3 SIRT1—A POSSIBLE MARKER FOR REPRODUCTIVE AGING OF IN VIVO-DERIVED BOVINE OOCYTES?

2017 ◽  
Vol 29 (1) ◽  
pp. 109 ◽  
Author(s):  
P. Kordowitzki ◽  
S. Klein ◽  
K.-G. Hadeler ◽  
P. Aldag ◽  
M. Nowak-Imialek ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Reproductive Aging ◽  
Standard Protocol ◽  
Blastocyst Formation ◽  
Fluorescent Intensity ◽  
Gene And Protein Expression ◽  
Maternal Aging

Maternal aging-associated reduction of oocyte viability is a common feature in mammals. Effective measures to counteract this process have not yet been developed. Cows are commonly used as a model of early human development, including maternal aging, because both species share a very high degree of similarity, including follicle selection, cleavage and blastocyst formation and a long reproductive lifespan. SIRT1, a member of the Sirtuin family, deacetylates transcriptional regulators localised in the nucleus and cytoplasm by a NAD+-dependent mechanism. Resveratrol (3,4′,5-trihydroxystilbene) is an antioxidant identified in various plant species and red wine which enhances SIRT1 activity. Based on these observations, the goal of the present study was to examine, if SIRT1 gene and protein expression is either affected by maternal age and/or can be modulated by resveratrol. Cumulus-oocyte-complexes of prepubertal (5–6 months old) and adult/aged (2 to 8 lactation) cows were collected by ovum pick-up twice a week. Medium for in vitro maturation (TCM 199) and in vitro fertilization (FertTalp) was supplemented with 20 µL of Resveratrol® (Sigma-Aldrich, Buchs, Switzerland) to get a final concentration of 2 µM Resveratrol respectively. Standard (TCM 199 and FertTalp) media without Resveratol were used as control. Cleavage rates and blastocyst formation were evaluated. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and blastocyst were conducted using next-generation sequencing technology. Finally, SIRT1 protein expression in oocytes and blastocysts were analysed by fluorescence immunostaining under a confocal microscope (LSM510, Zeiss, Germany) and relative fluorescent intensity was calculated. The cleavage rates of adult and prepubertal donors did not differ significantly among the treatments (standard protocol: 56.5 ± 5.4% for adult and 53.0 ± 4.7% for prepubertal donors, Resveratrol supplemented protocol: 62.1 ± 4.3% for cows and 63.6 ± 3.9% for calves). The blastocyst rates were slightly enhanced in the Resveratrol supplemented groups (cows: 34.2 ± 3.8% and calves: 33.1 ± 4.2%) compared to those of standard protocol (cows: 27.5 ± 4.8% and calves: 26.4 ± 3.3%). Relative mRNA abundance levels of SIRT1 were lower in oocytes and blastocysts derived from cows than in those derived from their younger counterparts (2.8-fold change; P = 0.05), but did not differ significantly among treatment groups. Protein expression profiles revealed that bovine SIRT1 was localised in the nucleus. The relative fluorescence levels of SIRT1 were significantly lower (221 ± 34 FIU) in control groups compared to the resveratrol treated groups (865 ± 45 FIU, respectively; P = 0.05). Additionally, SIRT1 protein levels were significantly higher in MII-oocytes (1255 ± 56 FIU) and blastocysts (984 ± 26 FIU) derived from calves compared with their older counterparts (442 ± 37 FIU and 310 ± 23 FIU, respectively, P = 0.05). In conclusion, these results indicate that resveratrol affects SIRT1 protein expression in oocytes and blastocysts of donors in different age. Thus, we hypothesise that SIRT1 is a reliable marker for reproductive aging, which could also be useful for better understanding of human infertility caused by aging.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals Alcohol induces TGFβ1 via downregulation of miR-1946a in murine lung fibroblast

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xian Fan ◽  
Stephen T. Mills ◽  
Mevelyn J. Kaalla ◽  
Viranuj Sueblinvong
Keyword(s):  
Protein Expression ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Direct Interaction ◽  
Lung Fibroblasts ◽  
Murine Lung ◽  
Gene And Protein Expression

Abstract Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.

MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1334-1334
Author(s):  
Robert W. Georgantas ◽  
Richard Hildreth ◽  
Jonathan Alder ◽  
Carlo M. Croce ◽  
George A. Calin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Hematopoietic Differentiation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.

scholarly journals Different molecular profiles are associated with breast cancer cell homing compared with colonisation of bone: evidence using a novel bone-seeking cell line

2014 ◽  
Vol 21 (2) ◽  
pp. 327-341 ◽  
Author(s):  
Faith Nutter ◽  
Ingunn Holen ◽  
Hannah K Brown ◽  
Simon S Cross ◽  
C Alyson Evans ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Metastasis ◽  
Expression Profiles ◽  
Molecular Profile ◽  
Calcium Signal ◽  
Cell Homing ◽  
Gene And Protein Expression ◽  
Breast Cancer Bone Metastasis

Advanced breast cancer is associated with the development of incurable bone metastasis. The two key processes involved, tumour cell homing to and subsequent colonisation of bone, remain to be clearly defined. Genetic studies have indicated that different genes facilitate homing and colonisation of secondary sites. To identify specific changes in gene and protein expression associated with bone-homing or colonisation, we have developed a novel bone-seeking clone of MDA-MB-231 breast cancer cells that exclusively forms tumours in long bones following i.v. injection in nude mice. Bone-homing cells were indistinguishable from parental cells in terms of growth ratein vitroand when grown subcutaneouslyin vivo. Only bone-homing ability differed between the lines; once established in bone, tumours from both lines displayed similar rates of progression and caused the same extent of lytic bone disease. By comparing the molecular profile of a panel of metastasis-associated genes, we have identified differential expression profiles associated with bone-homing or colonisation. Bone-homing cells had decreased expression of the cell adhesion molecule fibronectin and the migration and calcium signal binding protein S100A4, in addition to increased expression of interleukin 1B. Bone colonisation was associated with increased fibronectin and upregulation of molecules influencing signal transduction pathways and breakdown of extracellular matrix, including hRAS and matrix metalloproteinase 9. Our data support the hypothesis that during early stages of breast cancer bone metastasis, a specific set of genes are altered to facilitate bone-homing, and that disruption of these may be required for effective therapeutic targeting of this process.

scholarly journals Region between the Canine Distemper Virus M and F Genes Modulates Virulence by Controlling Fusion Protein Expression

2008 ◽  
Vol 82 (21) ◽  
pp. 10510-10518 ◽  
Author(s):  
Danielle E. Anderson ◽  
Veronika von Messling
Keyword(s):  
Protein Expression ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Transcriptional Control ◽  
Canine Distemper ◽  
F Protein ◽  
F Gene ◽  
Gene And Protein Expression

ABSTRACT Morbilliviruses, including measles and canine distemper virus (CDV), are nonsegmented, negative-stranded RNA viruses that cause severe diseases in humans and animals. The transcriptional units in their genomes are separated by untranslated regions (UTRs), which contain essential transcription and translation signals. Due to its increased length, the region between the matrix (M) protein and fusion (F) protein open reading frames is of particular interest. In measles virus, the entire F 5′ region is untranslated, while several start codons are found in most other morbilliviruses, resulting in a long F protein signal peptide (Fsp). To characterize the role of this region in morbillivirus pathogenesis, we constructed recombinant CDVs, in which either the M-F UTR was replaced with that between the nucleocapsid (N) and phosphoprotein (P) genes, or 106 Fsp residues were deleted. The Fsp deletion alone had no effect in vitro and in vivo. In contrast, substitution of the UTR was associated with a slight increase in F gene and protein expression. Animals infected with this virus either recovered completely or experienced prolonged disease and death due to neuroinvasion. The combination of both changes resulted in a virus with strongly increased F gene and protein expression and complete attenuation. Taken together, our results provide evidence that the region between the morbillivirus M and F genes modulates virulence through transcriptional control of the F gene expression.

scholarly journals Increased IL-1a expression is correlated with bladder cancer malignant progression

2020 ◽  
Author(s):  
Shi-Jie Yao ◽  
Hong-Shun Ma ◽  
Guang-Ming Liu ◽  
Yue Gao ◽  
Wei Wang
Keyword(s):  
Bladder Cancer ◽  
Protein Expression ◽  
Western Blot ◽  
Quantitative Polymerase Chain Reaction ◽  
Malignant Progression ◽  
Nuclear Antigen ◽  
Ki 67 ◽  
Gene And Protein Expression

IntroductionTo explore the function of interleukin 1α (IL-1α) in bladder cancer (BCa).Material and methodsImmunohistochemistry (IHC) was used to test the protein expression of IL-1α in BCa tissues. The relationship between IL-1α and clinical characteristics was analyzed by the Kaplan-Meier curve method. The gene and protein expression was tested by reverse transcription quantitative polymerase chain reaction (RT-q-PCR) and western blot, respectively. Colony formation and MTT assays were used to detect the potential of proliferation in vitro, and scratch and transwell chamber assays were used to detect the potential of invasion in vitro. Markers of proliferation such as Ki-67 and proliferating cell nuclear antigen (PCNA) and markers of invasion such as MMP-2 and MMP-9 were detected by western blot. Xenograft study was used for the in vivo experiment.ResultsWe found that IL-1α was highly expressed in BCa patients while highly expressed IL-1α was significantly related to short overall survival and progression-free survival in BCa as well. Moreover, knockdown of IL-1α might inhibit the ability of cancer cells to proliferate and invade or migrate both in vitro and in vivo.ConclusionsOur findings suggested that IL-1α might be a therapy target for BCa malignant progression.

Abstract O.21: Inositol 1,4,5 triphosphate 3- kinase C regulates NLRP3 Inflammasome activation in Kawasaki disease

Circulation ◽  
2015 ◽  
Vol 131 (suppl_2) ◽  
Author(s):  
Martin P Alphonse ◽  
Trang T Duong ◽  
Chisato Shimizu ◽  
Long T Hoang ◽  
Brian W McCrindle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Cell Lines ◽  
Nlrp3 Inflammasome ◽  
Ivig Therapy ◽  
Inflammasome Activation ◽  
Nlrp3 Inflammasome Activation ◽  
Gene And Protein Expression

Background: ITPKC controls calcium homeostasis and was identified in genome-wide studies to be associated with susceptibility and severity of KD. NLRP3 is activated by danger signals, leading to inflammasome activation and release of IL-1beta and IL-18. Ca 2+ mobilization mediates NLRP3 activation. We studied the contribution of ITPKC and NLRP3 inflammasome activation in KD. Methods: Cytokine levels were measured using ELISA. Gene expression was assayed using Illumina HumanHT-12v4. EBV-transformed B-cell lines from KD patients with different ITPKC genotypes were used to study the gene and protein expression and Ca 2+ flux using qRT-PCR, Western blot analysis, and FACS respectively. In vivo and in vitro analyses from ITPKC-deficient and wildtype mice were performed using ELISA, spinning disc confocal and Westerns, to determine cytokine release, Ca 2+ flux and protein expression responses respectively. Results: Children with KD show increased circulating IL-1beta, IL-18, IL-1RA and IL18BP protein during acute phase of KD (KD n=48, febrile controls n=41 p<0.001). NLRP3 and its associated inflammasome complex, and the IL-1[[Unsupported Character - Symbol Font &#61538;]] and IL18 receptor genes were up-regulated nearly two fold during the acute KD compared to matched convalescent controls (n=171, p<0.001). Pathway analysis showed specific up-regulation of NLRP3 related genes. The KD-associated genetic polymorphism in ITPKC (rs28493229) is functional, directly modulating [Ca 2+ ] i mobilization resulting in NLRP3 activation and increased production of IL-1beta and IL-18, as demonstrated by gene expression, circulating protein levels and functional assays of PBMCs and immortalized cell lines from affected children. These biologic data are partnered with evidence showing resistance to IVIG therapy in those with the ITPKC polymorphism associated with the highest basal and stimulated [Ca 2+ ] i levels. Studies using ITPKC-deficient mice in an animal model of KD support the molecular, cellular and clinical findings in affected children. Significance: ITPKC regulates NLRP3 activation via control of [Ca 2+ ] i mobilization, directing production of IL-1beta and IL-18, pointing to the key role of calcium mobilization in the immunopathogenesis of KD.

scholarly journals Effects of kiwifruit extracts on colonic gene and protein expression levels in IL-10 gene-deficient mice

2011 ◽  
Vol 108 (1) ◽  
pp. 113-129 ◽  
Author(s):  
Shelley J. Edmunds ◽  
Nicole C. Roy ◽  
Marcus Davy ◽  
Janine M. Cooney ◽  
Matthew P. G. Barnett ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dietary Intervention ◽  
Dietary Recommendations ◽  
Susceptible Host ◽  
Expression Levels ◽  
Background Strain ◽  
Gene And Protein Expression ◽  
Inflammatory Processes

Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity usingin vitromodels of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effectsin vivoagainst inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10− / −) mice (anin vivomodel of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While allIl10− / −mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated fromIl10− / −and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriatein vivomodels before making dietary recommendations, as a food that looks promisingin vitromay not be effectivein vivo.

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