69 QUALITY OF IN VITRO PRODUCED AND CRYOPRESERVED PORCINE EMBRYOS ASSESSED BY CELL NUMBER, TUNEL-POSITIVE NUCLEI, AND CASPASE-3 ACTIVITY

2011 ◽  
Vol 23 (1) ◽  
pp. 140
Author(s):  
M. Bryla ◽  
M. Trzcinska ◽  
B. Gajda
Keyword(s):  
In Vitro Culture ◽  
Caspase 3 ◽  
Preimplantation Embryos ◽  
Lower Percentage ◽  
Cell Detection ◽  
Large White ◽  
Number Of Cells

The aim of this experiment was to investigate the quality of in vitro cultured and cryopreserved porcine expanded blastocysts from Day 5, 6, and 7 of culture. The quality of the preimplantation embryos was determined by counting the number of cells, observing a TUNEL-positive reaction (TUNEL reagent; In Situ Cell Detection kit, Roche Diagnostics, Germany) and by caspase-3 labelling (PhiPhiLuxG2D2 Kit, Calbiochem, Germany). Embryos were collected from 32 superovulated donor gilts. All were crossbreds of Polish Landrace and Large White, age 6–8 months, weighing 90–100 kg. The experiment was done on 2–4 cell embryos produced in vivo and cultured in vitro for 7 days in NCSU-23 medium until expanding blastocyst stage. The embryos of this stage were obtained on Day 5, 6, and 7 of in vitro culture and divided into two groups: control-(1) 210 nonvitrified (NV) embryos and -(2) vitrified/thawed (VT) 169 embryos. The expanded blastocysts were vitrified the open pulled straw (OPS) method (Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). The results were analyzed by Student’s t-test, and all values were significant at P ≤ 0.05. The NV group of embryos showed significant differences in the number of cells (66.5 ± 24.0 v. 54.8 ± 15.9) and in TUNEL-positive nuclei (8.8 ± 12.5 v. 16.2 ± 14.9) between Day 5 and Day 7 of culture, respectively. Analysis of VT embryos also revealed significant differences in the number of cells (65.2 ± 17.4 v. 55.5 ± 14.3) and in TUNEL-positive nuclei (25.5 ± 16.4 v. 35.8 ± 19.3) between Day 5 and Day 7 of culture, respectively. Lower percentage of NT and VT blastocysts produced on Day 5 of culture revealed caspase-3 activity (51.3 v. 64.8%) compared with embryos on Day 7 (76.8 v. 89.3%), respectively. In conclusion, blastocysts cultured in vitro for 5 days consist of a high number of nuclei, have a low incidence of TUNEL-positive nuclei, and low caspase-3 activity compared with blastocysts cultured for 6 and 7 days in all analysed groups. Our results revealed that expanding blastocysts produced on Day 5 of in vitro culture had higher ability to survive vitrification/thawing procedure. This work was supported by Grant NR 12 0036 06 from the National Centre of Research and Development, Poland.

137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 182
Author(s):  
K. Imai ◽  
M. Ohtaku ◽  
Y. Aikawa ◽  
H. Matsuda ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Early Embryo ◽  
Lag Phase ◽  
Culture Dish

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P < 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

scholarly journals Tyrosol-Enriched Tomatoes by Diffusion across the Fruit Peel from a Chitosan Coating: A Proposal of Functional Food

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 335
Author(s):  
Silvia Tampucci ◽  
Antonella Castagna ◽  
Daniela Monti ◽  
Clementina Manera ◽  
Giuseppe Saccomanni ◽  
...  
Keyword(s):  
Tomato Fruit ◽  
Storage Period ◽  
Food Matrix ◽  
Fruit Peel ◽  
Fresh Food ◽  
In Vitro Permeation ◽  
Active Transfer

Chitosan is receiving increasing attention from the food industry for being a biodegradable, non-toxic, antimicrobial biopolymer able to extend the shelf life of, and preserve the quality of, fresh food. However, few studies have investigated the ability of chitosan-based coatings to allow the diffusion of bioactive compounds into the food matrix to improve its nutraceutical quality. This research is aimed at testing whether a hydrophilic molecule (tyrosol) could diffuse from the chitosan-tyrosol coating and cross the tomato peel. To this end, in vitro permeation tests using excised tomato peel and an in vivo application of chitosan-tyrosol coating on tomato fruit, followed by tyrosol quantification in intact fruit, peel and flesh during a seven-day storage at room temperature, were performed. Both approaches demonstrated the ability of tyrosol to permeate across the fruit peel. Along with a decreased tyrosol content in the peel, its concentration within the flesh was increased, indicating an active transfer of tyrosol into this tissue. This finding, together with the maintenance of constant tyrosol levels during the seven-day storage period, is very promising for the use of chitosan formulations to produce functional tomato fruit.

scholarly journals miR-146a-5p Promotes Chondrocyte Apoptosis and Inhibits Autophagy of Osteoarthritis by Targeting NUMB

Cartilage ◽  
2021 ◽  
pp. 194760352110235
Author(s):  
Hongjun Zhang ◽  
Wendi Zheng ◽  
Du Li ◽  
Jia Zheng
Keyword(s):  
Caspase 3 ◽  
Cartilage Tissue ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Knee Cartilage ◽  
Before And After ◽  
Related Proteins ◽  
Human And Mouse

Objective miR-146a-5p was found to be significantly upregulated in cartilage tissue of patients with osteoarthritis (OA). NUMB was shown to be involved in the autophagy regulation process of cells. We aimed to learn whether NUMB was involved in the apoptosis or autophagy process of chondrocytes in OA and related with miR-146a-5p. Methods QRT-PCR was used to detect miR-146a-5p level in 22 OA cartilage tissues and 22 controls. The targets of miR-146a-5p were analyzed using software and the luciferase reporter experiment. The apoptosis and autophagy, and related proteins were detected in chondrocytes treated with miR-146a-5p mimic/inhibitor or pcDNA3.1-NUMB/si-NUMB and IL-1β, respectively. In vivo experiment, intra-articular injection of miR-146a-5p antagomir/NC was administered at the knee of OA male mice before and after model construction. Chondrocyte apoptosis and the expression of apoptosis and autophagy-related proteins were also detected. Results miR-146a-5p was highly expressed in knee cartilage tissue of patients with OA, while NUMB was lowly expressed and negatively regulated by miR-146a-5p. Upregulation of miR-146a-5p can promote cell apoptosis and reduce autophagy of human and mouse chondrocytes by modulating the levels of cleaved caspase-3, cleaved PARP, Bax, Beclin 1, ATG5, p62, LC3-I, and LC3-II. Increasing the low level of NUMB reversed the effects of miR-146a-5p on chondrocyte apoptosis and autophagy. Intra-articular injection of miR-146a-5p antagomir can also reverse the effects of miR-146a-5p on the apoptosis and autophagy of knee joint chondrocytes in OA mice. Conclusion Downregulation of miR-146a-5p suppresses the apoptosis and promotes autophagy of chondrocytes by targeting NUMB in vivo and in vitro.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals In vitro characteristics and in vivo platelet quality of whole blood treated with riboflavin and UVA / UVB light and stored for 24 hours at room temperature

Transfusion ◽  
2021 ◽  
Vol 61 (S1) ◽  
Author(s):  
Turid Helen Felli Lunde ◽  
Lindsay Hartson ◽  
Shawn Lawrence Bailey ◽  
Tor Audun Hervig
Keyword(s):  
Whole Blood ◽  
Room Temperature

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

1994 ◽  
Vol 35 (1-2) ◽  
pp. 81-89 ◽  
Author(s):  
P.A. Pugh ◽  
J.G. Thompson ◽  
K. Logan ◽  
H.R. Tervit
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida

scholarly journals Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Peinkofer ◽  
Martina Maass ◽  
Kurt Pfannkuche ◽  
Agapios Sachinidis ◽  
Stephan Baldus ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Developmental Stage ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

scholarly journals Pharmacological Effects and Potential Clinical Usefulness of Polyphenols in Benign Prostatic Hyperplasia

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 450
Author(s):  
Kensuke Mitsunari ◽  
Yasuyoshi Miyata ◽  
Tomohiro Matsuo ◽  
Yuta Mukae ◽  
Asato Otsubo ◽  
...  
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Molecular Mechanisms ◽  
Prostatic Hyperplasia ◽  
Pathological Conditions ◽  
Urinary Tract Symptoms ◽  
Anti Inflammatory ◽  
Lower Urinary

Benign prostatic hyperplasia (BPH) is arguably the most common benign disease among men. This disease is often associated with lower urinary tract symptoms (LUTS) in men and significantly decreases the quality of life. Polyphenol consumption reportedly plays an important role in the prevention of many diseases, including BPH. In recent years, in addition to disease prevention, many studies have reported the efficacy and safety of polyphenol treatment against various pathological conditions in vivo and in vitro. Furthermore, numerous studies have also revealed the molecular mechanisms of the antioxidant and anti-inflammatory effects of polyphenols. We believe that an improved understanding of the detailed pharmacological roles of polyphenol-induced activities at a molecular level is important for the prevention and treatment of BPH. Polyphenols are composed of many members, and their biological roles differ. In this review, we first provide information regarding the pathological roles of oxidative stress and inflammation in BPH. Next, the antioxidant and anti-inflammatory effects of polyphenols, including those of flavonoids and non-flavonoids, are discussed. Finally, we talk about the results and limitations of previous clinical trials that have used polyphenols in BPH, with particular focus on their molecular mechanisms of action.

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