Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase Staphylococcus aureus Populations

ABSTRACT Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC+ conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC− conditions) for prolonged culture times (up to 8 days). VLEC+-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC+ conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC+ conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.

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Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

GSC Biological and Pharmaceutical Sciences ◽

10.30574/gscbps.2021.16.2.0221 ◽

2021 ◽

Vol 16 (2) ◽

pp. 001-013

Author(s):

Abwe Mercy Ngone ◽

Lawrence Monah Ndam ◽

Rita Mungfu Njilar ◽

Doungous Oumar ◽

Thomas Eku Njock

Keyword(s):

Culture Medium ◽

Culture Media ◽

Fungal Growth ◽

Growth Media ◽

Fungal Contamination ◽

Surface Sterilization ◽

Pure Cultures ◽

Nodal Cuttings ◽

Baseline Information

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

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Amino acid depletion and appearance during porcine preimplantation embryo development in vitro

Reproduction ◽

10.1530/rep.1.00727 ◽

2005 ◽

Vol 130 (5) ◽

pp. 655-668 ◽

Cited By ~ 37

Author(s):

Paul J Booth ◽

Peter G Humpherson ◽

Terry J Watson ◽

Henry J Leese

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Embryo Development ◽

Preimplantation Embryos ◽

Type I ◽

Preimplantation Embryo Development ◽

Type Iv ◽

Stage Of Development ◽

Amino Acid Depletion

Preimplantation embryos can consume and produce amino acids in a manner dependent upon the stage of development that may be predictive of subsequent viability. In order to examine these relationships in the pig, patterns of net depletion and appearance of amino acids byin vitroproduced porcine preimplantation embryos were examined. Cumulus oocyte complexes derived from slaughterhouse pre-pubertal pig ovaries were matured for 40 h in defined TCM-199 medium (containing PVA) before being fertilised (Day 0) with frozen-thawed semen in Tris–based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20, in NCSU-23 medium modified to contain 0.1 mM glutamine plus a mixture of 19 amino acids (aa) at low concentrations (0.02–0.11 mM) (NCSU-23aa). Groups of 2–20 embryos were removed (dependent on stage) on Day 0 (1 cell), Day 1 (two- and four-cells), Day 4 (compact morulae) and Day 6 (blastocysts) and placed in 4 μl NCSU-23aafor 24 h. After incubation, the embryos were removed and the spent media was analysed by HPLC. The net rate of amino acid depletion or appearance varied according to amino acid (P< 0.001) and, apart from serine and histidine, stage of development (P< 0.014). Glycine, isoleucine, valine, phenylalanine, tryptophan, methionine, asparagine, lysine, glutamate and aspartate consistently appeared, whereas threonine, glutamine and arginine were consistently depleted. Five types of stage-dependent trends could be observed: Type I: amino acids having high rates of net appearance on Day 0 that reached a nadir on Day 1 or 4 but subsequently increased by Day 6 (glycine, glutamate); Type II: those that exhibited lower rates of net appearance on Days 0 and 6 compared with the intermediate Days 1 and 4 (isoleucine, valine, phenylalanine, methionine, arginine); Type III: amino acids which showed a continuous fall in net appearance (asparagine, aspartate); Type IV: those that exhibited a steady fall in net depletion from Day 0 to Day 6 (glutamine, threonine); Type V: those following no discernable trend. Analysis of further embryo types indicated that presumptive polyspermic embryos on Day 0 had increased (P< 0.05) net rates of leucine, isoleucine, valine and glutamate appearance, and reduced (P< 0.05) net rates of threonine and glutamine depletion compared with normally inseminated oocytes. These data suggest that the net rates of depletion and uptake of amino acids by pig embryos vary between a) amino acids, b) the day of embryo development and, c) the type of embryos present at a given stage of development. The results also suggested that the net depletion and appearance rates of amino acids by early pig embryos might be more similar to those of the human than those of the mouse and cow.

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In vitro culture and non-invasive metabolic profiling of single bovine embryos

Reproduction Fertility and Development ◽

10.1071/rd17446 ◽

2019 ◽

Vol 31 (2) ◽

pp. 306

Author(s):

Monika Nõmm ◽

Rando Porosk ◽

Pille Pärn ◽

Kalle Kilk ◽

Ursel Soomets ◽

...

Keyword(s):

Molecular Weight ◽

Metabolic Profiling ◽

Culture Medium ◽

Culture Media ◽

Blastocyst Stage ◽

Growth Media ◽

Low Molecular Weight ◽

Bovine Embryos ◽

Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilized oocytes

Reproduction Fertility and Development ◽

10.1071/r98052 ◽

1998 ◽

Vol 10 (3) ◽

pp. 279 ◽

Cited By ~ 10

Author(s):

Y. G. Jung ◽

T. Sakata ◽

E. S. Lee ◽

Y. Fukui

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Metabolism ◽

Culture Medium ◽

Acid Metabolism ◽

Essential Amino Acids ◽

Fluid Medium ◽

Vitro Fertilization ◽

Oviduct Fluid

The uptake and synthesis of 19 amino acids by fresh or frozen–thawed bovine blastocysts produced by parthenogenesis (PT) or in vitro fertilization (IVF) were compared in the present study. Fresh blastocysts, 180 h after IVF or PT activation, and frozen–thawed blastocysts, 168 h old and cultured for 12 h post-thawing, were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA, respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2: M2). In Experiment 1, when fresh or frozen–thawed PT blastocysts were cultured in M1, the uptake of glutamate (in fresh only), aspartate and arginine, and the synthesis of glutamine and alanine were significantly enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine, glycine, arginine and alanine were significantly taken up. It was found that the glutamine concentrations was significantly higher (P < 0.001) in the culture medium drops containing embryos than in the drops without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the uptake of aspartate and synthesis of alanine were greater (P < 0.01) than those by IVF blastocysts. When M2 was used, a significant (P < 0.01) production of serine, asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured in M1, fresh blastocysts depleted more aspartate and glutamate, and produced more glutamine and alanine than frozen–thawed blastocysts. When cultured in M2, frozen–thawed blastocysts depleted more threonine (P < 0.01) than fresh blastocysts. These results indicate that the uptake and synthesis of amino acids were different in fresh or frozen–thawed bovine blastocysts derived from PT or IVF. These differences in amino acid metabolism may be related to the viability of the blastocysts.

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Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

Reproduction Fertility and Development ◽

10.1071/rd9960835 ◽

1996 ◽

Vol 8 (5) ◽

pp. 835 ◽

Cited By ~ 28

Author(s):

T Pinyopummintr ◽

BD Bavister

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Embryo Development ◽

Culture Medium ◽

Essential Amino Acids ◽

Bovine Embryo ◽

Bovine Embryos ◽

Development In Vitro ◽

Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

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Amino Acid Metabolism in Human Embryos

Physiological Research ◽

10.33549/physiolres.933240 ◽

2016 ◽

pp. 823-832 ◽

Cited By ~ 3

Author(s):

P. DRÁBKOVÁ ◽

L. ANDRLOVÁ ◽

R. HAMPL ◽

R. KANĎÁR

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Metabolism ◽

Culture Medium ◽

Acid Metabolism ◽

Culture Media ◽

Time Lapse ◽

Human Embryos ◽

Strongly Correlated ◽

Cell Stage

The aim of this study was to find some relationship between amino acid metabolism and the embryo morphokinetic parameters studied via time-lapse analysis. Study included 48 human embryo samples and their culture media. Two groups of embryos were identified: embryos reached the 8-cell stage on day 3 (n=34) and embryos failed to develop at any point during the incubation (n=14). Amino acids levels were measured on day 3 of embryo development; using time-lapse analysis, the precise timing of embryo cleavage, synchrony of division, grade of fragmentation etc. were established. No statistically significant differences between dividing and arresting embryos were observed in terms of amino acids production/consumption and turnover. Amino acids which were part of the culture medium did not exhibit any statistically significant correlation with kinetic parameters with the exception of the grade of fragmentation on day 3; there were negative correlation with glutamate, and positive with glutamine, glycine and taurine. In some dividing and in some arresting embryos appeared new amino acids which strongly correlated with each other, with methionine, but not with any other amino acid that is a regular part of the culture medium.

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Pre-formulation and systematic evaluation of amino acid assisted permeability of insulin across in vitro buccal cell layers

Scientific Reports ◽

10.1038/srep32498 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 11

Author(s):

Affiong Iyire ◽

Maryam Alaayedi ◽

Afzal R. Mohammed

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Glutamic Acid ◽

Ion Pairs ◽

Sodium Deoxycholate ◽

Confocal Imaging ◽

Systematic Evaluation ◽

Cell Integrity ◽

Insulin Transport

Abstract The aim of this work was to investigate alternative safe and effective permeation enhancers for buccal peptide delivery. Basic amino acids improved insulin solubility in water while 200 and 400 μg/mL lysine significantly increased insulin solubility in HBSS. Permeability data showed a significant improvement in insulin permeation especially for 10 μg/mL of lysine (p < 0.05) and 10 μg/mL histidine (p < 0.001), 100 μg/mL of glutamic acid (p < 0.05) and 200 μg/mL of glutamic acid and aspartic acid (p < 0.001) without affecting cell integrity; in contrast to sodium deoxycholate which enhanced insulin permeability but was toxic to the cells. It was hypothesized that both amino acids and insulin were ionised at buccal cavity pH and able to form stable ion pairs which penetrated the cells as one entity; while possibly triggering amino acid nutrient transporters on cell surfaces. Evidence of these transport mechanisms was seen with reduction of insulin transport at suboptimal temperatures as well as with basal-to-apical vectoral transport, and confocal imaging of transcellular insulin transport. These results obtained for insulin are the first indication of a possible amino acid mediated transport of insulin via formation of insulin-amino acid neutral complexes by the ion pairing mechanism.

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Callus induction and plant regeneration of two Cuban rice cultivars using different seed explants and amino acid supplements

Annals of Tropical Research ◽

10.32945/atr3121.2009 ◽

2009 ◽

pp. 1-15

Author(s):

Maylin Pérez-Bernal ◽

Magalis Delgado Rigo ◽

Carlos Alberto Hernández Díaz ◽

María Teresa Barceló Ávila ◽

Raúl Armas Ramos

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Culture Medium ◽

Callus Formation ◽

Callus Cultures ◽

Rice Cultivars ◽

Embryogenic Calli ◽

Rice Callus

Most of Cuban rice cultivars are classified into indica subspecies, and they are inclined to poor in vitro response. In this paper we studied the role of endosperm and amino acids on callus formation of two Cuban rice cultivars: J-104 and IACuba-28. Callus cultures were initiated from three treatments for mature seed: intact seed, embryo with scutellum but without endosperm, and endosperm alone. It demonstrated the direct incidence of endosperm on in vitro seed contamination. But the higher percentage of embryogenic calli was obtained from intact seeds, despite of 12.94 % of seed contamination. Callus formation from endosperm alone did not occur. The role of endosperm to successful callus formation from scutellum was discussed. Effect of amino acids on rice callus growth from intact seeds was examined by supplying callus formation medium with glutamine and proline, separately or in combination, in both cultivars. Callus formation of J-104 was improved considerably with 500 mg/l of proline and glutamine in the culture medium, but in IACuba-28 were not observed significant changes. The percentage of embryogenic callus and the increase of fresh weight of calli were correlated with genotype and amino acid supplement in culture medium.

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Competitive Inhibition of Amino Acid Uptake Suppresses Chlamydial Growth: Involvement of the Chlamydial Amino Acid Transporter BrnQ

Journal of Bacteriology ◽

10.1128/jb.01240-07 ◽

2007 ◽

Vol 190 (5) ◽

pp. 1822-1830 ◽

Cited By ~ 28

Author(s):

Peter R. Braun ◽

Hesham Al-Younes ◽

Joscha Gussmann ◽

Jeannette Klein ◽

Erwin Schneider ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Growth Inhibition ◽

Competitive Inhibition ◽

Culture Media ◽

Functional Characterization ◽

Mammalian Target Of Rapamycin ◽

Signal Transduction Pathway ◽

Acid Uptake

ABSTRACT Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the mTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydial transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation.

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Comprehensive analysis of whole genome methylation in mouse blastocysts cultured with four different constituents following in vitro fertilization

Middle East Fertility Society Journal ◽

10.1186/s43043-019-0012-z ◽

2019 ◽

Vol 24 (1) ◽

Author(s):

Yu Horibe ◽

Kazuhiko Nakabayashi ◽

Miyuki Arai ◽

Kohji Okamura ◽

Kazunori Hashimoto ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mouse Model ◽

Culture Medium ◽

Bisulfite Sequencing ◽

Culture Media ◽

Essential Amino Acids ◽

Imprinted Genes ◽

Reduced Representation ◽

Reduced Representation Bisulfite Sequencing

Abstract Background With the development of assisted reproductive technology (ART), diseases believed to be caused by ART have begun to be identified as imprinted disease. However, no conclusion has been reached. So we sought to determine whether ART procedures disturb gene methylation and whether imprinted genes alone are selectively disturbed. To examine whether the constituents of the culture medium contribute to the changes in methylation, we used a mouse model to conduct IVF and comprehensively analyzed 5′–C–phosphate–G–3′ (CpG) by reduced representation bisulfite sequencing (RRBS) using a second-generation sequencer to determine changes in methylation using four types of culture media with different amino acid constituents. Results We cultured ova to the blastocyst stage in a mouse model in culture media with four different amino acid constituents. Each culture medium included (1) KSOM culture medium (NoAA), (2) KSOM media + essential amino acids (EAAs), (3) KSOM medium + non-essential amino acids (NEAAs), or (4) KSOM medium + EAAs + NEAAs (AllAA) analyzed by reduced representation bisulfite sequencing. The results showed that (1) there were many regions that maintained hypermethylation with NEAAs, (2) there was little effect of demethylation on reprogramming in the 5′UTR and promoter regions, and (3) specific changes were observed in imprinted genes such as Nnat and Nespas. Conclusions Compared with EAAs, NEAAs could protect genes from demethylation caused by reprogramming. On the imprinted genes, methylation of the promoter region of H19 was decreased by NEAAs, suggesting that specific genes were prone to changes in methylation. It was suggested that these changes could provide similar results in humans. Further studies are needed to understand how changes in methylation may affect gene expression profiles.

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