Hydroxyl-Functionalized DNAs with Different Linkers and Their Complmentary Duplex Stability

Tert-butyldiphenylsilyl (TBDPS) was testified to be an appropriate orthogonal protecting group for novel 7-hydroxyl-functionalized 8-aza-7-deaza-2′-deoxyadenosine analogues. It was stable in partial and complete hydrogenation reactions used for the different linker preparation. The corresponding phosphoramidites and hydroxyl-functionalized oligodeoxynucleotides were synthesized and identified. The thermal effect of the hydroxyl group with different linkers on DNA duplexes was evaluated. It provided a feasible strategy for the preparation of hydroxyl-functionalized DNAs for the nucleic acid research.     

Possible Roles of mmu-miR-141 in the Endometrium of Mice in Early Pregnancy Following Embryo Implantation

Objective

Embryo implantation is directly affected by genes related to uterine receptivity. Studies have demonstrated the important roles of miRNAs in the regulation of gene expression. Our early miRNA chip analyses revealed that the mmu-miR-141 expression in endometrial tissue is lower after embryo implantation than before it. However, the possible roles of miR-141 in embryo implantation have not yet been elucidated. Here, mmu-miR-141 was designed to detect the expression and role of miR-141 in the endometria of mice in early pregnancy following embryo implantation.

Methods

Real-time PCR and in-situ hybridization were used to study mmu-miR-141 expression in mouse uterus. Cell proliferation was detected by tetrazolium dye (MTT) assay and flow cytometry. Real-time PCR and Western blot analysis were used to confirm the mRNA and protein levels of phosphatase and tensin homolog (PTEN) to determine whether it was the target gene of mmu-miR-141. Enhanced green fluorescent protein (EGFP) fluorescence reporter vector analysis was also performed. A functional study was performed by injecting mice uteri with mmu-miR-141 inhibitor or mimic vectors.

Results

mmu-miR-141 expression was lower on day 6 (D6) than day 4 (D4) and could be increased by progesterone. Reduced mmu-miR-141 could decrease the proliferation activity of stromal cells and promote apoptosis. Upregulation of mmu-miR-141 inhibited PTEN protein expression but downregulation of mmu-miR-141 increased it, while the mRNA level remained unchanged. EGFP fluorescence reporter vector analysis showed that miR-141 targets the 3′-untranslated region of the PTEN mRNA. In addition, when the physiological mmu-miR-141 level was altered on D2 by injecting with inhibitor or mimic, the embryo implantation sites were significantly decreased on D7.

Conclusions

This study demonstrated that mmu-miR-141 might influence cell proliferation and apoptosis in the endometrium by negatively regulating PTEN expression, and could also influence the number of embryo implantation sites. mmu-miR-141 plays an essential role in embryo implantation.     

ATL3 Is a Tubular ER-Phagy Receptor for GABARAP-Mediated Selective Autophagy

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Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg⁻¹·day ⁻¹ BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.     

南极地区苔藓地衣植物的地球化学元素营养富集特征

研究南极苔藓地衣中地球化学元素的营养富集特征,发现K,Ca为苔藓地衣中最活跃元素,主要以主动吸收的方式累积于苔藓地衣植物中,P极易富集在地衣的藻层,参与藻类的有机合成过程,苔藓容易富集环境中的S,Al,Si以被动吸收的方式累积于地衣中,同时Fe,Mg以被动吸收的方式累积于苔藓体内,根据元素的含量和营养作用,研究认为K,Ca苔地衣的大量无机营养元素,S,P为苔藓地衣的中等营养元素,Al,Si为苔藓地衣的环境累积元素。     

MAP2 is required for dendrite elongation,PKA anchoring in dendrites,and proper PKA signal transduction

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.     

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

Background

Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.

Methods

The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.

Results

Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.

Conclusion

Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes rpoB and gyrB1 were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree rpoB proved to be reliable phylogenetic marker for the genus Prevotella.     

Pseudomonas sihuiensis sp. nov., isolated from a forest soil in South China

A Gram-stain negative, motile, rod-shaped bacterium, designated strain WM-2^T, was isolated from a forest soil in Sihui City, South China, and characterized by means of a polyphasic approach. Growth occurred with 0–5 % (w/v) NaCl (optimum 0–1 %) and at pH 5.0–10.5 (optimum pH 8.5) and 4–40 °C (optimum 30 °C) in Luria–Bertani medium. Comparative 16S rRNA gene sequence analyses showed that strain WM-2^T is a member of the genus Pseudomonas and most closely related to P. guguanensis, P. oleovorans subsp. lubricantis, P. toyotomiensis, P. alcaliphila and P. mendocina with 97.1–96.6 % sequence similarities. In terms of gyrB and rpoB gene sequences, strain WM-2^T showed the highest similarity with the type strains of the species P. toyotomiensis and P. alcaliphila. The DNA–DNA relatedness values of strain WM-2^T with P. guguanensis and P. oleovorans subsp. lubricantis was 48.7 and 37.2 %, respectively. Chemotaxonomic characteristics (the main ubiquinone Q-9, major fatty acids C_18:1 ω7c/C_18:1 ω6c, C_16:0 and C_16:1 ω7c/C_16:1 ω6c and DNA G+C content 65.2 ± 0.7 mol%) were similar to those of members of the genus Pseudomonas. Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown aminophospholipid, an unknown phospholipid and five unknown lipids. According to the results of polyphasic analyses, strain WM-2^T represents a novel species in the genus Pseudomonas, for which the name Pseudomonas sihuiensis sp. nov. is proposed. The type strain is WM-2^T (=KCTC 32246^T=CGMCC 1.12407^T).     

杨树胞外蛋白的提取分离及2-D电泳体系的建立

该研究以美洲黑杨杂种优良无性系NL895杨(Populus deltoides×Populus euramericana cv.)组培苗叶片和茎段为研究对象,对NL895杨叶片细胞壁蛋白(cell wall proteins,CWPs)和茎段质外体蛋白(apoplastic proteins,APPs)的提取、分离和双向电泳等技术进行了系统研究。结果表明:10g以上叶片、超声波破碎10min、CaCl2法提取的细胞壁蛋白效果较好,经G-6-PDH酶活性检测,提取的细胞壁蛋白胞质污染率较低;真空渗透法提取的茎段质外体蛋白胞质污染率较前者高,但在允许范围之内。TCA/丙酮沉淀法纯化提取的细胞壁蛋白、pH 4~7的24cm胶条、上样量为500μg的双向电泳体系,其蛋白电泳图谱中的斑点多而清晰,斑点数达550多个,是叶片细胞壁蛋白电泳分析较适合的体系;pH 3~10胶条对茎段质外体蛋白的电泳分离效果较好。该研究初步建立了杨树胞外蛋白的提取、分离及2-DE电泳体系,为木本植物胞外蛋白的研究奠定了基础。