Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.08.015 Byline: Paula Stein, Fanyi Zeng, Hua Pan, Richard M. Schultz Keywords: RNAi; dsRNA; Oocyte; Interferon response; Off-targeting Abstract: RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA. Author Affiliation: Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA Article History: Received 12 July 2005; Revised 11 August 2005; Accepted 12 August 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.03.038 Byline: Fanyi Zeng, Richard M. Schultz Keywords: Microarray; Gene expression; Zygotic gene activation; Preimplantation mouse embryo Abstract: Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on [alpha]-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is mediated at the level of chromatin structure. The identity of the genes that are expressed during the 1- and 2-cell stages, however, is poorly described, as are those genes involved in mediating the transcriptionally repressive state. Using the Affymetrix MOE430 mouse GeneChip set, we characterized the set of [alpha]-amanitin-sensitive genes expressed during the 1- and 2-cell stages, and we used Expression Analysis Systematic Explorer (EASE) and Ingenuity Pathway Analysis (IPA) to identify biological and molecular processes represented by these genes, as well as interactions among them. We find that although the 1-cell embryo is transcriptionally active, we did not detect any transcripts present on the MOE430 GeneChip set to be [alpha]-amanitin-sensitive. Thus, what the BrUTP incorporation represents remains elusive. About 17% of genes expressed in the 2-cell embryo are [alpha]-amanitin-sensitive. EASE analysis reveals that genes involved in ribosome biogenesis and assembly, protein synthesis, RNA metabolism and transcription are over-represented, suggesting that genome activation during 2-cell stage may not be as global and promiscuous as previously proposed. IPA implicated Myc and Hdac1 as candidate genes involved in genome activation and the development of the transcriptionally repressive state, respectively. Author Affiliation: Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA Article History: Received 4 January 2005; Revised 28 March 2005; Accepted 29 March 2005

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach. Keywords: Microarray; Gene expression; Genome activation; Maternal mRNA; Compaction; Blastocyst

[Display omitted] • The detection of salted sea cucumber was realized by near infrared hyperspectral imaging and low field nuclear magnetic resonance. • Cuckoo optimization algorithm was proved to be effective in extracting salient features from the data, which was helpful to improve the accuracy of the models. • Deep learning framework was used to establish moisture content prediction models of salted sea cucumber based on single-data and multiple-data. The accurate control of moisture content (MC) during the processing of sea cucumber is beneficial to improve the taste of sea cucumber and maintain its nutritional value, which is directly related to the quality and shelf life of sea cucumber. The purpose of this study is to explore the feasibility using deep learning (DL) to realize rapid nondestructive detection of MC in salted sea cucumbers based on hyperspectral imaging (HSI) and low field nuclear magnetic resonance (LF-NMR) data. Firstly, three Cuckoo Search (CS) dimensionality reduction algorithms (Traditional-CS, Binary-CS and Chaotic-CS) were combined with DL framework respectively using HSI and LF-NMR data to establish prediction models, which proved the feasibility of DL framework in predicting the MC of sea cucumbers, and Chaotic-CS algorithm was selected as the optimal dimensionality reduction algorithm. Then, the MC visualization based on HSI and LF-NMR data was realized respectively to detect the migration and decrease of MC. Finally, using both HSI and LF-NMR data, the advantages of the models based on Fusion-net DL (FDL) framework were discussed, which showed better performance than the single-data models, with R C 2 of 0.9929, RMSE C of 0.0016, R P 2 of 0.9936 and RPD of 12.5041. In summary, the rapid nondestructive detection of MC in salted sea cucumbers could be realized by HSI and LF-NMR data based on DL framework, and the advantage of data fusion detection based on FDL framework was verified. [ABSTRACT FROM AUTHOR]

Objective: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. Study design: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45–55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. Results: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 ± 1.10% and 2.80 ± 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 × 104, 2.9 × 104, and 3.2 × 104 copies of human GPA DNA per μg of genomic DNA. FISH experiments showed that cells containing human specific α-satellite DNA sequence were present in the peripheral blood of the transplanted goats. Conclusions: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment. [Copyright &y& Elsevier]

An established technology to create cloned animals is through the use of somatic cell nuclear transfer (SCNT), in which reprogramming the somatic cell nucleus to a totipotent state by enucleated oocyte cytoplasm is a necessary process, including telomere length reprogramming. The limitation of this technology; however, is that the live birth rate of offspring produced through SCNT is significantly lower than that of IVF. Whether and how telomere length play a role in the development of cloned animals is not well understood. Only a few studies have evaluated this association in cloned mice, and fewer still in cloned cows. In this study, we investigated the difference in telomere length as well as the abundance of some selected molecules between newborn deceased cloned calves and normal cows of different ages either produced by SCNT or via natural conception, in order to evaluate the association between telomere length and abnormal development of cloned cows. The absolute telomere length and relative mitochondrial DNA (mtDNA) copy number were determined by real-time quantitative PCR (qPCR), telomere related gene abundance by reverse-transcription quantitative PCR (RT-qPCR), and senescence-associated β-galactosidase (SA-β-gal) expression by SA-β-gal staining. The results demonstrate that the newborn deceased SCNT calves had significantly shortened telomere lengths compared to newborn naturally conceived calves and newborn normal SCNT calves. Significantly lower mtDNA copy number, and significantly lower relative abundance of LMNB1 and TERT , higher relative abundance of CDKN1A , and aberrant SA-β-gal expression were observed in the newborn deceased SCNT calves, consistent with the change in telomere length. These results demonstrate that abnormal telomere shortening, lower mtDNA copy number and abnormal abundance of related genes were specific to newborn deceased SCNT calves, suggesting that abnormally short telomere length may be associated with abnormal development in the cloned calves. • The association of telomere length and related genes with developmental abnormalities in cloned calves was investigated. • Shortened telomere length and reduced mtDNA copy number were specific to newborn deceased SCNT calves. • Abnormal telomere shortening may be associated with abnormal development in cloned cows. • To help further understand the role of telomeres in developmental barriers to cloned calves. • Telomeres and related genes may be able to serve as molecular targets to enhance live birth rates in cloned calves. [ABSTRACT FROM AUTHOR]

Two-dimensional polymer/nanoparticle (NP) composite superlattice monolayers have attracted great attention, due to their applications in flexible optical, sensing, and electronic devices. The large-scale fabrication of these devices requires large-area superlattice monolayers. However, the preparation of large-area superlattice monolayers of polymer-grafted NP still faces great challenges. Herein, we report a modified Langmuir-Blodgett method for preparing large-area superlattice monolayers of polystyrene-grafted gold NPs (AuNP@PS). This method involves two key steps: pre-assembling the AuNP@PS on a liquid surface using mixed high/low-boiling-point solvent as the spreading solvent and then compressing the pre-assembled film in low-boiling-point solvent vapor. The discrete monolayer islands of AuNP@PS are formed in the pre-assembly process. During the surface compression process, the introduction of solvent vapor facilitates the fusion of AuNP@PS islands to form the continuous superlattice monolayers. Otherwise, discrete films with nonuniform thickness are obtained by compression without solvent vapor. This strategy enables the preparation of large superlattice monolayer films over 100 cm2, which are useful in the manufacture of microelectronic devices. [Display omitted] • A modified Langmuir-Blodgett method for preparing superlattice films is reported. • Superlattice films of polymer-grafted nanoparticles over 100 cm2 can be obtained. • Mixed solvent and solvent vapor facilitate the formation of superlattice monolayers. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7±0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos. [Copyright &y& Elsevier]

• lacZ codons optimized with 7 highly abundant cow milk proteins to construct olacZ. • olacZ expression increased in vitro, in cell lines 293T, HeLa, Cos7, NIH 3T3, HC11. • olacZ expression increased in vivo, in breast tissue in transgenic lactating female mice. • olacZ expression enhancement observed at the translational level. • System-preferential codon optimization can be used with other organs or bioreactors. lacZ encodes for β-galactosidase within the galactose operon of bacterial cells. When used as a reporter gene, bacterial "β-galactosidase" expression is often insufficient for detection in mammalian cells. We intended to optimize the lacZ codon usage according to the most frequently used codons for the seven major proteins in cow's milk, in order to pave a way for the enhancement of transgenic genes expression in eukaryotes. We constructed modified lacZ (named olacZ) according to optional codons used for proteins expressed in cow's milk. The expression of lacZ and olacZ was then compared in HC11 (a murine mammary gland epithelial line), 293T, HeLa, Cos7, and NIH 3T3 cells. While there was no significant difference at the mRNA level between lacZ and olacZ (P > 0.05). The quantification of β-galactosidase activity and in situ staining experiments showed a 1.2-fold to 3.3-fold expression improvement when comparing olacZ with lacZ. The levels of β-galactosidase expression at the protein levels from olacZ were approximately 9.2-fold and 2.4-fold respectively for Cos7 and HC11 cells. Furthermore, a 1.9-fold tendency of enhanced expression of olacZ in mammary gland during lactation was observed in transgenic- olacZ mice. This study demonstrates an alternative choice for improving lacZ reporter expression in eukaryotes, especially in the mammary gland of cattle or goats. [ABSTRACT FROM AUTHOR]

The fast charge carrier recombination and slow oxidation kinetics of ternary semiconductor InGaN as promising photocatalyst impede the PEC performance. Herein, we fabricate transition metal zinc (Zn) doped InGaN nanorods arrays by radio-frequency plasma-assisted molecular beam epitaxy. The doping obviously reduces indium atoms composition, the aggregation of In–In and induces the deep energy level. This greatly decreases the defects and improves the valence band potential of InGaN nanorods, which is beneficial for the rapid carrier separation efficiency with the decreased photogenerated carrier recombination rate and improved water oxidation kinetics. Significantly, Zn doped InGaN nanorods photoanode shows three times higher photocurrent density of 1.65 mA/cm2 at 1.23 V versus reversible hydrogen electrode (RHE) compared to undoped InGaN nanorods (0.58 mA/cm2). More importantly, after loading Au nanoparticles, the maximum applied bias photo-to-current efficiency of Zn-doped nanorods photoanode reaches 1.33%, which is superior to the recent results of reported Zn doped based photoanodes. This efficient doping strategy not only bridges the gaps of heteroatom doped InGaN nanorods based photoelectrodes, but also provides deep insights into controlling the electronic structure, and crystallinity of photoelectrodes for enhanced solar converting efficiency. Image 1 • Highly efficient Zn-doped InGaN NRs photoanodes are prepared. • Photogenerated carrier recombination is suppressed for Zn-doped InGaN NRs. • Zn doping contributes to the positive shift of valence band. • High photovoltage for Zn-doped InGaN NRs is achieved. [ABSTRACT FROM AUTHOR]