Probabilistic Results on the Architecture of Mathematical Reasoning Aligned by Cognitive Alternation

We envision a machine capable of solving mathematical problems. Dividing the quantitative reasoning system into two parts: thought processes and cognitive processes, we provide probabilistic descriptions of the architecture

Cloning animals by somatic cell nuclear transfer – biological factors

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle

Hypoxia-Regulated miR-146a Targets Cell Adhesion Molecule 2 to Promote Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma

Background/Aims: miR-146a has recently been shown to promote cell proliferation, migration, and invasion in many cancers, but the role of miR-146a in clear cell renal cell carcinoma (ccRCC) remains unclear. Methods: Reverse transcription quantitative PCR (RT-qPCR) was performed to investigate the mRNA expression of miR-146a and CADM2 in ccRCC tissues. The luciferase reporter assay, Western blotting, and ChIP assay were carried out to explore the promoter and the transcription factor of miR-146a. Moreover, the effect of miR-146a and CADM2 on ccRCC cells was explored using methyl thiazolyl tetrazolium, colony formation, and migration and invasion assays. The luciferase reporter assay, RT-qPCR, western blotting, and immunofluorescence assay were carried out to investigate whether CADM2 is directly regulated by miR-146a. A tumor xenograft model and immunohistochemical staining were used to examine the carcinogenic effect of miR-146a and CADM2 in vivo. Results: miR-146a has been shown to promote cell proliferation, migration, and invasion. Here, we found that miR-146a is highly expressed in ccRCC tissues, whereas CADM2 is down-regulated. Hypoxia can induce the expression of miR-146a by stimulating its promoter. In addition, we demonstrated that miR-146a promoted and CADM2 inhibited proliferation, migration, and invasion of ccRCC cells. The 3’ untranslated region (UTR) luciferase reporter assay identified that miR-146a targeted the 3’ UTR of CADM2 and negatively regulated its expression. Ectopic expression of CADM2 counteracted the promoting effect of miR-146a on cell proliferation, migration, invasion, and the epithelial–mesenchymal transition process. Conclusion: Together, the finding of down-regulation of CADM2 by miR-146a can provide new insights into ccRCC pathogenesis and might contribute to the development of novel therapeutic strategies

Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells

<p>Abstract</p> <p>Background</p> <p>Dengue viruses (DENs) are the wildest transmitted mosquito-borne pathogens throughout tropical and sub-tropical regions worldwide. Infection with DENs can cause severe flu-like illness and potentially fatal hemorrhagic fever. Although RNA interference triggered by long-length dsRNA was considered a potent antiviral pathway in the mosquito, only limited studies of the value of small interfering RNA (siRNA) have been conducted.</p> <p>Results</p> <p>A 21 nt siRNA targeting the membrane glycoprotein precursor gene of DEN-1 was synthesized and transfected into mosquito C6/36 cells followed by challenge with DEN. The stability of the siRNA in cells was monitored by flow cytometry. The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. The presence of cells containing siRNA at 0.25, 1, 3, 5, 7 days after transfection were 66.0%, 52.1%, 32.0%, 13.5% and 8.9%, respectively. After 7 days incubation with DEN, there was reduced cytopathic effect, increased cell survival rate (76.9 ± 4.5% <it>vs </it>23.6 ± 14.6%) and reduced viral RNA copies (Ct value 19.91 ± 0.63 <it>vs </it>14.56 ± 0.39) detected in transfected C6/36 cells.</p> <p>Conclusions</p> <p>Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. siRNA may offer a potential new strategy for prevention and treatment of DEN infection.</p

OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors both in vitro and in vivo

AbstractGenerating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However, the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study, we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly, the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore, the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo

The Cell Agglutination Agent, Phytohemagglutinin-L, Improves the Efficiency of Somatic Nuclear Transfer Cloning in Cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P \u3c 0.05), and from 59 to 88% (P \u3c 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P \u3c 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer

Beneficial Effect of Young Oocytes for Rabbit Somatic Cell Nuclear Transfer

Abstract This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p<0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R=0.326; blastocyst rate vs. MAPK, R=0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n=117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12h after hCG injection, was beneficial for the development of NT embryos.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78139/1/clo.2008.0042.pd

Oocyte Source and Hormonal Stimulation for In Vitro

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, P = .001). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, P = .001). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries