30 SCRIPTAID TREATMENT IMPROVES POST-IMPLANTATION DEVELOPMENT OF SHEEP CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 121 ◽  
Author(s):  
V. Bordignon ◽  
M. Albornoz ◽  
C. Colato ◽  
N. El-Beyrouthi ◽  
J. I. Mellano ◽  
...  
Keyword(s):  
Transrectal Ultrasound ◽  
Fibroblast Cell ◽  
Media Control ◽  
Reconstructed Embryos ◽  
Deacetylase Inhibitor ◽  
Fetal Bovine ◽  
Skin Biopsies ◽  
Electric Fusion ◽  
Post Implantation

Increased histone acetylation by exposure to inhibitors of deacetylase enzymes has been reported to improve development of embryos produced by somatic cell nuclear transfer (SCNT). However, the response to such treatment seems to vary according to the species, cell line, and type of inhibitor used. The main objective of this study was to evaluate if treatment with the histone deacetylase inhibitor Scriptaid could improve the development to term of sheep SCNT-embryos. The 2 fibroblast cell lines used in this study were obtained from skin biopsies collected from 2 adult rams of the Santa Ines breed. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 30 crossbred sheep that were hormonally stimulated as described previously (Baldassarre et al. 2002 Theriogenology 57, 275). Oocyte maturation, cell transfer, fusion and activation, culture and transfer to recipients were conducted following procedures previously described (Baldassarre et al. 2003 Cloning Stem Cells 5, 279). Briefly, oocytes were matured in vitro for 24 h in TCM 199 supplemented with hormones and 10% fetal bovine serum, at 38.5°C in 5% CO2. Cells were transferred into enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6 kV cm–1 for 70 μs. The reconstructed embryos were then activated using ionomycin (5 μM/5 min) followed by cycloheximide (10 μg mL–1) and cytochalasin B (7.5 μg mL–1) for 4 to 5 h and then cultured in mSOF media (control); while half of the reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Subsequent to culture in mSOF ± Scriptaid as above, selected embryos were finally transferred into the oviducts of synchronized recipients within 24 h from fusion. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. A total of 258 oocytes were recovered (8.6/donor), of which 203 resulted in fused embryos after micromanipulation (79%) and 178 (69%) were selected for transfer into the oviducts of 18 synchronized recipients (avg. 10 embryos/recipient). Initial pregnancy was significantly higher in the Scriptaid group (40 v. 12.5%; P < 0.01). Interestingly, pregnancy was maintained through gestation Day 90 in the Scriptaid group, while the pregnant recipient carrying the control embryos lost her pregnancy by Day 60. All 4 pregnant recipients are due in early August. Our results are consistent with a previous report from (Zhao et al. 2010 Cel. Reprog. 12, 75) working with pig embryos and suggest that Scriptaid treatment can improve post-implantation development of SCNT sheep embryos. The results above will be further evaluated when data from births becomes available.

33 PRODUCTION OF CLONED BOER GOATS AND DORPER SHEEP IN ARGENTINA

2011 ◽  
Vol 23 (1) ◽  
pp. 123 ◽  
Author(s):  
C. Colato ◽  
M. Albornoz ◽  
M. L. Mellano ◽  
P. H. Mellano ◽  
J. I. Mellano ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transrectal Ultrasound ◽  
Fibroblast Cell ◽  
Incurable Disease ◽  
Reconstructed Embryos ◽  
Electric Fusion ◽  
Normal Offspring ◽  
Genetic Value ◽  
Boer Goats

Somatic cell nuclear transfer (SCNT) has been proposed as an outstanding tool for expanding the dissemination capacity of animals of extreme genetic value, as well as for the genetic resurrection of elite animals affected by incurable disease or that died suddenly. Numbers of outstanding males of meat-specialised breeds of goats (Boer) and sheep (Dorper) recently imported into Argentina were expanded using SCNT technology. Oocytes were collected by laparoscopic ovum pickup (LOPU) from 40 Raza Criolla goats and 38 crossbreed sheep that were hormonally stimulated, as described previously (Baldassarre et al. 2002 Theriogenology 57, 275). Oocyte maturation, cell transfer, fusion and activation, culture, and transfer to recipients were conducted following procedures previously described (Baldassarre et al. 2003 Cloning Stem Cells 5, 279). Briefly, oocytes were matured in vitro for 24 h in TCM 199 supplemented with hormones and 10% serum, at 38.5°C and 5% CO2. 2 caprine fetal fibroblast cell lines (FF1 and FF2) were established from purebred Boer fetuses generated by selective breeding of elite animals, while a fibroblast ovine line was established from a skin biopsy from an elite Dorper ram. Cells were transferred into previously enucleated oocytes, followed by electric fusion using a single DC pulse of 1.6 kV cm–1 for 70 μs. Finally, the reconstructed embryos were activated using ionomycin (5 μM/5 min) followed by cycloheximide (10 μg mL–1) and cytochalasin B (7.5 μg mL–1) for 4 to 5 h, followed by in vitro culture in mSOF media before transfer into the oviducts of synchronized recipients within 24 h after fusion. An average of 10.4 and 12.8 reconstructed embryos were transferred to each of 21 and 12 recipient goats and sheep, respectively. Pregnancy was detected and monitored for the first 3 months by transrectal ultrasound scanning. Initial pregnancy (4 recipients, 33%) was maintained from gestation Day 30 to term in sheep, while goats exhibited a dramatic drop from 9 recipients pregnant (41%) on Day 30 to only 2 (9%) giving birth. Deliveries were by elective C-section. The number of normal offspring with good postpartum survival was 2/2 in goats (100%) and 3/5 (60%) in sheep. Substantial differences were observed between the 2 cell lines used in goats, where pregnancy was 4/11 (36%) for FF1 and 5/10 (50%) for FF2 at Day 30; however, only 2 goats carrying FF2 pregnancies carried. These results are in agreement with previous reports suggesting that cell line may be the largest source of result variation in SCNT. At the time of writing this abstract these clones are ∼4 months of age, healthy and growing normally (>40 kg weight). To the best of our knowledge, these are the first cloned goats produced by SCNT technology in Latin America, and the second group to produce cloned sheep in the region.

27 CLONING OF ADULT PIGS USING SCRIPTAID TREATMENT AND PREOVULATORY EMBRYO TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 120
Author(s):  
M. Albornoz ◽  
C. Colato ◽  
N. El-Beyrouthi ◽  
F. Mellano ◽  
A. Mellano ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Expected Time ◽  
Reconstructed Embryos ◽  
Adult Cell ◽  
Skin Biopsies

There is growing interest in the use of swine in biomedical research. Cloning from cultured somatic cells (SCNT) has been the preferred method to generate genetically modified swine models. In a recent report, swine cloning efficiency was increased by treatment of reconstructed embryos with the inhibitor of deacetylase enzymes Scriptaid (Zhao et al. 2010 Cel. Reprog. 12, 75). Also, the timing of SCNT-embryo transfer with respect to the recipient’s expected time of ovulation was shown to affect cloning efficiency, whereas preovulatory embryo transfer resulted in a higher rate of cloned piglets born compared to postovulatory embryo transfer (Petersen et al. 2008 Cloning Stem Cells 10, 355). Therefore, our objective was to combine Scriptaid treatment and preovulatory embryo transfer in the same protocol for swine cloning. Cumulus–oocyte complexes aspirated from 3- to 6-mm diameter follicles were matured in vitro under standard conditions (Martinez Diaz et al. 2010 Cel. Reprog. 12, 85) and used as host oocytes for SCNT. Fibroblast cell lines were established from skin biopsies collected from 2 adult boars and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Oocytes were micromanipulated in Tyrode’s lactate-pyruvate-HEPES medium supplemented with 7.5 μg mL–1 cytochalasin B (CB) and electrically fused using a single DC pulse of 1.6 kV cm–1 for 70 μs. Activation was performed using ionomycin (15 μM/5 min) followed by exposure to CB (7.5 μg mL–1) and cyclohexemide (10 μg mL–1) for 5 h in porcine zygote medium (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112). Reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Oocytes were then washed and cultured in PZM-3 medium until transfer. Peripubertal recipient gilts were synchronized by oral administration of altrenogest (Regu-Mate®; 20 mg day–1) for 12 days, followed by 1.000 IU eCG injected on the last day of altrenogest treatment and 500 IU hCG 72 h later. 1-cell stage embryos were transferred into the oviduct after ∼20 h from hCG injection or 22 h before the expected ovulation time. Pregnancy was confirmed and monitored by ultrasonography and parturition was induced by injecting PGF2α at Day 115 of pregnancy. A total of 840 reconstructed embryos were transferred into 10 gilts [average 84 (range 60–110) embryos/gilt]. 4 gilts (40%) were detected to be pregnant 4 weeks after transfer, and 2 (20%) delivered 1 (1100 g) and 2 (950 and 850 g) healthy cloned piglets. The number of embryos transferred to these 2 gilts was 85 and 70. These results confirm that Scriptaid treatment and preovulatory embryo transfer can be applied in the same cloning protocol to produce cloned piglets from adult cell lines. To our knowledge, these are the first cloned pigs produced in Latin America.

Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda (Ailurus fulgens)

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 117-124 ◽  
Author(s):  
Yong Tao ◽  
Jianming Liu ◽  
Yunhai Zhang ◽  
Meiling Zhang ◽  
Junshun Fang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Fibroblast Cell ◽  
Basic Medium ◽  
Fibroblast Cell Line ◽  
Cell Line Establishment ◽  
Red Panda ◽  
Fetal Bovine ◽  
Ailurus Fulgens

SummaryIn evolution, the red panda (Ailurus fulgens) plays a pivotal role in the higher level phylogeny of arctoides carnivore mammals. The red panda inhabits certain Asian countries only and its numbers are decreasing. Therefore, the development of feasible ways to preserve this species is necessary. Genetic resource cryopreservation and somatic cell nuclear transfer (SCNT) have been used extensively to rescue this endangered species. The present study describes the establishment, for the first time, of a red panda ear fibroblast cell line, which was then cryopreserved, thawed and cultured. Through micromanipulation, interspecies embryos were reconstructed using the cryopreserved–thawed fibroblasts of the red panda as the donor and rabbit oocytes as recipients. A total of 194 enucleated rabbit oocytes were reconstructed with red panda ear fibroblasts; enucleated oocytes were activated without fusion as the control. The results show that the fibroblast cell line was established successfully by tissue culture and then cryopreserved in liquid nitrogen. Supplementation with 20% fetal bovine serum and 8% dimethyl sulphoxide in basic medium facilitated the cryopreservation. The interspecies embryos were successfully reconstructed. The cleavage, morulae and blastocyst rates after in vitro culture were 71, 47 and 23% (31/194), respectively. This study indicated that a somatic cell line could be established and cryopreserved from red panda and that rabbit cytoplast supports mitotic cleavage of the red panda karyoplasts and is capable of reprogramming the nucleus to achieve blastocysts.

370 HISTONE PHOSPHORYLATION PATTERNS AND CHROMOSOMAL STABILITY OF CULTURED BOVINE FIBROBLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 290
Author(s):  
A. Giraldo ◽  
J. Lynn ◽  
R. Godke ◽  
J. Jenkins ◽  
K. Bondioli
Keyword(s):  
Cell Lines ◽  
Histone H3 ◽  
Fibroblast Cell ◽  
Culture Conditions ◽  
Chromosome Loss ◽  
Early Passage ◽  
Multinucleated Cells ◽  
Fetal Bovine ◽  
Population Doublings

The low percentage of cloned offspring produced by nuclear transfer has been attributed to a variety of factors, including aneuploidy of the donor cells. Previous reports indicate that cultured bovine fibroblasts have a significantly higher level of aneuploidy in late passages than in early passages (Giraldo et al. 2005 J. Reprod. Fert. Dev. 17, 167). Phosphorylation of histone H3 at Serine 10 (Ser10) has been shown to be involved in chromosome compaction during cell division.Abnormal phosphorylation of this histone residue during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation (HP) patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine if the high percentage of aneuploid bovine fibroblast cells observed after long-term culture is associated with an abnormal HP pattern. Four bovine fibroblast cell lines were established from 40- to 60-day fetuses. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37°C and passaged at confluence. Relative levels of HP were determined in three different replicates at population doublings (PD) 2, 10, and 20. Cells were fixed and incubated with an anti-phosphorylated histone H3 (Ser10) antibody, labeled with a secondary antibody, counterstained with propidium iodide, and analyzed for HP fluorescence by flow cytometry. The number of chromosomes was also determined in counts of 800 metaphases. Differences in aneuploidy and HP fluorescence of cells in metaphase among PD were analyzed by χ2 two-way ANOVA, respectively (P < 0.05). The percentages of aneuploid cells in each of the cell lines increased progressively with duration of culture and were elevated from the start. Multinucleated cells were frequently observed after prolonged time in culture in all of the cell lines. The mean phosphorylated histone levels (relative fluorescence intensity) in cells during metaphase were 180.0, 131.5, 174.7, and 157.6 in PD 2; 170.4, 105.72, 145.8, and 152.7 in PD 10; and 274.0, 251.6, 191.4, and 308.3 in PD 20 for the four cell lines, respectively. No difference in HP levels was observed between PD 2 and PD 10. The average of HP during metaphase for the cell lines increased significantly from 160.9 at early passage to 256.3 at late passage (see Table 1). The increase in levels of HP occurred concurrently with the high percentage of aneuploid cells after extended time in culture. These data are consistent with the hypothesis that aneuploid cells observed after long in vitro culture are associated with abnormal HP patterns. Table 1. Relative HP fluorescence and aneuploidy at different PD of fetal bovine fibroblasts This study was supported by Grants in Aid of Research Program (GIAR) from Sigma Xi to A.G.

scholarly journals EXTH-33. VALPROIC ACID ENHANCES THE ANTI-PROLIFERATIVE EFFECT OF TUMOR-TREATING FIELDS ON PATIENT-DERIVED GLIOSARCOMA CELLS IN VITRO

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii94-ii94
Author(s):  
Sharon Michelhaugh ◽  
Sandeep Mittal
Keyword(s):  
Valproic Acid ◽  
Fetal Bovine Serum ◽  
Tumor Treating Fields ◽  
Proliferative Effect ◽  
Anti Epileptic Drug ◽  
Deacetylase Inhibitor ◽  
The Media ◽  
Microtubule Formation ◽  
Fetal Bovine

Abstract BACKGROUND Valproic acid (VPA) is an anti-epileptic drug often prescribed for glioma-associated seizures. VPA is also investigated as a potential anti-proliferative cancer treatment due to its histone deacetylase inhibitor activity. Tumor-treating fields (TTFields) are an FDA-approved therapy that reduce proliferation of tumor cells by interfering with microtubule formation during mitosis. In this study, we determined whether the combination of VPA with TTFields in comparison to each treatment alone would alter proliferation or cytotoxicity in patient-derived gliosarcoma cells. METHODS Gliosarcoma cells were grown in DMEM/F12 media with 10% fetal bovine serum and gentamicin. Prior to TTFields application and VPA incubation, cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. TTFields were applied to cells at 200 kHz optimal frequency (field intensity of ~1.6 V/cm) for 14 days and/or incubated with VPA at concentrations of 0, 0.25, 0.5, or 1mM. Proliferation was assessed by quantifying total RNA from each coverslip (n=3–4/group). Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) released into the media (n=8/group). Groups were compared by t-tests. RESULTS RNA yields from cells treated with only VPA were unchanged compared to control. TTFields treatment alone reduced RNA yield from 14.85±4.23 to 8.92±1.39 µg (mean± SD; P&lt; 0.05). The addition of 1mM VPA to TTFields further reduced RNA yield to 5.48±1.5 µg (P&lt; 0.03), although VPA alone had no effect. Additionally, 1mM VPA increased media LDH from 4.00±1.3 to 6.68±1.7 AU (mean± SD; P&lt; 0.01), while TTFields alone also increased media LDH to 5.67±0.99 AU (P&lt; 0.02). Media LDH levels due to combined TTFields and 1mM VPA treatment was 5.86±1.2 AU (not statistically different from either alone). CONCLUSIONS In patient-derived gliosarcoma cells, TTFields and VPA individually elicited modest cytotoxicity. However, TTFields plus VPA combination treatment reduced proliferation more than TTFields alone, though VPA treatment alone did not induce an anti-proliferative effect.

scholarly journals Cytotoxicity Evaluation of Root Canal Sealers Using an In Vitro Experimental Model with Roots

2017 ◽  
Vol 28 (2) ◽  
pp. 165-171 ◽  
Author(s):  
Ligia Teixeira ◽  
Fernanda Gonçalves Basso ◽  
Josimeri Hebling ◽  
Carlos Alberto de Souza Costa ◽  
Graziela Garrido Mori ◽  
...  
Keyword(s):  
Root Canal ◽  
Root Apex ◽  
Fibroblast Cell ◽  
Control Group ◽  
Primary Human Fibroblast ◽  
Root Canals ◽  
Ah Plus ◽  
Fetal Bovine ◽  
Root Canal Sealers

Abstract The aim of the present study was to evaluate the cytotoxicity of root canal sealers under conditions closely resembling a clinical reality. A primary human fibroblast cell line was seeded in 24-well acrylic plates with Dulbecco’s modified Eagle’s medium supplemented with 10% serum fetal bovine (SFB) and incubated for 24 h. Root canals from premolars were filled and individually attached to nylon devices to be stabilized in the wells with the already seeded cells. Specimens were divided into groups as follows: Control: gutta-percha cones (GPC); AH Plus+GPC; Sealapex+GPC; MTA Fillapex+GPC and Endofill+GPC. After 24 and 48 h, cell viability and morphology were evaluated by MTT assay and scanning electron microscopy (SEM), respectively. Statistical analysis was performed by Mann-Whitney test, complemented by Kruskal Wallis test (p<0.05). Only Endofill presented cytotoxicity after 24 h. MTA Fillapex and Endofill reduced the production of succinic desidrogenase after 48 h. AH Plus was non-toxic at any time point. SEM showed that the AH Plus and MTA Fillapex groups presented fibroblasts with morphology close to the control group, while the Endofill group presented few cells with thin extensions cells. The present study showed that good results were present in AH Plus and Sealapex, but not the Endofill group after 48 h. The method used enabled evaluation of the cytotoxicity of the studied sealers that diffused through the root apex.

49 EFFECTS OF CULTURE CONDITIONS AND GENE TRANSFECTION ON THE DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 172
Author(s):  
P. Pärn ◽  
M. Plaas ◽  
M. Nõmm ◽  
Ü. Jaakma ◽  
S. Kõks
Keyword(s):  
Cell Lines ◽  
Gene Transfection ◽  
Fibroblast Cell ◽  
Culture Conditions ◽  
Fetal Fibroblast ◽  
Reconstructed Embryos ◽  
A Cell ◽  
Fetal Fibroblast Cell ◽  
Fibroblast Cell Lines

Somatic cell nucleus transfer (SCNT) and in vitro culture of reconstructed embryos are the pivotal steps for successful cloning and generation of transgenic cattle. The aim of the study was to determine the influence of different cell fusion parameters, maturation, and culture conditions and the type of a cell line (bovine fetal fibroblast cell lines with or without gene transfection) on SCNT blastocyst development. Slaughterhouse-derived oocytes were matured for 17 h in TCM-199 (Sigma, St. Louis, MO, USA) supplemented with 0.05 µg mL–1 of epidermal growth factor (EGF) and 15 IU mL–1 of hCG/eCG (Intervet, PG600) or 10 µg mL–1 of FSH and 12.5 mU mL–1 of LH (Sioux Biochemical Inc., Sioux Center, IA, USA). Four fetal fibroblast cell lines (4 to 5 passages) and identical cell lines transfected with plasmid containing either human erythropoietin, FSH, growth hormone, or insulin-coding cDNA under β-casein promoter (7 to 9 passages) were used for SCNT. Cell fusion was induced by 2 direct-current pulses in 0.5 or 0.2 micro fusion chambers (Eppendorf Multiporator) using one of the following treatments: 100V for 15 µs (F1), 65V for 25 µs (F2), 65V for 20 µs (F3; all in a 0.5-mm chamber), or 36V for 25 µs (F4; 0.2-mm chamber). Fused complexes were activated with 4 µg mL–1 of Ca-ionophore for 4 min and then incubated for 5 h in 2 mM DMAP. The embryos were cultured in SOFaaci medium (Holm et al. 1999) or in commercial SOF medium (Minitüb GmbH, Tiefenbach, Germany) for 7 days. Data were analysed by ANOVA and the chi-square test. The results of the study showed that the cleavage rate of the reconstructed embryos was influenced by the fusion regimen (P < 0.05) but not by the donor cell type (P < 0.05). Treatments F2 and F3 resulted in cleavage rates higher (P < 0.05) than F1 and F4 (77.2, 82.0, 62.8, and 63.1%, respectively). Blastocyst yield was not significantly influenced by the different in vitro maturation (IVM) media – altogether, addition of FSH/LH resulted in 14.6% (158/1079) and EGF + hCG/eCG in 13.2% (73/554) of blastocysts (P < 0.05). The combination of TCM-199 + FSH/LH and SOFaaci resulted in 19.6% (79/403) blastocysts compared with 12.4% (74/596) when the same IVM medium and commercial SOF were used (P < 0.05). The use of transgenic cell lines for cloning led to a lower overall blastocyst rate (10.9%, 38/348) than use of non-transfected cell lines (17.7%, 115/651; P < 0.05), whereas the differences were 5.6 and 4.1 percentage points for SOF and SOFaaci, respectively. There were no significant differences between the individual cell lines within a cell line type. In conclusion, the optimization of the fusion parameters and in vitro culture (IVC) conditions led to improved blastocyst yields. In vivo development potential of the generated embryos still has to be evaluated in further studies. This study was supported by Project EU29023 of Enterprise Estonia, CCRMB, targeted grant SF1080045s07, and grant P8001 from the Estonian University of Life Sciences.

scholarly journals Overexpression of ubiquitin-specific peptidase 15 in systemic sclerosis fibroblasts increases response to transforming growth factor β

Rheumatology ◽  
2019 ◽  
Vol 58 (4) ◽  
pp. 708-718
Author(s):  
Christine Galant ◽  
Joel Marchandise ◽  
Maria S Stoenoiu ◽  
Julie Ducreux ◽  
Aurélie De Groof ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Transforming Growth Factor ◽  
Cell Metabolism ◽  
Fibroblast Cell ◽  
Transforming Growth Factor Β ◽  
Primary Fibroblast ◽  
Skin Biopsies ◽  
Fibroblast Cell Lines

Abstract Objective Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-β signalling in primary fibroblast cell lines. Methods High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-β stimulation. Results Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-β signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-β induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-β-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action. Conclusion Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-β, and is a potential therapeutic target in SSc.

In vitro validation of a 3-dimensional transrectal ultrasound system for prostate biopsiess

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Derek Cool ◽  
Shi Sherebrin ◽  
Jonathan Izawa ◽  
Joseph Chin ◽  
Aaron Fenster
Keyword(s):  
Prostate Biopsy ◽  
3D Model ◽  
Transrectal Ultrasound ◽  
Surface Topology ◽  
Sufficient Information ◽  
Needle Guidance ◽  
3 Dimensional ◽  
High Incidence ◽  
3D Information

Introduction: Transrectal ultrasound (TRUS) prostate biopsy (Bx) is currently confined to 2D information to both target and record 3D Bx locations. Accurate placement of Bx needles cannot be verified without 3D information, and recording Bx sites in 2D does not provide sufficient information to accurately guide the high incidence of repeat Bx. We have designed a 3D TRUS prostate Bx system that augments the current 2D TRUS system and provides tools for biopsy-planning, needle guidance, and recording of the biopsy core locations entirely in 3D. Methods: Our Bx system displays a 3D model of the patient’s prostate, which is generated intra-procedure from a collection of 2D TRUS images, representative of the particular prostate shape. Bx targets are selected, needle guidance is facilitated, and 3D Bx sites are recorded within the 3D context of the prostate model. The complete 3D Bx system was validated, in vitro, by performing standard ten-core Bx on anatomical phantoms of two patient’s prostates. The accuracy of the needle-guidance, Bx location recording, and 3D model volume and surface topology were validated against a CT gold standard. Results: The Bx system successfully reconstructed the 3D patient prostate models with a mean volume error of 3.2 ± 7.6%. Using the 3D system, needles were accurately guided to the pre-determined targets with a mean error of 2.26 ± 1.03 mm and the 3D locations of the Bx cores were accurately recorded with a mean distance error of 1.47 ± 0.79 mm. Conclusion: We have successfully developed a 3D TRUS prostate biopsy system and validated the system in vitro. A pilot study has been initiated to apply the system clinically.

Design, Synthesis, In vitro and In silico Evaluation of New Hydrazonebased Antitumor Agents as Potent Akt Inhibitors

2020 ◽  
Vol 17 (11) ◽  
pp. 1380-1392
Author(s):  
Emine Merve Güngör ◽  
Mehlika Dilek Altıntop ◽  
Belgin Sever ◽  
Gülşen Akalın Çiftçi
Keyword(s):  
Cell Line ◽  
Anticancer Agents ◽  
In Silico ◽  
Antitumor Agents ◽  
Colorimetric Assay ◽  
Fibroblast Cell ◽  
Lipinski’S Rule ◽  
In Silico Docking ◽  
Embryonic Fibroblast

Background: Akt is overexpressed or activated in a variety of human cancers, including gliomas, lung, breast, ovarian, gastric and pancreatic carcinomas. Akt inhibition leads to the induction of apoptosis and inhibition of tumor growth and therefore extensive efforts have been devoted to the discovery of potent antitumor drugs targeting Akt. Objectives: The objective of this work was to identify potent anticancer agents targeting Akt. Methods: New hydrazone derivatives were synthesized and investigated for their cytotoxic effects on 5RP7 H-ras oncogene transformed rat embryonic fibroblast and L929 mouse embryonic fibroblast cell lines. Besides, the apoptotic effects of the most active compounds on 5RP7 cell line were evaluated using flow cytometry. Their Akt inhibitory effects were also investigated using a colorimetric assay. In silico docking and Absorption, Distribution, Metabolism and Excretion (ADME) studies were also performed using Schrödinger’s Maestro molecular modeling package. Results and Discussion: Compounds 3a, 3d, 3g and 3j were found to be effective on 5RP7 cells (with IC50 values of <0.97, <0.97, 1.13±0.06 and <0.97 μg/mL, respectively) when compared with cisplatin (IC50= 1.87±0.15 μg/mL). It was determined that these four compounds significantly induced apoptosis in 5RP7 cell line. Among them, N'-benzylidene-2-[(4-(4-methoxyphenyl)pyrimidin- 2-yl)thio]acetohydrazide (3g) significantly inhibited Akt (IC50= 0.5±0.08 μg/mL) when compared with GSK690693 (IC50= 0.6±0.05 μg/mL). Docking studies suggested that compound 3g showed good affinity to the active site of Akt (PDB code: 2JDO). According to in silico ADME studies, the compound also complies with Lipinski's rule of five and Jorgensen's rule of three. Conclusion: Compound 3g stands out as a potential orally bioavailable cytotoxic agent and apoptosis inducer targeting Akt.

Sign in / Sign up

Export Citation Format

Share Document