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Background: Peripheral blood carries a reservoir of mRNAs that regulate cardiac structure and function potential. Although it is well recognized that the typical symptoms of Myxomatous Mitral Valve Disease (MMVD) stage B2 are long-standing hemodynamic disorder and cardiac structure remodeling caused by mitral regurgitation, the transcriptomic alterations in blood from such dogs are not understood. Results: In the present study, comparative high-throughput transcriptomic profiling of blood was performed from normal control (NC) and naturally-occurring MMVD stage B2 (MMVD) dogs. Using Weighted Gene Co-expression Network Analyses (WGCNA), Gene Ontology (GO), and Kyoto Encyclopedia of Gene and Genomes (KEGG), we identified that the turquoise module was the most highly correlated with echocardiographic features and found 64 differentially expressed genes (DEGs) that were significantly enriched in platelet activation related pathways. Therefore, from the turquoise module, we selected five DEGs (MDM2, ROCK1, RIPK1, SNAP23, and ARHGAP35) that, according to real-time qPCR, exhibited significant enrichment in platelet activation related pathways for validation. The results showed that the blood transcriptional abundance of MDM2, ROCK1, RIPK1, and SNAP23 differed significantly (P < 0.01) between NC and MMVD dogs. On the other hand, Correlation Analysis revealed that MDM2, ROCK1, RIPK1, and SNAP23 genes negatively regulated the heart structure parameters, and followed the same trend as observed in WGCNA. Conclusion: We screened four platelet activation related genes, MDM2, ROCK1, RIPK1, and SNAP23, which may be considered as the candidate biomarkers for the diagnosis of MMVD stage B2. These findings provided new insights into MMVD pathogenesis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) has a strong genetic predisposition. However, its risk genes remain incompletely identified. We developed an Alzheimer's brain gene network-based approach to predict AD-associated genes by leveraging the functional pattern of known AD-associated genes. Our constructed network outperformed existing networks in predicting AD genes. We then systematically validated the predictions using independent genetic, transcriptomic, proteomic data, neuropathological and clinical data. First, top-ranked genes were enriched in AD-associated pathways. Second, using external gene expression data from the Mount Sinai Brain Bank study, we found that the top-ranked genes were significantly associated with neuropathological and clinical traits, including the Consortium to Establish a Registry for Alzheimer's Disease score, Braak stage score and clinical dementia rating. The analysis of Alzheimer's brain single-cell RNA-seq data revealed cell-type-specific association of predicted genes with early pathology of AD. Third, by interrogating proteomic data in the Religious Orders Study and Memory and Aging Project and Baltimore Longitudinal Study of Aging studies, we observed a significant association of protein expression level with cognitive function and AD clinical severity. The network, method and predictions could become a valuable resource to advance the identification of risk genes for AD. [ABSTRACT FROM AUTHOR]

Motivation Alzheimer's disease (AD) is a complex brain disorder with risk genes incompletely identified. The candidate genes are dominantly obtained by computational approaches. In order to obtain biological insights of candidate genes or screen genes for experimental testing, it is essential to assess their relevance to AD. A platform that integrates different types of omics data and approaches would facilitate the analysis of candidate genes and is in great need. Results We report AlzCode, a platform for multiview analysis of genes related to AD. First, this platform integrates a rich collection of functional genomic data, including expression data of AD samples (gene expression, single-cell RNA-seq data and protein expression), AD-specific biological networks (co-expression networks and functional gene networks), neuropathological and clinical traits (CERAD score, Braak staging score, Clinical Dementia Rating, cognitive function and clinical severity) and general data such as protein–protein interaction, regulatory networks, sequence similarity and miRNA-target interactions. These data provide basis for analyzing genes from different views. Second, the platform integrates multiple approaches designed for the various types of data. We implement functions to analyze both individual genes and gene sets. We also compare AlzCode with two existing platforms for AD analysis, which are Agora and AD Atlas. We pinpoint the features of each platform and highlight their differences. This platform would be valuable to the understanding of AD genetics and pathological mechanisms. Availability and implementation AlzCode is freely available at: http://www.alzcode.xyz. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Some genes tend to cluster and be co-expressed. Multiple factors affect gene co-expression. In this study, we investigated the relationships between multiple factors and the correlations of expression levels of neighboring genes, which were divided into four kinds of pattern genes and one type of non-pattern gene. Our results indicate that the correlation between expression levels of neighboring non-pattern genes is related to multiple factors with the exception of transcriptional orientations of neighboring genes. The correlation between expression levels of neighboring specific genes or neighboring repressed genes is likely to be dependent on the co-functions of neighboring genes. The correlation between expression levels of neighboring housekeeping genes is associated with histone modifications in intergenic regions. [ABSTRACT FROM AUTHOR]

Abstract: It has been reported that the sedative component of pentobarbital is mediated by GABA receptors in an endogenous sleep pathway and the ventrolateral preoptic area (VLPO)-tuberomammillary nucleus (TMN) or VLPO-dorsal raphe nucleus (DRN) neural circuit is important in the sedative response to pentobarbital. Our previous findings indicated that the VLPO-TMN neuronal circuit may play crucial part in the augmentative effect of diltiazem on pentobarbital sleep and the serotonergic system may be involved. This study was designed to investigate the role of DRN and the serotonergic receptors 5-HT1A and 5-HT2A/2C in the augmentative effect of diltiazem on pentobarbital-induced hypnosis in rats. The results showed that diltiazem (5mg/kg, i.g.) significantly reversed pentobarbital-induced (35mg/kg, i.p.) reduction of c-Fos expression in 5-HT neurons of DRNV (at −7.5mm Bregma), DRND, DRNVL and MRN (at −8.0mm Bregma). However it did not influence this reducing effect of pentobarbital on non-5-HT neurons either in DRN or in MRN. Moreover, the effect of diltiazem (1 or 2mg/kg, i.g.) on pentobarbital-induced (35mg/kg, i.p.) hypnosis was significantly inhibited by 5-HT1A agonist 8-OH-DPAT (0.5mg/kg, i.p.) and 5-HT2A/2C agonist DOI (0.5mg/kg, i.p.), and potentiated by 5-HT1A antagonist p-MPPI (2mg/kg, i.p.) and 5-HT2A/2C antagonist ritanserin (2mg/kg, i.p.), respectively. From these results, it should be presumed that the augmentative effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT1A and 5-HT2A/2C receptors, and DRN may be involved. In addition, it also suggested that the DRN may play a multi-modulating role in sleep–wake regulation rather than being recognized simply as arousal nuclei. [Copyright &y& Elsevier]

Abstract: The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63–70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT). [Copyright &y& Elsevier]

Abstract: To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components. [Copyright &y& Elsevier]

Abstract: Dicer1, an RNAse III enzyme, is a key factor for the production of microRNAs involved in post-transcriptional gene silencing. To elucidate the roles of Dicer1 and the microRNA pathway in early embryo development, we initially evaluated its gene expression in mouse oocytes and embryos in vitro. The transcript levels in GV stage oocytes steadily decreased up to the 2-cell embryo stage, and expression remained stable during morulae and blastocyst formation. DICER1 protein synthesis was additionally observed in mouse oocytes and early embryos. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit development up to the blastocyst stage. Real-time RT-PCR experiments confirmed the decreased expression of selected transcription factors, including POU domain, class 5, transcription factor 1 (Pou5f1), SRY-box containing gene 2 (Sox2), and Nanog homeobox (Nanog). Moreover, POU5F1 protein expression was suppressed by Dicer1 siRNA. The results suggest that Dicer1 gene expression is associated with the levels of transcription factors, Pou5f1, Sox2, and Nanog which possibly regulate differentiation processes at the blastocyst stage. [Copyright &y& Elsevier]

The aim of this study was to determine the effects of mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase cell numbers of embryos. Addition of 2 ng/ml GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes developing in vitro. However, total cell numbers were not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcriptase polymerase chain reaction revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium. [Copyright &y& Elsevier]

Y‐box binding protein 1 (YBX1) is a member of the family of DNA‐ and RNA‐binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one‐cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four‐cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6‐methyladenosine (m6A) writer N6‐adenosine‐methyltransferase 70 kDa subunit (METTL3) and reader insulin‐like growth factor 2 mRNA‐binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process. [ABSTRACT FROM AUTHOR]

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs. [ABSTRACT FROM AUTHOR]

Shugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear. We analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments. The chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1β, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway. Quercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1β were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice. SGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction. [Display omitted] • Non-alcoholic steatohepatitis (NASH) is a kind of fatty liver disease. • Shugan Xiaozhi (SGXZ) decoction has the effect of protecting liver function. • SGXZ decoction could improve NASH by inhibiting apoptosis signalling pathways. • SGXZ decoction might alleviate NASH though suppressing TLR4/MyD88/NF-κB pathway. [ABSTRACT FROM AUTHOR]

Being an essential mineral nutrient, potassium (K+) plays numerous important roles in plant growth and development and determines the yield and quality of crop products. The cellular level of K+ is controlled to a large extent by the K+ transporter, which belongs to the KT/HAK/KUP (HAK) family. However, little is known about these genes in tobacco. In this study, we surveyed the tobacco genome and identified 41 putative NtHAK genes (NtHAKS1–NtHAKS21 and NtHAKT1–NtHAKT20). Investigation of the cis-elements in upstream regions of these NtHAK genes suggests that members of this family respond to environmental cues and phytohormones. Expression data mining reveals that NtHAK genes showed clear sub-genome dominance. In all, these results will provide molecular insights into K+ transporter research in tobacco. [ABSTRACT FROM AUTHOR]

Background Ductal carcinoma in situ (DCIS) is a common type of non-invasive breast cancer and can sometimes progress into invasive breast cancer (IBC). Identification of the critical genes and biological processes specifically and/or commonly changed in DCIS or IBC can help us understand more about breast cancer development and provide more critical targets and signal transduction pathways for the diagnosis and treatments for breast cancer patients. Aim and methods We aimed to gain more understanding about the whole process of IBC development, especially in the early stage. Here we systematically analyzed an online breast cancer patient database to identify those significantly changed genes and biological processes in epithelium from normal stage to DCIS stage or from DCIS stage to IBC stage. Results 344 specific genes, such as FN1, AURKA and HSPA8, were found to be significantly changed (both upregulated and downregulated) in DCIS group in comparison with normal tissue group, which represents the gene profile changes in early stage of breast cancer development. Meanwhile, 304 specific genes were significantly changed (both upregulated and downregulated) in IBC group in comparison with normal tissue group, which represents the gene profile changes in late stage of breast cancer development. Importantly, seven genes were identified to have consistent changes in both early stage and late stage, indicating they might play “driving” roles in the breast cancer development. Of these 7 genes, 5 have been shown to be involved in breast cancer progression by previous studies, which demonstrates the validity of our analyses. Notably, DNAPTP3 was identified for the first time to play an oncogenic role in breast cancer development. In the GO term analyses, cell cycle genes was found to play more important roles in the early stage while biological adhesion was indicated to be more specifically involved in late stage of breast cancer development. Significance Our systematic analyses provide better understanding of the unique gene profiles and biological processes during the breast cancer development and identify more potentially important targets for future studies, such as DNAPTP3. [ABSTRACT FROM AUTHOR]

Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi) that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152). Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos. [ABSTRACT FROM AUTHOR]

Abstract: The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning. [Copyright &y& Elsevier]

The feasibility of using the modified-cut standard straw (M-CSS) method for the vitrification of immature mouse oocytes has been tested. The effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs), and apoptosis have also been compared. Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and cultured to meiosis II (MII) stage. Oocyte development, cytoskeletal organization, CG distribution, and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7 vs. 74.9%) and survival (89.0 vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs. 35.1%, respectively). Abnormal release of CGs also frequently occurred in EM grid groups (42.6 vs. 32.7%, respectively). Pro-apoptosis-related gene expression levels of Bax, caspase 3 were expressed lower than control in MII stage oocytes derived from M-CSS group; anti apoptosis-related genes, survivin and heat shock factor-1 (Hsf-1) were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces the survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes. [ABSTRACT FROM AUTHOR]

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems. [ABSTRACT FROM AUTHOR]

Objective: To investigate the molecular mechanism of mouse oocyte polarity loss during aging. Design: Experimental study. Setting: Academic basic research laboratory. Animal(s): Mice. Intervention(s): Oocytes were collected 16 hours after injection of hCG and cultured in M16 medium for an additional 14 hours with or without caffeine. Main Outcome Measure(s): Expression and localizations of actin nucleators actin-related protein 2/3 complex, JMY, and WAVE2 were examined by immunofluorescence staining, and their messenger RNA levels were examined by real-time reverse transcription–polymerase chain reaction. Result(s): The protein and messenger RNA levels of actin-related protein 2/3 complex, JMY, and WAVE2 were decreased in aged oocytes, but the levels were normal in caffeine-treated aged oocytes. Conclusion(s): Our data indicated that the loss of oocyte polarity may be due to the degradation of actin nucleators in aged oocytes. [Copyright &y& Elsevier]

Abstract: Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41°C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P <0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30°C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability. [Copyright &y& Elsevier]

Starch biosynthesis, a very critical event during pollen development, is not well understood in terms of gene regulation and intracellular controls. Using the rice HL-type cytoplasmic male sterile (HL-CMS) line with starch deficiency in pollen and two related male-fertile lines ( Oryza sativa L.), we have studied two specific developmental stages: ‘early’, that is, before or during pollen mitosis I, without starch accumulation, and ‘late’, the actively starch-filling postpollen mitosis I phase. Real-time reverse transcription-PCR analysis showed temporal changes in the expression patterns of six metabolic genes involved in the sucrose–starch pathway, i.e. sucrose synthase (EC 2.4.1.13), Rsus3; acid invertase (EC 3.2.1.26), OSINV2; phosphoglucomutase (EC 5.4.2.2), OsPGM; uridine diphosphate-glucose pyrophosphorylase (EC 2.7.7.9), OsUGP; a subunit of adenosine diphosphate-glucose pyrophosphorylase (AGPase) (EC 2.2.7.2), OsAGPL3; soluble starch synthase (EC 2.4.1.21), OsSSI and three transporter genes [sucrose transporter, OsSUT3; hexose transporter, OSMST7 and plasma membrane H+-adenosine triphosphatase (ATPase) (EC 3.6.1.35), OSA2]. Regarding metabolic enzyme activities, lower activity of sucrose synthase was detected at ‘late’ compared with ‘early’ stage in the three rice lines; invertase and other downstream enzymes had higher activities at ‘late’ stage in male-fertile but not male-sterile lines. Meanwhile, significantly reduced sugars and ATP levels were detected at ‘early’ and ‘late’ stages in male-sterile compared with male-fertile tissues. Furthermore, transgenic data showed that AGPase gene can stimulate starch accumulation in pollen of male-sterile host lines, showing the importance of AGPase in the metabolic pathway. Taken together, these data suggest that the combined effects of the reduced energy supply and sugar levels in starch biosynthesis may lead to the observed temporal changes in gene expression, and eventually to pollen sterility. [ABSTRACT FROM AUTHOR]

Luman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines - macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) - causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell-cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis. [ABSTRACT FROM AUTHOR]