 values in the Artemia assay were 2–16% of the corresponding values in the ATP-leakage assay. The greatly increased toxicity of the two compounds in the animals may be related to systemic effects. FeSO4 was very toxic to Artemia salina at a concentration of lug/ml, but it did not cause leakage of ATP from cultured cells, even at a concentration of 8,000μg/ml, showing that widely different mechanisms of interaction with FeSO4 are measured by the two assays. A lower toxicity of polygodial, a sesquiterpenoid unsaturated dialdehyde, in cell culture medium, was obvious in the Artemia assay. Thus, some factors in cell culture medium must, by interacting with the toxic molecule, protect the animals against the toxicity of polygodial, as was previously found in cultured cells.

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A combination of electrochemistry and mass spectrometry to monitor the interaction of reactive species with supported lipid bilayers

Scientific Reports ◽

10.1038/s41598-020-75514-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

M. Ravandeh ◽

H. Kahlert ◽

H. Jablonowski ◽

J.-W. Lackmann ◽

J. Striesow ◽

...

Keyword(s):

Mass Spectrometry ◽

Lipid Bilayers ◽

Mammalian Cells ◽

Reactive Species ◽

Oxidation Products ◽

Supported Lipid Bilayers ◽

Resonance Spectroscopy ◽

Membrane Composition ◽

Physical Plasma ◽

The Impact

Abstract Reactive oxygen and nitrogen species (RONS), e.g. generated by cold physical plasma (CPP) or photodynamic therapy, interfere with redox signaling pathways of mammalian cells, inducing downstream consequences spanning from migratory impairment to apoptotic cell death. However, the more austere impact of RONS on cancer cells remains yet to be clarified. In the present study, a combination of electrochemistry and high-resolution mass spectrometry was developed to investigate the resilience of solid-supported lipid bilayers towards plasma-derived reactive species in dependence of their composition. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was undisturbed by 200 µM H2O2 (control) but showed full permeability after CPP treatment and space-occupying oxidation products such as PoxnoPC, PAzePC, and POPC hydroperoxide were found. Electron paramagnetic resonance spectroscopy demonstrated the presence of hydroxyl radicals and superoxide anion/hydroperoxyl radicals during the treatment. In contrast, small amounts of the intramembrane antioxidant coenzyme Q10 protected the bilayer to 50% and LysoPC was the only POPC derivative found, confirming the membrane protective effect of Q10. Such, the lipid membrane composition including the presence of antioxidants determines the impact of pro-oxidant signals. Given the differences in membrane composition of cancer and healthy cells, this supports the application of cold physical plasma for cancer treatment. In addition, the developed model using the combination of electrochemistry and mass spectrometry could be a promising method to study the effect of reactive species or mixes thereof generated by chemical or physical sources.

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Ultrastructural Alterations in Hela Cells Induced by Spider Venom

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060118 ◽

1967 ◽

Vol 25 ◽

pp. 20-21

Author(s):

Dale E. McClendon ◽

Paul N. Morgan ◽

Bernard L. Soloff

Keyword(s):

Growth Medium ◽

Mammalian Cells ◽

Carcinoma Cells ◽

Venom Protein ◽

Sterile Saline ◽

Carbon Coated ◽

Available Information ◽

Loxosceles Reclusa ◽

Essential Growth ◽

Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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Constructing well-being from hedonic building blocks: Evidence that depression distorts the impact of daily pleasure and pain

PsycEXTRA Dataset ◽

10.1037/e529412014-209 ◽

2005 ◽

Author(s):

Michael F. Steger ◽

Todd B. Kashdan ◽

Shigehiro Oishi

Keyword(s):

Well Being ◽

Building Blocks ◽

The Impact

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IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

Acta Endocrinologica ◽

10.1530/acta.0.068s027 ◽

1971 ◽

Vol 68 (1_Suppl) ◽

pp. S27-S40 ◽

Cited By ~ 1

Author(s):

T. Kobayashi ◽

T. Kigawa ◽

M. Mizuno ◽

T. Watanabe

Keyword(s):

Cell Culture ◽

Organ Culture ◽

Cultured Cells ◽

Cell Sheet ◽

Short Term ◽

Hypothalamic Extract ◽

Numerous Cell ◽

Single Cell Culture ◽

Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

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Mechanically-Tunable Quantum Interference in Ferrocene-Based Single-Molecule Junctions

10.26434/chemrxiv.12252059 ◽

2020 ◽

Author(s):

María Camarasa-Gómez ◽

Daniel Hernangómez-Pérez ◽

Michael S. Inkpen ◽

Giacomo Lovat ◽

E-Dean Fung ◽

...

Keyword(s):

Single Molecule ◽

Quantum Interference ◽

Degrees Of Freedom ◽

Building Blocks ◽

Ferrocene Derivative ◽

Single Molecule Junctions ◽

Molecule Junction ◽

Single Molecule Junction ◽

The Impact ◽

D Orbitals

Ferrocenes are ubiquitous organometallic building blocks that comprise a Fe atom sandwiched between two cyclopentadienyl (Cp) rings that rotate freely at room temperature. Of widespread interest in fundamental studies and real-world applications, they have also attracted some interest as functional elements of molecular-scale devices. Here we investigate the impact of the configurational degrees of freedom of a ferrocene derivative on its single-molecule junction conductance. Measurements indicate that the conductance of the ferrocene derivative, which is suppressed by two orders of magnitude as compared to a fully conjugated analog, can be modulated by altering the junction configuration. Ab initio transport calculations show that the low conductance is a consequence of destructive quantum interference effects that arise from the hybridization of metal-based d-orbitals and the ligand-based π-system. By rotating the Cp rings, the hybridization, and thus the quantum interference, can be mechanically controlled, resulting in a conductance modulation that is seen experimentally.

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Model Pembelajaran Teams Games Tournaments (TGT) untuk Meningkatkan Hasil Belajar Siswa

PHILANTHROPY Journal of Psychology ◽

10.26623/philanthropy.v3i2.1622 ◽

2019 ◽

Vol 3 (2) ◽

pp. 110

Author(s):

Suwarno Suwarno

Keyword(s):

Social Capital ◽

Academic Success ◽

Service Learning ◽

Learning Outcomes ◽

School Effectiveness ◽

Social Trust ◽

Building Blocks ◽

Learning Model ◽

Committee Report ◽

The Impact

Abstract. This study aims to determine the effectiveness of the Teams Games Tournament (TGT) learning model to improve student learning outcomes. This research is important because the lecture learning model makes learning meaningless so it impacts on low learning outcomes. This research uses quasi experiment using control class and experimental class. Respondents in this study were students of class X SMK 8 Semarang Academic Year 2017/2018. Sample selection using random sampling, class X1 is used as a control class and X2 is an experimental class. The experimental class was given an intervention by learning Teams Games Tournaments (TGT), while the control class used lecture learning. The effectiveness of the model was measured by the student test analysis method. then analyzed by completeness test and average difference test. The findings of this study are the learning outcomes of experimental class students achieving better learning outcomes than classes using the lecture method.Keywords: Learning Model, Teams Games Tournaments (TGT). Students Daftar Pustaka Bofota, Y. B., & Bofota, Y. B. (2017). The impact of social capital on children educational outcomes : the case of Tanzania The impact of social capital on children educational outcomes : The case of Tanzania.Cahuc, P., Shleifer, A., & Algan, Y. (2014). Teaching Practices and Social Capital. (6052).Catts, R., & Ozga, J. (2015). What is Social Capital and how might it be used in Scotland ’ s Schools ? (36).Flint, N. (2017). Full report Schools , communities and social capital : building blocks in the ’ Big Society ’ Contents.Goddard, R. D. (2016). Relational Networks , Social Trust , and Norms : A Social Capital Perspective on Students ’ Chances of Academic Success. 25(1), 59–74.Eddy Prasongko, 2017. Team Game Tournament. Bandung. Jawa BaratEndang Poerwanti, dkk. 2008. Asesmen Pembelajaran SD. Jakarta: Direktorat Jendral Pendidikan Tinggi Departemen Pendidikan NasionalHargreaves, A. (2015). School Social Capital and School Effectiveness. 37, 119–136.Kurnia, Inggridwati. dkk. 2018. Perkembangan belajar peserta didik. Jakarta: Direktorat Jendral Pendidikan Tinggi Departemen Pendidikan NasionalPurwanto. M Ngalim. 2015. Psikologi Pendidikan. Bandung: PT Remaja RosdakaryaSiddiq, M. Djauhar. 2018. Pengembangan Bahan Pembelajaran SD. Jakarta: Direktorat Jendral Pendidikan Tinggi Departemen Pendidikan Nasional.Sugiyono, 2005. Metode Penelitian Kuantitatif, Kualitatif dan Rn D, Bandung AftabetaLash, D., & Belfiore, G. (2017). 5 Essentials in Building Social Capital Report 4 of the MyWays Student Success Series. (October).Mikiewicz, P., Jonasson, J. T., Gudmundsson, G., Blondal, K. S., & Korczewska, D. M. (2011). Comparative research between Poland and Iceland.Schlesselman, L., Borrego, M., Bloom, T. J., Mehta, B., Drobitch, R. K., & Smith, T. (2015). An Assessment Of Service-Learning In 34 US Schools Of Pharmacy Follow Up On The 2001 Professional Affairs Committee Report. American Journal of Pharmaceutical Education, 79(8). https://doi.org/10.5688/ajpe798116

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Development of Strategies for Glycopeptide Synthesis: An Overview on the Glycosidic Linkage

Current Organic Chemistry ◽

10.2174/1385272824999200701121037 ◽

2020 ◽

Vol 24 (21) ◽

pp. 2475-2497

Author(s):

Andrea Verónica Rodríguez-Mayor ◽

German Jesid Peralta-Camacho ◽

Karen Johanna Cárdenas-Martínez ◽

Javier Eduardo García-Castañeda

Keyword(s):

Chemical Synthesis ◽

Solid Phase ◽

Building Blocks ◽

Heterogeneous Environments ◽

Phase Synthesis ◽

Low Concentrations ◽

Biological Sources ◽

Synthetic Routes ◽

The Impact ◽

Potential Use

Glycoproteins and glycopeptides are an interesting focus of research, because of their potential use as therapeutic agents, since they are related to carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions, which are commonly involved in biological processes. It has been established that natural glycoconjugates could be an important source of templates for the design and development of molecules with therapeutic applications. However, isolating large quantities of glycoconjugates from biological sources with the required purity is extremely complex, because these molecules are found in heterogeneous environments and in very low concentrations. As an alternative to solving this problem, the chemical synthesis of glycoconjugates has been developed. In this context, several methods for the synthesis of glycopeptides in solution and/or solid-phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for both solution and solid-phase synthesis. The synthetic viability of glycoconjugates is a critical parameter for allowing their use as drugs to mitigate the impact of microbial resistance and/or cancer. However, the chemical synthesis of glycoconjugates is a challenge, because these molecules possess multiple reaction sites and have a very specific stereochemistry. Therefore, it is necessary to design and implement synthetic routes, which may involve various protection schemes but can be stereoselective, environmentally friendly, and high-yielding. This review focuses on glycopeptide synthesis by recapitulating the progress made over the last 15 years.

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