Disturbance of suprachiasmatic nucleus function improves cardiac repair after myocardial infarction by IGF2-mediated macrophage transition.

Suprachiasmatic nucleus (SCN) in mammals functions as the master circadian pacemaker that coordinates temporal organization of physiological processes with the environmental light/dark cycles. But the causative links between SCN and cardiovascular diseases, specifically the reparative responses after myocardial infarction (MI), remain largely unknown. In this study we disrupted mouse SCN function to investigate the role of SCN in cardiac dysfunction post-MI. Bilateral ablation of the SCN (SCNx) was generated in mice by electrical lesion; myocardial infarction was induced via ligation of the mid-left anterior descending artery (LAD); cardiac function was assessed using echocardiography. We showed that SCN ablation significantly alleviated MI-induced cardiac dysfunction and cardiac fibrosis, and promoted angiogenesis. RNA sequencing revealed differentially expressed genes in the heart of SCNx mice from D0 to D3 post-MI, which were functionally associated with the inflammatory response and cytokine-cytokine receptor interaction. Notably, the expression levels of insulin-like growth factor 2 (Igf2) in the heart and serum IGF2 concentration were significantly elevated in SCNx mice on D3 post-MI. Stimulation of murine peritoneal macrophages in vitro with serum isolated from SCNx mice on D3 post-MI accelerated the transition of anti-inflammatory macrophages, while antibody-mediated neutralization of IGF2 receptor blocked the macrophage transition toward the anti-inflammatory phenotype in vitro as well as the corresponding cardioprotective effects observed in SCNx mice post-MI. In addition, disruption of mouse SCN function by exposure to a desynchronizing condition (constant light) caused similar protective effects accompanied by elevated IGF2 expression on D3 post-MI. Finally, mice deficient in the circadian core clock genes (Ckm-cre; Bmal1f/f mice or Per1/2 double knockout) did not lead to increased serum IGF2 concentration and showed no protective roles in post-MI, suggesting that the cardioprotective effect observed in this study was mediated particularly by the SCN itself, but not by self-sustained molecular clock. Together, we demonstrate that inhibition of SCN function promotes Igf2 expression, which leads to macrophage transition and improves cardiac repair post-MI.

Establishment of an in vitro safety assessment model for lipid-lowering drugs using same-origin human pluripotent stem cell-derived cardiomyocytes and endothelial cells.

Cardiovascular safety assessment is vital for drug development, yet human cardiovascular cell models are lacking. In vitro mass-generated human pluripotent stem cell (hPSC)-derived cardiovascular cells are a suitable cell model for preclinical cardiovascular safety evaluations. In this study, we established a preclinical toxicology model using same-origin hPSC-differentiated cardiomyocytes (hPSC-CMs) and endothelial cells (hPSC-ECs). For validation of this cell model, alirocumab, a human antibody against proprotein convertase subtilisin kexin type 9 (PCSK9), was selected as an emerging safe lipid-lowering drug; atorvastatin, a common statin (the most effective type of lipid-lowering drug), was used as a drug with reported side effects at high concentrations, while doxorubicin was chosen as a positive cardiotoxic drug. The cytotoxicity of these drugs was assessed using CCK8, ATP, and lactate dehydrogenase release assays at 24, 48, and 72 h. The influences of these drugs on cardiomyocyte electrophysiology were detected using the patch-clamp technique, while their effects on endothelial function were determined by tube formation and Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake assays. We showed that alirocumab did not affect the cell viability or cardiomyocyte electrophysiology in agreement with the clinical results. Atorvastatin (5-50 µM) dose-dependently decreased cardiovascular cell viability over time, and at a high concentration (50 µM, ~100 times the normal peak serum concentration in clinic), it affected the action potentials of hPSC-CMs and damaged tube formation and Dil-Ac-LDL uptake of hPSC-ECs. The results demonstrate that the established same-origin hPSC-derived cardiovascular cell model can be used to evaluate lipid-lowering drug safety in cardiovascular cells and allow highly accurate preclinical assessment of potential drugs.

Decidual expression and regulation of fatty acid desaturase 3 during mouse decidualization.

Decidualization is required for the successful establishment of pregnancy in rodents and primates. Fatty acid desaturase 3 (Fads3) belongs to the fatty acid desaturase family, which is a crucial enzyme for highly unsaturated fatty acid biosynthesis. However, the expression, regulation and function of Fads3 during early pregnancy in mice are still unknown. In this study, we examined Fads3 expression, regulation and function during mouse decidualization. The expression of Fads3 is detected in the subluminal stromal cells at implantation site on day 5 of pregnancy, but not at inter-implantation site and in day 5 pseudopregnant uteri. Compared to delayed implantation, Fads3 is strongly expressed after delayed implantation is activated by estrogen treatment. From days 6 to 8, Fads3 mRNA signals are significantly detected in the decidua. In ovariectomized mice, estrogen significantly stimulates Fads3 expression. However, estrogen has no effect on Fads3 expression in ovariectomized ERα-deficient mice, suggesting that estrogen regulation on Fads3 expression is ERα dependent. When ovariectomized mice were treated with progesterone, Fads3 expression is significantly increased by progesterone. Progesterone stimulation on Fads3 expression is also detected in cultured stromal cells, which is abrogated by RU486 treatment. These data indicate that progesterone upregulation on Fads3 expression is progesterone receptor-dependent. Fads3 knockdown by siRNA reduces in vitro decidualization of mouse stromal cells. Taken together, Fads3 may play an important role during mouse decidualization.

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction.

[Effects of simulated insect herbivory on defense traits of Galinsoga parviflora.] / 模拟昆虫取食对牛膝菊防御特征的影响.

To understand mechanisms underlying Galinsoga parviflora invasion and its responses to simulated insect herbivory, individuals of Galinsoga parviflora were treated with different concentrations of methyl jasmonate (MeJA) before blooming. We measued plant height, abundance of leaves and inflorescences, biomass, specific leaf area, trichome density, condensed tannins, total polyphenols, and flavonoids in leaves and inflorescences. The growth and reproduction parameters of G. parviflora treated with 5 mmol·L-1 MeJA were not significantly different from those of control, higher than those of control when treated with 10 mmol·L-1 MeJA, with significant difference except plant height, and declined when treated with 20 mmol·L-1 MeJA. The trichome density of leaf upper epidermis increased and specific leaf area decreased with increasing MeJA concentration, with both being significantly different from that of control. The contents of flavonoids, total polyphenols, and condensed tannins in leaves treated with 5 mmol·L-1MeJA were not significantly different from those of control. These defensive substances in leaves and inflorescences were highest under 10 mmol·L-1MeJA treatment. The contents of flavonoids and total polyphenols in inflorescences being higher than those of leaves, while condensed tannins was opposite. The defensive substances in leaves declined under 20 mmol·L-1MeJA treatment. The results suggested that G. parviflora could use tolerance and resistance strategies comprehensively, and adopted a variety of defense strategies such as compensatory growth, physical defense, and chemical defense, which was conducive to its success in invasion.

Case Report: A Young Man With Giant Pericardial Synovial Sarcoma.

Pericardial synovial sarcomas are sporadic tumors. Herein, we report a case of primary pericardial synovial sarcoma originating from the right pericardium. Missed diagnosis delayed surgical treatment. Eventually, the tumor occupied the almost entire pericardial cavity. The pericardial tumor was surgically removed as soon as possible after admission. In this paper, we aim to provide details that can help further understand the differing symptoms and presentations of pericardial synovial sarcoma and highlight the importance of consideration of this disease in similar cases where the etiology of pericardial effusion is unknown.

[Effects of "3414" fertilizer application on the yield of Fagopyrum cymosum].

OBJECTIVE: To study the effects of different fertilizer applications on the yield of Fagopyrum cymosum and explore the hese scheme for getting the maximum yield on purple soil in the Chongqing-west. METHODS: Experiment with implementing plan of "3414"; The quality Assessment by the contents of bipoly-chrysanthemin; The data process program under the Excel 2003, SPSS 13.0, MatlaB 7.0, Word 2003 environments. RESULTS: Various fertilizer combinations had different transformation efficiency which the N3P2K2 combination was the maximum 97.09% and the NOP2K2 combination was the minimum 4.32%; The NOP2K2 combination had the lowest yield except of the bland group which was 186 kg/667 m2; When the N fertilizer Rate was controlled in the level of 15 kg/667 m2 The yied had no obvious change as the increase of another two kinds fertilizer rate; Three kinds of function could better reflect the relationships between fertilizer and yields, which all of the R2 value were above 0.88; The best one was N K function with the maximum R2; The blank group had maximum content 8.67% of bipoly-chrysanthemin and the content had a little decrease as the increase of N or K, but all higher than 7.14% which were planted in Bei Jing area. CONCLUSIONS: Various fertilizer combinations influenced the transformation efficiency of N, P, K;N is the key fertilizer on purple soil; Reconmentation funtion was N,K function which could be as the guiding function; F; Fertilizer would not influence the quality of Fagopyrum cymosum.

Global analysis of differential luminal epithelial gene expression at mouse implantation sites.

Although implantation types differ between species, the interaction between blastocyst trophectoderm and apical surface of the uterine epithelium is a common event during the implantation process. In this study, uterine luminal epithelium at implantation and inter-implantation sites was isolated by enzymatic digestion and used for microarray analysis. In a mouse microarray containing 12 345 unigenes, we found 136 genes upregulated more than twofold at the implantation site, while 223 genes were downregulated by at least twofold. Reverse transcription-PCR was used to verify the differential expression of seven upregulated and six downregulated genes chosen randomly from our microarray analysis, and the expression trends were similar. The differential expression patterns of eight upregulated genes were verified by in situ hybridization. Compared with the inter-implantation site on day 5 of pregnancy and the uterus on day 5 of pseudopregnancy, protease, serine, 12 neurotrypsin, endothelin-1, gamma-glutamyl hydrolase, Ras homolog gene family, member U, T-cell immunoglobulin, and mucin domain containing 2, proline-serine-threonine phosphatase-interacting protein 2, 3-monooxgenase/tryptophan 5-monooxgenase activation protein, gamma-polypeptide, and cysteine-rich protein 61 (Cyr61) were upregulated in the luminal epithelium at implantation site on day 5 of pregnancy. These genes may be related to embryo apposition and adhesion during embryo implantation. Cyr61, a gene upregulated at the implantation site, was chosen to examine its expression and regulation during the periimplantation period by in situ hybridization. Cyr61 mRNA was specifically localized in the luminal epithelium surrounding the implanting blastocyst at day 4 midnight and on day 5 of pregnancy, and in the activated uterus, but not expressed in inter-implantation sites and under delayed implantation, suggesting a role for Cyr61 in mediating embryonic-uterine dialog during embryo attachment. Our data could be a valuable source for future study on embryo implantation.

Tissue-specific distribution of serine/threonine protein phosphatase 1 of Toxocara canis.

Serine/threonine protein phosphatase 1 (PP1) is expressed in developing and reproductively active male Toxocara canis. To investigate the tissue-specific expression of PP1 in T. canis, the PP1 protein was expressed in Escherichia coli, and the recombinant protein was used to generate a rabbit polyclonal antiserum. Indirect fluorescence immunohistochemical analysis of adult male T. canis showed that PP1 was expressed in the germ line tissues, primarily in the testis, seminal vesicle, vas deferens, and sperm cells, indicating the potential roles of PP1 in spermatogenesis. What's more, structural predictions of PP1 in T. canis were performed. The predictions of the structure indicated that PP1 may be a potential target for antihelmintic drugs. This is the first report of the tissue distributions and structural prediction of PP1 in T. canis, which might lead to the development of novel, innovative strategies for controlling T. canis infestations.

Role of 5-aminolevulinic acid in the salinity stress response of the seeds and seedlings of the medicinal plant Cassia obtusifolia L.

BACKGROUND: Soil salinity, one of the major abiotic stresses affecting germination, crop growth, and productivity, is a common adverse environmental factor. The possibility of enhancing the salinity stress tolerance of Cassia obtusifolia L. seeds and seedlings by the exogenous application of 5-aminolevulinic acid (ALA) was investigated. RESULT: To improve the salinity tolerance of seeds, ALA was applied in various concentrations (5, 10, 15, and 20 mg/L). To improve the salinity tolerance of seedlings, ALA was applied in various concentrations (10, 25, 50, and 100 mg/L). After 10 mg/L ALA treatment, physiological indices of seed germination (i.e., germination vigor, germination rate, germination index, and vigor index) significantly improved. At 25 mg/L ALA, there was a significant protection against salinity stress compared with non-ALA-treated seedlings. Chlorophyll content, total soluble sugars, free proline, and soluble protein contents were significantly enhanced. Increased thiobarbituric acid reactive species and membrane permeability levels were also inhibited with the ALA treatment. With the treatments of ALA, the levels of chlorophyll fluorescence parameters, i.e., the photochemical efficiency of photosystem II (Fv/Fm), photochemical efficiency (Fv'/Fm'), PSII actual photochemical efficiency (ΦPSII), and photochemical quench coefficient (qP), all significantly increased. In contrast, the non-photochemical quenching coefficient (NPQ) decreased. ALA treatment also enhanced the activities of superoxide dismutase, peroxidase, and catalase in seedling leaves. The highest salinity tolerance was obtained at 25 mg/L ALA treatment. CONCLUSION: The plant growth regulator ALA could be effectively used to protect C. obtusifolia seeds and seedlings from the damaging effects of salinity stress without adversely affecting plant growth.

The integrative analysis of microRNA and mRNA expression in mouse uterus under delayed implantation and activation.

BACKGROUND: Delayed implantation is a developmental arrest at the blastocyst stage and a good model for embryo implantation. MicroRNAs (miRNAs) have been shown to be involved in mouse embryo implantation through regulating uterine gene expression. This study was to have an integrative analysis on global miRNA and mRNA expression in mouse uterus under delayed implantation and activation through Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: By deep sequencing and analysis, we found that there are 20 miRNAs up-regulated and 42 miRNAs down-regulated at least 1.2 folds, and 268 genes up-regulated and 295 genes down-regulated at least 2 folds under activation compared to delayed implantation, respectively. Many different forms of editing in mature miRNAs are detected. The percentage of editing at positions 4 and 5 of mature miRNAs is significantly higher under delayed implantation than under activation. Although the number of miR-21 reference sequence under activation is slightly lower than that under delayed implantation, the total level of miR-21 under activation is higher than that under delayed implantation. Six novel miRNAs are predicted and confirmed. The target genes of significantly up-regulated miRNAs under activation are significantly enriched. CONCLUSIONS: miRNA and mRNA expression patterns are closely related. The target genes of up-regulated miRNAs are significantly enriched. A high level of editing at positions 4 and 5 of mature miRNAs is detected under delayed implantation than under activation. Our data should be valuable for future study on delayed implantation.

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China.

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.

MicroRNA expression and regulation in mouse uterus during embryo implantation.

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.

Differential expression and regulation of prostaglandin transporter and metabolic enzymes in mouse uterus during blastocyst implantation.

OBJECTIVE: To examine the spatiotemporal expression and regulation of prostaglandin transporter (PGT), 15-hydroxy-PG dehydrogenase (15-PGDH), and carbonyl reductase 1 (CBR1) messenger RNA (mRNA) and protein in the mouse uterus during embryo implantation and in related models. DESIGN: Experimental animal study. SETTING: University research laboratory. ANIMAL(S): Sexually mature female Kunming strain white mice. INTERVENTION(S): Delayed and activated implantation, artificial decidualization, and subcutaneous injection of progesterone (P) and E(2) in ovariectomized mouse. MAIN OUTCOME MEASURE(S): The expression of mRNA and protein were detected by in situ hybridization and immunohistochemistry in mouse uterus. RESULT(S): Prostaglandin transporter mRNA and protein were expressed in the subluminal stroma at implantation site on day 5 of pregnancy and then in decidua but were not detected at the interimplantation sites and in preimplantation or pseudopregnant uterus. The presence of an active blastocyst was required for PGT expression at the implantation site. Both 15-PGDH and CBR1 mRNA were detected in glandular epithelium on day 4 of pregnancies. The expression of 15-PGDH and CBR1 mRNA was also detected in postimplantation embryos. CONCLUSION(S): These data suggest that differentially expressed PGT and 15-PGDH may participate in PG signaling in mouse uterus during implantation and decidualization.

Differential expression of transcriptional repressor snail gene at implantation site in mouse uterus.

The snail superfamily of zinc-finger transcription factors is involved in pronounced cell movements during both embryonic development and tumor progression. This study was to examine snail expression in mouse uterus during early pregnancy and its regulation under pseudopregnancy, delayed implantation, steroid hormone treatment, and artificial decidualization by in situ hybridization and immunohistochemistry. There was a low level of snail mRNA signal and immunostaining in mouse uteri on day 1-4 of pregnancy. When embryo implanted on day 5, both snail mRNA signal and immunostaining were strongly detected in the subluminal stroma immediately surrounding the implanting blastocyst, but not detected in the inter-implantation sites. Under delayed implantation, there was no detectable snail expression. After delayed implantation was terminated by estrogen treatment and embryo implanted, there was a strong level of snail mRNA and immunostaining in the subluminal stroma surrounding the implanting blastocyst, which was similar to that on day 5 of pregnancy. Furthermore, there was no detectable snail expression in mouse uterus on day 5 of pseudopregnancy. From day 6-8 of pregnancy, both snail mRNA signal and immunostaining were detected in the decidua. Our data suggest that snail may play an important role during mouse embryo implantation.

Serial analysis of gene expression in mouse uterus at the implantation site.

Although oligonucleotide chips, cDNA microarrays, differential display reverse transcription-PCR, and other approaches have been used to screen implantation-related molecules, the mechanism by which embryo implantation occurs is still unknown. The aim of this study was to profile the differential gene expression between interimplantation site and implantation site in mouse uterus on day 5 of pregnancy by serial analysis of gene expression (SAGE). In our two SAGE libraries of 11-bp tags, the total numbers of tags sequenced were 48,121 for the interimplantation site and 50,227 for the implantation site. There were 1,039 tags specifically expressed at interimplantation site, and 1,252 tags specifically expressed at the implantation site. Based on the p value, there were 195 tags significantly up-regulated at the interimplantation site and 261 tags significantly up-regulated at the implantation site, of which 100 genes were single matched at the interimplantation site and 127 genes were single matched at the implantation site, respectively. By reverse transcription-PCR, the tag ratio between the implantation site and interimplantation site was verified on 14 significantly changed genes. Using in situ hybridization, 1810014L12Rik, Psmb5, Cd63, Npm1, Fads3, and Tagln2 were shown to be highly expressed at the implantation site compared with the interimplantation site. Compared with the interimplantation site, Ddx39 was strongly expressed in the subluminal stromal cells at the implantation site on day 5 of pregnancy. Ddx39 expression at the implantation site was specifically induced by active blastocysts. Additionally, Ddx39 expression was significantly up-regulated by estrogen in the ovariectomized mice. In our SAGE data, many implantation-related genes were identified in mouse uterus. Our data could be a valuable source for future study on embryo implantation.