In Vitro, In Vivo Comparison of Cyclosporin A Induced Hepatic Protein Expression Profiles

2016 ◽  
Vol 6 (3) ◽  
Author(s):  
Freek G Bouwman ◽  
Anke Van Summeren
Keyword(s):  
Protein Expression ◽  
Cyclosporin A ◽  
Expression Profiles ◽  
Protein Expression Profiles

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 Temporal Profiling of the Cortical Synaptic Mitochondrial Proteome Identifies Ageing Associated Regulators of Stability

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3403
Author(s):  
Laura C. Graham ◽  
Rachel A. Kline ◽  
Douglas J. Lamont ◽  
Thomas H. Gillingwater ◽  
Neil A. Mabbott ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Mitochondrial Protein ◽  
Wild Type Mouse ◽  
Proteomic Profiling ◽  
Temporal Regulation ◽  
Mitochondrial Proteome ◽  
Age Dependent ◽  
Protein Expression Profiles

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.

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.

Identification of differentially expressed genes to predict radioresistant prostate carcinomas.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 57-57
Author(s):  
Tim Nestler ◽  
Maike Wittersheim ◽  
Martin Hellmich ◽  
David J. K. P. Pfister ◽  
Margarete Odenthal ◽  
...  
Keyword(s):  
High Risk ◽  
Protein Expression ◽  
Risk Group ◽  
Expression Profiles ◽  
Gleason Pattern ◽  
Cohen’S D ◽  
Bonferroni Test ◽  
Protein Expression Profiles ◽  
Cohen's D

57 Background: Although relapses after radiotherapy are common in prostate cancer (PCa) patients, there are no clinical models or markers to identify patients at high risk for radioresistance. So far, only in vitro studies and xenograft models have been performed to identify gene expression patterns associated with radioresistance. However, studies which address the protein pattern to predict radioresistance in humans are completely missing. In order to determine potential biomarkers for radioresistance, we compared protein expression profiles of radioresistant PCa patients with PCa of primary prostatectomized patients. Methods: Two study groups consisting of: I) 30 patients who were treated by salvage prostatectomy and II) 94 patients treated by primary prostatectomy were formed. Tissue microarrays were constructed and immunostained for 15 proteins which are suggested to be associated with radioresistance by in vitro findings. Kruskal-Wallis test was used for multiple group comparison and followed by Dunn-Bonferroni-Test to detect intergroup differences. Cohen’s d was used to calculate the intergroup effect size. Results: Most proteins studied did not show any relevant differences between radioresistant PCa and primary PCa, except for two (AR and AKR1C3). On comparing immunostaining patterns between radioresistant PCa and primary PCa separated by Gleason risk groups, we observed only AR (androgen receptor) to be most expressed in radioresistant PCa (89.7%) and, in 87.8% of primary PCa of the high-risk group ( > 7a) (p = 0.851, Cohen’s d = 0.05), while only 67.3% PCa of the low-risk group (≤7a) (p = 0.017, Cohen’s d = 0.55) were positive. Considering the highest Gleason pattern per patient, only AKR1C3 (Aldo-Keto Reductase Family 1 Member C3) was seen to be similarly expressed in radiation-resistant PCa and patients with Gleason patterns 4 and 5 (p = 0.827, Cohen’s d = 0.05 and p = 0.893, Cohen’s d = 0.10) as compared to Gleason pattern 3 (p = 0.20, Cohen’s d = 0.69) in primary PCa. Conclusions: This is the first study evaluating protein expression profiles to predict radioresistance in PCa, where AR and AKR1C3 were identified to be the most promising protein markers.

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.

330 THE PROTEIN EXPRESSION PROFILE (SECRETOME) OF INDIVIDUAL CUMULUS - OOCYTE COMPLEXES DURING IVM IN THE MOUSE IS AFFECTED BY FSH

2006 ◽  
Vol 18 (2) ◽  
pp. 272 ◽  
Author(s):  
M. G. Katz-Jaffe ◽  
C. Sheehan ◽  
W. B. Schoolcraft ◽  
D. K. Gardner
Keyword(s):  
Protein Expression ◽  
Expression Profile ◽  
Expression Profiles ◽  
Surrounding Medium ◽  
Protein Expression Profile ◽  
Separate Branch ◽  
Maturation Medium ◽  
The Individual ◽  
Protein Expression Profiles

Studies of the protein expression profile into the surrounding medium (secretome) of in vitro-matured cumulus-oocyte complexes (COCs) have the potential to elucidate biochemical pathways involved in oogenesis, including the complex dialogue between the oocyte and its supporting cells. The understanding of these processes should assist in improving IVM success and fertility outcome, as early embryo development reflects the quality of the oocyte and its cumulus cells. Through the analysis of the individual COC secretome, we have investigated the effects of adding follicle stimulating hormone (FSH) to a defined maturation medium during IVM. COCs were collected from 3-week-old female mice (C57BL/6 � CBA) 48 h post-pregnant mare serum gonadotropin (PMSG) (5/iu) injection. Individual COCs were cultured in 5-�L drops of a defined maturation medium (0.25 mg/mL recombinant albumin) with the addition of 0, 2, 20, or 200 ng/mL FSH, under oil for 17 h. Oocytes were denuded and maturity recorded. Each microdrop of media (n = 8 oocytes per group) was collected, processed through an optimized series of buffers and washes prior to analysis by time-of-flight mass spectrometry (TOF-MS). Differential protein expression profiles were obtained from the secretome of individual COCs producing MII oocytes after maturation in differing doses of FSH. Statistical analysis revealed significant differences observed across 10 proteins/biomarkers with mass-to-charge (m/z) ratios ranging from 2.7 to 6 kDa (Mann-Whitney non-parametric test; P < 0.05). In addition, hierarchical and horizontal clustering analysis identified unique clusters of both up-regulated and down-regulated proteins/biomarkers within the m/z range of 2 to 18 kDa in the 2 ng/mL FSH group. Several of the individual COCs from the 20 ng/mL FSH group were also clustered alongside the 2 ng/mL group with similar protein expression profiles. In contrast, COCs cultured in the presence of 0 ng/mL and 200 ng/mL FSH were observed to cluster as a separate branch with distinctly different protein expression profiles. This study has determined for the first time the secretome profiles of individual COCs after IVM. These data have shown that the FSH dose in a defined maturation medium affects the secretome of an individual COC. Further investigation is currently underway to characterize these protein differences. The development of this proteomics approach will assist in revealing the intricate cellular function of an individual COC and elucidate critical pathways involved in mammalian oocyte maturation.

scholarly journals Cytoplasmic and nuclear maturation of oocytes in mammals – living in the shadow of cells developmental capability

2018 ◽  
Vol 6 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Marta Rybska ◽  
Sandra Knap ◽  
Maurycy Jankowski ◽  
Michal Jeseta ◽  
Dorota Bukowska ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Expression Profiles ◽  
Ovarian Follicle ◽  
Single Layer ◽  
Cumulus Cells ◽  
Protein Accumulation ◽  
Corona Radiata ◽  
Protein Expression Profiles

AbstractThe pig is a polyestrous animal in which the ovarian cycle lasts about 21 days and results in ovulation of 10-25 oocytes. Ovum reaches 120-150 μm in diameter, with the surrounding corona radiata providing communication with the environment. The zona pellucida is composed of glycoproteins: ZP1, ZP2, ZP3. In the course of oogenesis, RNA and protein accumulation for embryonic development occurs. Maternal mRNA is the template for protein production. Nuclear, cytoplasmic and genomic maturity condition the ability of the ovum to undergo fertilization. There are several differences in protein expression profiles observed between in vitro and in vivo conditions. Oogenesis is the process of differentiating female primary sex cells into gametes. During development gonocytes migrate from the yolk sac into the primary gonads with TGF-1, fibronectin, and laminin regulating this process. Cell cycle is blocked in dictyotene. Primary oocyte maturation is resumed before each ovulation and lasts until the next block in metaphase II. At the moment of penetration of the sperm into the ovum, the metaphase block is broken. The oocytes, surrounded by a single layer of granular cells, form the ovarian follicle. The exchange of signals between the oocyte and the cumulus cells done by gap-junctions, as well as various endo and paracrine signals. The contact between the corona radiata cells provides substances necessary for growth, through the same gap junctions. Studies on follicular cells can be used to amplify the knowledge of gene expression in these cells, in order to open way for potential clinical applications.

scholarly journals The PAK Inhibitor PF-3758309 Promotes the Inhibitory Effects of Multiple Chemotherapeutic Reagents on Patient-Derived Pancreatic Cancer Cell Lines

2018 ◽  
Author(s):  
Kai Wang ◽  
Nhi Huynh ◽  
Xiao Wang ◽  
Marina Pajic ◽  
Ashleigh Parkin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Tumour Growth ◽  
Pancreatic Cancer Cell ◽  
Expression Profiles ◽  
Ductal Adenocarcinoma ◽  
Inhibitory Effects ◽  
Protein Levels ◽  
Protein Expression Profiles

Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play key roles in PDA growth, and the PAK inhibitor PF-3758309 synergistically reduced PDA growth with gemcitabine. The aim of this study was to determine the effect of PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell proliferation was determined. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with PF-3758309, gemcitabine, PF-3758309 plus gemcitabine, or gemcitabine plus abraxane. Tumour growth was measured by volume and weight. PF-3758309 enhanced the inhibitory effects of 5-FU, gemcitabine and abraxane on a panel of patient-derived PDA cells, inhibited HIF-1 protein expression and reduced the protein levels of palladin and -SMA in these cells. The combination of PF-3758309 with gemcitabine maximally inhibited PDA growth in vivo, which was comparable to the combination of gemcitabine with abraxane. PF-3758309 enhanced the suppressive effects of multiple chemotherapeutic reagents on the growth of a panel of patient-derived PDA cell lines. The combination of PF-3758309 with gemcitabine provides a potential treatment option with less toxicity than gemcitabine plus abraxane.

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