Comparison of the effects of in vitro and in situ storage on the viability of mouse ovarian tissue collected after death

2001 ◽  
Vol 13 (6) ◽  
pp. 389 ◽  
Author(s):  
M. Snow ◽  
M. Cleary ◽  
S-L. Cox ◽  
J. Shaw ◽  
M. Paris ◽  
...  
Keyword(s):  
Room Temperature ◽  
Storage Temperature ◽  
Ovarian Tissue ◽  
The Body ◽  
Tissue Storage ◽  
Duration Of Storage ◽  
The Impact ◽  
Phosphate Buffered Saline

Ovarian tissues, collected or salvaged from endangered species at the time of gonadectomy or following their death, are being transported to genebanks for storage with the assumption that they will (subsequently) yield sufficient numbers of germ cells to help preserve the species. The present study aimed to quantify the impact of delays in collecting and/or processing ovarian tissue on the number of follicles in this tissue that remained normal after grafting. The study compared the viability of ovarian tissue stored in vitro (in phosphate-buffered saline) versus in situ (in the body) either on ice or at room temperature for 0 (non-stored fresh grafts), 3, 6, 12, 24 or 48 h. The conditions of storage had significant effects on the total number of morphologically normal follicles, with significantly more follicles in grafts developing from in vitro-stored tissue than in situ-stored tissue. Storage temperature and duration of storage, but not the storage temperature alone, influenced follicle survival. Tissue that was grafted immediately after collection (0 h) was best, but normal follicles were recovered in grafts stored in vitro (on ice or at room temperature) or in situ (on ice only) for up to 48 h before grafting. The rate of follicle loss over time was very rapid, with approximately 50% fewer follicles in grafts derived from tissue stored for only 3 h compared with non-stored fresh grafts (0 h). The results show that viable ovarian tissue can be salvaged from animals up to 48 h after death; however, in order to best protect the follicle population, the ovaries should be removed from the animal’s body as soon as possible.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

scholarly journals Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aiyan Guan ◽  
Inge Van Damme ◽  
Frank Devlieghere ◽  
Sarah Gabriël
Keyword(s):  
Enzymatic Digestion ◽  
Infected Fish ◽  
Effect Of Temperature ◽  
Video Recordings ◽  
Different Temperatures ◽  
Semi Solid ◽  
Zoonotic Parasites ◽  
The Impact ◽  
Phosphate Buffered Saline

AbstractAnisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

scholarly journals 695 Oral delivery of a microbial extracellular vesicle induces potent anti-tumor immunity in mice

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Loise Francisco-Anderson ◽  
Loise Francisco-Anderson ◽  
Mary Abdou ◽  
Michael Goldberg ◽  
Erin Troy ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Extracellular Vesicles ◽  
Tumor Immunity ◽  
Extracellular Vesicle ◽  
The Body ◽  
Checkpoint Inhibition ◽  
Small Intestinal ◽  
The Impact

BackgroundThe small intestinal axis (SINTAX) is a network of anatomic and functional connections between the small intestine and the rest of the body. It acts as an immunosurveillance system, integrating signals from the environment that affect physiological processes throughout the body. The impact of events in the gut in the control of tumor immunity is beginning to be appreciated. We have previously shown that an orally delivered single strain of commensal bacteria induces anti-tumor immunity preclinically via pattern recognition receptor-mediated activation of innate and adaptive immunity. Some bacteria produce extracellular vesicles (EVs) that share molecular content with the parent bacterium in a particle that is roughly 1/1000th the volume in a non-replicating form. We report here an orally-delivered and gut-restricted bacterial EV which potently attenuates tumor growth to a greater extent than whole bacteria or checkpoint inhibition.MethodsEDP1908 is a preparation of extracellular vesicles produced by a gram-stain negative strain of bacterium of the Oscillospiraceae family isolated from a human donor. EDP1908 was selected for its immunostimulatory profile in a screen of EVs from a range of distinct microbial strains. Its mechanism of action was determined by ex vivo analysis of the tumor microenvironment (TME) and by in vitro functional studies with murine and human cells.ResultsOral treatment of tumor-bearing mice with EDP1908 shows superior control of tumor growth compared to checkpoint inhibition (anti-PD-1) or an intact microbe. EDP1908 significantly increased the percentage of IFNγ and TNF producing CD8+ CTLs, NK cells, NKT cells and CD4+ cells in the tumor microenvironment (TME). EDP1908 also increased tumor-infiltrating dendritic cells (DC1 and DC2). Analysis of cytokines in the TME showed significant increases in IP-10 and IFNg production in mice treated with EDP1908, creating an environment conducive to the recruitment and activation of anti-tumor lymphocytes.ConclusionsThis is the first report of striking anti-tumor effects of an orally delivered microbial extracellular vesicle. These data point to oral EVs as a new class of immunotherapeutic drugs. They are particularly effective at harnessing the biology of the small intestinal axis, acting locally on host cells in the gut to control distal immune responses within the TME. EDP1908 is in preclinical development for the treatment of cancer.Ethics ApprovalPreclinical murine studies were conducted under the approval of the Avastus Preclinical Services’ Ethics Board. Human in vitro samples were attained by approval of the IntegReview Ethics Board; informed consent was obtained from all subjects.

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 711-720 ◽  
Author(s):  
H.V. Isaacs ◽  
D. Tannahill ◽  
J.M. Slack
Keyword(s):  
Specific Activity ◽  
Biological Properties ◽  
The Body ◽  
Mesoderm Induction ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Mesoderm Formation ◽  
Low Levels

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.

A Mechanistic Motor-Clutch Model That Explains Cell Shape Dynamics to Cyclic Stretch

2022 ◽  
Author(s):  
Benjamin W. Scandling ◽  
Jia Gou ◽  
Jessica Thomas ◽  
Jacqueline Xuan ◽  
Chuan Xue ◽  
...  
Keyword(s):  
Computational Model ◽  
Computational Models ◽  
The Body ◽  
Cyclic Stretch ◽  
Substrate Stiffness ◽  
Cell Alignment ◽  
Cellular Mechanisms ◽  
Actin Bundles ◽  
The Impact

Many cells in the body experience cyclic mechanical loading, which can impact cellular processes and morphology. In vitro studies often report that cells reorient in response to cyclic stretch of their substrate. To explore cellular mechanisms involved in this reorientation, a computational model was developed by utilizing the previous computational models of the actin-myosin-integrin motor-clutch system developed by others. The computational model predicts that under most conditions, actin bundles align perpendicular to the direction of applied cyclic stretch, but under specific conditions, such as low substrate stiffness, actin bundles align parallel to the direction of stretch. The model also predicts that stretch frequency impacts the rate of reorientation, and that proper myosin function is critical in the reorientation response. These computational predictions are consistent with reports from the literature and new experimental results presented here. The model suggests that the impact of different stretching conditions (stretch type, amplitude, frequency, substrate stiffness, etc.) on the direction of cell alignment can largely be understood by considering their impact on cell-substrate detachment events, specifically whether detachment occurs during stretching or relaxing of the substrate.

scholarly journals Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

Nutrients ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1601 ◽  
Author(s):  
Hiroki Saito ◽  
Yu Toyoda ◽  
Tappei Takada ◽  
Hiroshi Hirata ◽  
Ami Ota-Kontani ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Human Health ◽  
Serum Urate ◽  
The Body ◽  
Omega 3 ◽  
Beneficial Effects ◽  
Uricosuric Agents ◽  
Transport Assay ◽  
The Impact

The beneficial effects of fatty acids (FAs) on human health have attracted widespread interest. However, little is known about the impact of FAs on the handling of urate, the end-product of human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia, a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway. URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renal urate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly contained in foods or produced in the body. For this purpose, we conducted an in vitro transport assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especially long-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among the tested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0, 14.2, and 15.2 μM, respectively. Although further studies are required to investigate whether the ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as nutritionally important substances influencing human health.

scholarly journals O-Vanillin Attenuates the TLR2 Mediated Tumor-Promoting Phenotype of Microglia

2020 ◽  
Vol 21 (8) ◽  
pp. 2959
Author(s):  
Paul Triller ◽  
Julia Bachorz ◽  
Michael Synowitz ◽  
Helmut Kettenmann ◽  
Darko Markovic
Keyword(s):  
Malignant Gliomas ◽  
Experimental Models ◽  
Brain Macrophages ◽  
Human Microglia ◽  
Glioma Growth ◽  
Toll Like Receptor 2 ◽  
Brain Slice Cultures ◽  
The Impact

Malignant gliomas are primary brain tumors with poor prognoses. These tumors are infiltrated by brain intrinsic microglia and peripheral monocytes which promote glioma cell invasion. In our previous studies, we discovered that the activation of Toll-like receptor 2 (TLR2) on microglia/brain macrophages converts them into a protumorigenic phenotype through the induction of matrix metalloproteinases (MMP) 9 and 14. In the present study, we used in vitro and in situ microglia-glioma interaction experimental models to test the impact of a novel inhibitor of TLR 2, ortho vanillin (O-Vanillin) to block TLR2 mediated microglia protumorigenic phenotype. We demonstrate that O-Vanillin inhibits the TLR2 mediated upregulation of MMP 9, MMP 14, IL 6 and iNOS expression. Similarly, the glioma supernatant induced MMP 9 and MMP 14 expression in murine and human microglia is abrogated by O-Vanillin treatment. O-Vanillin is not toxic for microglia, astrocytes or oligodendrocytes. Glioma growth in murine brain slice cultures is significantly reduced after treatment with O-Vanillin, and this reduced glioma growth depends on the presence of microglia. In addition, we also found that O-Vanillin inhibited the glioma induced proliferation of murine primary microglia. In summary, O-Vanillin attenuates the pro-tumorigenic phenotype of microglia/brain macrophages and thus qualifies as a candidate for glioma therapy.

scholarly journals Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach

2020 ◽  
Vol 21 (20) ◽  
pp. 7690
Author(s):  
Tigran Harutyunyan ◽  
Ahmed Al-Rikabi ◽  
Anzhela Sargsyan ◽  
Galina Hovhannisyan ◽  
Rouben Aroutiounian ◽  
...  
Keyword(s):  
Human Lymphocytes ◽  
Nuclear Genome ◽  
Biological Phenomenon ◽  
Molecular Cytogenetic ◽  
Chromosome Breaks ◽  
Pathological Conditions ◽  
The Impact ◽  
In Vitro Treatment

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42–8.76, p < 0.05) and 3.02 (95% CI: 1.19–7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.

Frequency-force relationships of mammalian ventricular muscle in vivo and in vitro

1976 ◽  
Vol 230 (3) ◽  
pp. 631-636 ◽  
Author(s):  
ML Kahn ◽  
F Kavaler ◽  
VJ Fisher
Keyword(s):  
Heart Rate ◽  
Left Ventricle ◽  
Room Temperature ◽  
Physiological Role ◽  
Ventricular Muscle ◽  
Contractile State ◽  
Force Relationship

The change in contractility with increasing heart rate was studied in the left ventricle of dogs and in isolated trabeculae carneae of cats. For some of the studies in situ a transient isovolumic state was created by aortic occlusion. At physiological temperatures the frequency-force relationship is flatter than at room temperature and at the same temperature it is flatter in vivo than in vitro. The frequency-(dF/dt)max relationship is steeper than the frequency-force relationship at both temperatures in vivo and in vitro. The frequency-(dF/dt)max relationship is steeper in vitro than it is in situ, although the discrepancy is less marked than in the case of the frequency-force relationship. It is concluded that "staircase" plays less of a physiological role in adjustment of contractile state in situ than might be inferred from studies of isolated tissue.

The impact of storage temperature and sperm number on the fertility of liquid-stored bull semen

2016 ◽  
Vol 28 (9) ◽  
pp. 1349 ◽  
Author(s):  
Craig Murphy ◽  
Shauna A. Holden ◽  
Edel M. Murphy ◽  
Andrew R. Cromie ◽  
Patrick Lonergan ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Storage Temperature ◽  
Osmotic Resistance ◽  
Sperm Number ◽  
Ambient Temperatures ◽  
Progressive Motility ◽  
Bull Semen ◽  
Duration Of Storage ◽  
The Impact ◽  
Storage Temperatures

In Ireland, liquid bull semen is stored at unregulated ambient temperatures, typically at 5 × 106 spermatozoa per dose, and inseminated within 2.5 days of collection. In Experiment 1, the effect of storage temperature (5, 15, 22, 32°C and fluctuations (Flux) between these temperatures) on progressive motility, viability, acrosomal status, DNA fragmentation and osmotic resistance was assessed. In Experiment 2, the field fertility of liquid semen at 5, 4 and 3 × 106 spermatozoa per dose, up to Day 2 after collection, was assessed in comparison to frozen–thawed semen at 20 × 106 spermatozoa per dose (n = 35 328 inseminations). In Experiment 1, storage at 15°C resulted in the highest progressive motility (P < 0.01). The osmotic resistance of spermatozoa declined with duration of storage; however, after Day 3 this decline was reduced in the 5°C and Flux 15°C treatments (P < 0.01). In Experiment 2, the non-return rate of liquid semen stored at 4 and 3 × 106 spermatozoa per dose on Day 2 of storage was reduced in comparison to frozen–thawed semen (P < 0.01). In conclusion, liquid semen is versatile between storage temperatures of 5 and 22°C, but demonstrates reduced fertility on Day 2 of storage at lower sperm numbers in comparison to frozen–thawed semen.

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