scholarly journals Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro

2020 ◽  
Vol 21 (15) ◽  
pp. 5326 ◽  
Author(s):  
Charles Banliat ◽  
Daniel Le Bourhis ◽  
Ophélie Bernardi ◽  
Daniel Tomas ◽  
Valérie Labas ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Intact Cell ◽  
Phospholipid Composition ◽  
Cell Number ◽  
Phospholipid Content ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Before And After ◽  
Oviduct Fluid

Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization—Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

O-glycans on human high endothelial CD34 putatively participating in L-selectin recognition

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2609-2611 ◽  
Author(s):  
Tero Satomaa ◽  
Ossi Renkonen ◽  
Jari Helin ◽  
Juha Kirveskari ◽  
Antti Mäkitie ◽  
...  
Keyword(s):  
Laser Desorption ◽  
Sialyl Lewis X ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Sialyl Lewis ◽  
Candidate Structure ◽  
Selectin Ligands ◽  
Matrix Assisted Laser

Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands. We report here on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of N- and O-linked oligosaccharide fractions from human tonsillar endothelial CD34. All detected O-glycans were sialylated; some were also monosulfated or monosulfated and monofucosylated. If a given CD34-glycan may carry all requirements for L-selectin recognition, that is, both 6-sulfo-sLex and MECA-79 epitopes, only one O-glycan fraction, O-9, SA2Hex3HexNAc3- Fuc1(SO3)1, meets the criteria. A candidate structure is SAα2-3Galβ1-4(Fucα1-3)(6-sulfo)GlcNAcβ1-3Galβ1-3(SAα2-3Galβ1-4GlcNAcβ1-6)GalNAc. However, if sulfo sLex glycans are supplemented with separate sulfated, nonfucosylated O-glycans, saccharides in O-6, O-8, or O-9, putatively carrying MECA-79 epitopes, could form multiglycan binding epitopes for L-selectin.

scholarly journals Identification of a Protein That Inactivates the Competence-Stimulating Peptide of Streptococcus pneumoniae

2002 ◽  
Vol 184 (2) ◽  
pp. 610-613 ◽  
Author(s):  
Mathieu Bergé ◽  
Hanno Langen ◽  
Jean-Pierre Claverys ◽  
Bernard Martin
Keyword(s):  
Streptococcus Pneumoniae ◽  
Laser Desorption ◽  
Physiological Property ◽  
Laser Desorption Ionization ◽  
Treatment Results ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Licl Treatment

ABSTRACT Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.

scholarly journals In Vitro Susceptibility of Fusarium to Isavuconazole

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
A. Broutin ◽  
J. Bigot ◽  
Y. Senghor ◽  
A. Moreno-Sabater ◽  
J. Guitard ◽  
...  
Keyword(s):  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
In Vitro Susceptibility ◽  
Ionization Time ◽  
Valuable Alternative ◽  
Flight Mass Spectrometry ◽  
Microdilution Method ◽  
Species Complexes ◽  
Broth Microdilution Method

ABSTRACT To evaluate the in vitro susceptibility of Fusarium to isavuconazole, 75 clinical isolates were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry and then tested with a broth microdilution method (EUCAST) and the gradient concentration strip (GCS) technique. The activity of isavuconazole overall was shown to be limited, with an MIC50 of >16 μg/ml, without significant differences between the species complexes. The categorical agreement between GCS and EUCAST was 97.4% to 100%, making the GCS as a valuable alternative.

scholarly journals Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II

2020 ◽  
Vol 25 (4) ◽  
pp. 329-344
Author(s):  
Malcolm A. O’Neill ◽  
Ian Black ◽  
Breeanna Urbanowicz ◽  
Vivek Bharadwaj ◽  
Mike Crowley ◽  
...  
Keyword(s):  
Plant Species ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Pectic Polysaccharide ◽  
Ionization Time ◽  
Methyl Esterification ◽  
Flight Mass Spectrometry ◽  
Borate Ester ◽  
Rhamnogalacturonan Ii

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4- O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

scholarly journals Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

2011 ◽  
Vol 55 (7) ◽  
pp. 3465-3475 ◽  
Author(s):  
Christopher R. Bethel ◽  
Magdalena Taracila ◽  
Teresa Shyr ◽  
Jodi M. Thomson ◽  
Anne M. Distler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Active Site ◽  
Positive Charge ◽  
Evolutionary Strategy ◽  
Ionization Mass ◽  
Ionization Time ◽  
Esi Ms ◽  
Flight Mass Spectrometry ◽  
Before And After ◽  
Enzyme Intermediate

ABSTRACTCurrently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1.Kivalues ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C3carboxylate of inhibitors and that the localization of this positive charge to a “region of the active site” rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

scholarly journals Hierarchically electrospraying a PLGA@chitosan sphere-in-sphere composite microsphere for multi-drug-controlled release

2020 ◽  
Vol 7 (4) ◽  
pp. 381-390
Author(s):  
Zhu Liu ◽  
Weilong Ye ◽  
Jingchuan Zheng ◽  
Qindong Wang ◽  
Guowu Ma ◽  
...  
Keyword(s):  
Controlled Release ◽  
Drug Loading ◽  
Uv Spectrophotometry ◽  
Composite Microsphere ◽  
Ionization Time ◽  
Composite Microspheres ◽  
Flight Mass Spectrometry ◽  
Sequential Administration ◽  
Cellular Behaviors

Abstract Sequential administration and controlled release of different drugs are of vital importance for regulating cellular behaviors and tissue regeneration, which usually demands appropriate carriers like microspheres (MS) to control drugs releases. Electrospray has been proven an effective technique to prepare MS with uniform particle size and high drug-loading rate. In this study, we applied electrospray to simply and hierarchically fabricate sphere-in-sphere composite microspheres, with smaller poly(lactic-co-glycolic acid) MS (∼8–10 μm in diameter) embedded in a larger chitosan MS (∼250–300 μm in diameter). The scanning electron microscopy images revealed highly uniform MS that can be accurately controlled by adjusting the nozzle diameter or voltage. Two kinds of model drugs, bovine serum albumin and chlorhexidine acetate, were encapsulated in the microspheres. The fluorescence-labeled rhodamine-fluoresceine isothiocyanate (Rho-FITC) and ultraviolet (UV) spectrophotometry results suggested that loaded drugs got excellent distribution in microspheres, as well as sustained, slow release in vitro. In addition, far-UV circular dichroism and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) results indicated original secondary structure and molecular weight of drugs after electrospraying. Generally speaking, our research proposed a modified hierarchically electrospraying technique to prepare sphere-in-sphere composite MS with two different drugs loaded, which could be applied in sequential, multi-modality therapy.

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