233 DIFFERENTIAL GENE EXPRESSION IN BOVINE BLASTOCYSTS AND ELONGATING CONCEPTUSES DERIVED IN VIVO OR IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 274 ◽  
Author(s):  
M. Clemente ◽  
I. Lopez-Vidriero ◽  
P. O'Gaora ◽  
J. de la Fuente ◽  
A. Gutierrez-Adan ◽  
...  
Keyword(s):  
Fatty Acid Desaturase ◽  
Temporal Changes ◽  
Embryonic Mortality ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Transcript Profile ◽  
Rt Pcr ◽  
Embryo Survival

The majority of embryonic mortality in cattle occurs before maternal recognition of pregnancy at Day 16 postconception. In vitro-derived embryos exhibit a greater incidence of loss than their in vivo-derived counterparts. To better understand the causes of such embryonic loss, the aim of the current study was to compare transcript profiles of Days 7 and 13 bovine embryos derived in vitro or in vivo using the bovine Affymetrix microarray. We wanted to answer 3 questions: (1) what genes differ on Day 7 between blastocysts derived in vivo or in vitro, (2) what genes differ between Day 13 embryos derived from in vitro or in vivo embryos, and (3) what genes change between the blastocyst stage (Day 7) and the initiation of elongation (Day 13) and how are these temporal changes affected by the origin of the embryo. Day 7 bovine blastocysts were produced either in vitro by maturation, fertilization, and culture or in vivo by superovulation, AI, and nonsurgical embryo recovery. Half of the Day 7 blastocysts were snap frozen in liquid nitrogen in pools of 25 (microarray) or 10 (quantitative RT-PCR), and the other half were transferred in groups of 10 to synchronized heifers (10 recipients per group) ipsilateral to the corpus luteum and recovered on Day 13 by flushing the uterus after slaughter. Day 13 conceptuses were snap frozen individually. Three replicate pools of 25 Day 7 blastocysts and 5 Day 13 conceptuses were used for microarray analysis. Of the 24 128 probe-sets on the array, approximately 9500 genes were actively expressed in Days 7 and 13 embryos, irrespective of source. In Day 7 blastocysts, 50 genes were found to be differentially expressed (≥ 1.5-fold; P ≤ 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared with in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (≥1.5-fold; P ≤ 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared with in vitro embryos. The comparison between Days 7 and 13 embryos revealed significant temporal changes in transcript profile, with 1806 and 909 transcripts differentially expressed in in vitro and in vivo-derived embryos, respectively. Across the 3 array comparisons between Days 7 and 13 embryos, 444 genes were consistently exclusively present in in vivo embryos, whereas 1341 were exclusively present in in vitro embryos. Array validation was done by quantitative RT-PCR analysis of fatty acid desaturase 1 (FADS1), cytochrome P450, family 51, subfamily A, polypeptide I (CYP51), and hyaluronan binding protein 2 (HABP2) genes. In conclusion, these results indicate that the origin of the blastocyst can have a significant effect on the transcript profile of the conceptus at the initiation of elongation and might be associated with the likelihood of embryo survival/loss subsequently. Further hierarchical clustering analysis and quantitative RT-PCR data will address the functional roles for certain known genes and novel candidate genes related to embryonic mortality. This work was supported by a grant (AGL2006-05616) from the Spanish Ministry of Science and Technology.

scholarly journals Expression and Localization of Plasma Transglutaminase Factor XIIIA in Bone

2007 ◽  
Vol 55 (7) ◽  
pp. 675-685 ◽  
Author(s):  
Yukiko Nakano ◽  
Hadil F. Al-Jallad ◽  
Aisha Mousa ◽  
Mari T. Kaartinen
Keyword(s):  
Transglutaminase 2 ◽  
Pcr Primers ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Factor Xiiia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Osteoblast Cell Line

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immuno-histochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immuno-reactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.

scholarly journals The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

2005 ◽  
Vol 73 (7) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sarika Agarwal ◽  
Carol A. King ◽  
Ellen K. Klein ◽  
David E. Soper ◽  
Peter A. Rice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Iron Concentration ◽  
Pcr Analysis ◽  
Positive Trend ◽  
Gonococcal Infection ◽  
Rt Pcr ◽  
Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

4 GENE EXPRESSION IN CULTURES OF INNER CELL MASSES ISOLATED FROM IN VITRO-PRODUCED AND IN VIVO-DERIVED BOVINE BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 110 ◽  
Author(s):  
D. Pant ◽  
C. Keefer
Keyword(s):  
Es Cells ◽  
Primary Cultures ◽  
Embryonic Stem ◽  
Pcr Analysis ◽  
Beta Catenin ◽  
Rt Pcr ◽  
Self Renewal ◽  
Domestic Species

Genetic modification of embryonic stem (ES) cells derived from domestic species could be exploited to produce transgenic animals; however, fully validated ES have not been obtained in domestic species. Recent findings regarding key transcription factors and regulation of pluripotency and self-renewal in murine ES cells may provide keys to enable the derivation of ES in domestic species. The aim of this study was to identify and monitor the expression of candidate genes, which are known to be involved in the maintenance of self-renewal and pluripotency in mouse and human ES cells, during the critical first steps in establishment of primary cultures. Inner cell masses (ICMs) were isolated via manual dissection of 25 to 30 commercial in vitro-produced (IVP) blastocysts (Bomed, Inc., Madison, WI, USA) in each of three separate replicates and from 10 in vivo-derived Day 7-8 bovine blastocysts. On the day of ICM isolation (Day 0), 4-5 ICM clumps were collected for RT-PCR analysis. The remaining isolated ICMs were cultured (4-5 ICM clumps per well) on mitomycin C (Sigma-Aldrich, St. Louis, MO, USA)-inactivated mouse embryonic fibroblasts (STO, ATCC, Manassas, VA, USA). The ICM clumps were cultured in 12-well tissue culture dishes in ES medium consisting of Knockout DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% FCS (Hyclone, Logan, UT, USA), 2 mM l-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (MP Biomedicals, Irvine, CA, USA), and non-essential amino acids (Sigma). Two to four cultured ICM clumps were collected for RT-PCR analysis on Days 1-4 from IVP embryos and on Days 2, 4, and 6 from in vivo-derived embryos. Total RNA was extracted from the collected samples using the Absolutely RNA Nanoprep Kit (Strategene, La Jolla, CA, USA). First-strand DNAs were synthesized using Superscript III (Invitrogen) and cDNAs were amplified with PfuUltra hotstart PCR mastermix (Stratagene). Primers were designed based on homology between human and mouse sequences and were validated using bovine tissues. In experiments spanning these critical first few days of culture, the pluripotency-related genes (Nanog, Oct-4, Sox-2) and components of the LIF (LIFR, Gp130), BMP (Bmpr1a, Id-1), and Wnt (Beta-catenin, Frizzled) pathways were expressed in the ICM cultures over the 4 days of IVP-ICM cultures and the 6 days of in vivo-derived ICM cultures. These results indicate that the markers of pluripotency and the components of signaling pathways implicated in the maintenance of murine embryonic stem cells are present in ICMs of Day 7-8 bovine blastocysts and continue to be expressed at least during the initial days of culture. Genes (NCAM, Lef1) associated with early differentiation, however, were also expressed. Whether their expression is an indicator of ICM differentiation or of residual contamination with trophectoderm remains to be determined. Further studies will determine whether stimulation of these pathways can facilitate efficient derivation and maintenance ruminant ES cells.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

scholarly journals CircFAM13B promotes the proliferation of hepatocellular carcinoma by sponging miR-212, upregulating E2F5 expression and activating the P53 pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Xie ◽  
Xiaofeng Hang ◽  
Wensheng Xu ◽  
Jing Gu ◽  
Yuanjing Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Gene Expression Omnibus ◽  
Differentially Expressed ◽  
In Vivo Studies ◽  
Circular Rnas ◽  
Novel Target ◽  
Underlying Mechanisms

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

scholarly journals Interaction of periodontitis and orthodontic tooth movement—an in vitro and in vivo study

2021 ◽  
Author(s):  
Birgit Rath-Deschner ◽  
Andressa V. B. Nogueira ◽  
Svenja Beisel-Memmert ◽  
Marjan Nokhbehsaim ◽  
Sigrun Eick ◽  
...  
Keyword(s):  
Alveolar Bone ◽  
Tooth Movement ◽  
Mechanical Strain ◽  
Orthodontic Tooth Movement ◽  
Fusobacterium Nucleatum ◽  
In Vivo Study ◽  
Rt Pcr ◽  
Periodontal Inflammation

Abstract Objectives The aim of this in vitro and in vivo study was to investigate the interaction of periodontitis and orthodontic tooth movement on interleukin (IL)-6 and C-X-C motif chemokine 2 (CXCL2). Materials and methods The effect of periodontitis and/or orthodontic tooth movement (OTM) on alveolar bone and gingival IL-6 and CXCL2 expressions was studied in rats by histology and RT-PCR, respectively. The animals were assigned to four groups (control, periodontitis, OTM, and combination of periodontitis and OTM). The IL-6 and CXCL2 levels were also studied in human gingival biopsies from periodontally healthy and periodontitis subjects by RT-PCR and immunohistochemistry. Additionally, the synthesis of IL-6 and CXCL2 in response to the periodontopathogen Fusobacterium nucleatum and/or mechanical strain was studied in periodontal fibroblasts by RT-PCR and ELISA. Results Periodontitis caused an increase in gingival levels of IL-6 and CXCL2 in the animal model. Moreover, orthodontic tooth movement further enhanced the bacteria-induced periodontal destruction and gingival IL-6 gene expression. Elevated IL-6 and CXCL2 gingival levels were also found in human periodontitis. Furthermore, mechanical strain increased the stimulatory effect of F. nucleatum on IL-6 protein in vitro. Conclusions Our study suggests that orthodontic tooth movement can enhance bacteria-induced periodontal inflammation and thus destruction and that IL-6 may play a pivotal role in this process. Clinical relevance Orthodontic tooth movement should only be performed after periodontal therapy. In case of periodontitis relapse, orthodontic therapy should be suspended until the periodontal inflammation has been successfully treated and thus the periodontal disease is controlled again.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

scholarly journals Influence of estradiol on bovine trophectoderm and uterine gene transcripts around maternal recognition of pregnancy

2021 ◽  
Author(s):  
Emmalee J Northrop-Albrecht ◽  
Jerica J J Rich ◽  
Robert A Cushman ◽  
Runan Yao ◽  
Xijin Ge ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Mrna Level ◽  
Fixed Time ◽  
Beef Cows ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Rt Pcr ◽  
Embryo Survival ◽  
Uterine Fluid ◽  
Gene Transcripts

Abstract Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time artificial insemination (AI), but there are no differences in conceptus survival to d16. The objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm transcripts, and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-Time PCR (RT-PCR) was performed on trophectoderm (TE; n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D LC–MS/MS based iTRAQ method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in mRNA abundances in TE, but there were 432 differentially expressed genes (DEGs) (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (DEPs; 19 upregulated, 29 downregulated), 6 of these were differentially expressed (FDR &lt; 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin releasing hormone (CRH) signaling. These differences in uterine function, may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals.

scholarly journals miR-142-3p Suppresses Cell Growth by Targeting CDK4 in Colorectal Cancer

2018 ◽  
Vol 51 (4) ◽  
pp. 1969-1981 ◽  
Author(s):  
Xiangyu Zhu ◽  
Si-ping Ma ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Yong-peng Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Tumor Cell ◽  
Nude Mice ◽  
Colony Formation ◽  
Tumor Cell Growth ◽  
Rt Pcr ◽  
Tumor Tissues

Background/Aims: Deregulation of microRNAs (miRNAs) has been associated with a variety of cancers, including colorectal cancer (CRC). Here, we investigated anomalous miR-142-3p expression and its possible functional consequences in primary CRC samples. Methods: The expression of miR-142-3p was measured by quantitative RT-PCR in 116 primary CRC tissues and adjacent non-tumor tissues. The effect of miR-142-3p up- or down-regulation in CRC-derived cells was evaluated in vitro by cell viability and colony formation assays and in vivo by growth assays in xenografted nude mice. Results: Using quantitative RT-PCR, we found that miR-142-3p was down-regulated in 78.4 % (91/116) of the primary CRC tissues tested when compared to the adjacent non-tumor tissues. We also found that the miR-142-3p mimic reduced in vitro cell viability and colony formation by inducing cell cycle arrest in CRC-derived cells, and inhibited in vivo tumor cell growth in xenografted nude mice. Inversely, we found that the miR-142-3p inhibitor increased the viability and colony forming capacity of CRC-derived cells and tumor cell growth in xenografted nude mice. In addition, we identified CDK4 as a potential target of miR-142-3p by predictions and dual-luciferase reporter assays. Concordantly, we found that miR-142-3p mimics and inhibitors could decrease and increase CDK4 protein levels in CRC-derived cells, respectively. Conclusion: From our results we conclude that miR-142-3p may act as a tumor suppressor in CRC and may serve as a tool for miRNA-based CRC therapy.

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