High-Resolution 3D Genome Map of Brucella Chromosomes in Exponential and Stationary Phases.

The three-dimensional (3D) genome structure of an organism or cell is highly relevant to its biological activities, but the availability of 3D genome information for bacteria, especially intracellular pathogens, is still limited. Here, we used Hi-C (high-throughput chromosome conformation capture) technology to determine the 3D chromosome structures of exponential- and stationary-phase Brucella melitensis at a 1-kb resolution. We observed that the contact heat maps of the two B. melitensis chromosomes contain a prominent diagonal and a secondary diagonal. Then, 79 chromatin interaction domains (CIDs) were detected at an optical density at 600 nm (OD600) of 0.4 (exponential phase), with the longest CID being 106 kb and the shortest being 12 kb. Moreover, we obtained 49,363 significant cis-interaction loci and 59,953 significant trans-interaction loci. Meanwhile, 82 CIDs of B. melitensis at an OD600 of 1.5 (stationary phase) were detected, with the longest CID being 94 kb and the shortest being 16 kb. In addition, 25,965 significant cis-interaction loci and 35,938 significant trans-interaction loci were obtained in this phase. Furthermore, we found that as the B. melitensis cells grew from the logarithmic to the plateau phase, the frequency of short-range interactions increased, while that of long-range interactions decreased. Finally, combined analysis of 3D genome and whole-genome transcriptome (RNA-seq) data revealed that the strength of short-range interactions in Chr1 is specifically and strongly correlated with gene expression. Overall, our study provides a global view of the chromatin interactions in the B. melitensis chromosomes, which will serve as a resource for further study of the spatial regulation of gene expression in Brucella. IMPORTANCE The spatial structure of chromatin plays important roles in normal cell functions and in the regulation of gene expression. Three-dimensional genome sequencing has been performed in many mammals and plants, but the availability of such data for bacteria, especially intracellular pathogens, is still limited. Approximately 10% of sequenced bacterial genomes contain more than one replicon. However, how multiple replicons are organized within bacterial cells, how they interact, and whether these interactions help to maintain or segregate these multipartite genomes are unresolved issues. Brucella is a Gram-negative, facultative intracellular, and zoonotic bacterium. Except for Brucella suis biovar 3, Brucella species have two chromosomes. Here, we applied Hi-C technology to determine the 3D genome structures of exponential- and stationary-phase Brucella melitensis chromosomes at a 1-kb resolution. Combined analysis of the 3D genome and RNA-seq data indicated that the strength of short-range interactions in B. melitensis Chr1 is specifically and strongly correlated with gene expression. Our study provides a resource to achieve a deeper understanding of the spatial regulation of gene expression in Brucella.

Comprehensive Evaluation of Quality of Camellia semiserrata Seed Oils from Various Harvest Dates.

The ripening degree of camellia fruit is one of the key factors affecting the quality of camellia seed oil. In this study, taking Camellia semiserrata as the research object, the oil content, physicochemical indexes, nutritional indexes, fatty acid composition, and volatile compounds of camellia seed oils from various harvest dates (from September to October) were determined. The results showed that with the increase of the ripening degree of camellia fruit, the oil content of camellia seed increased at first and then decreased and reached the highest (58.74%) on September 30, while the acid value, peroxide value, ß-sitosterol, α-tocopherol, and polyphenols of camellia seed oil showed a downward trend. Among them, the highest contents of ß-sitosterol, α-tocopherol, and polyphenols were observed on September 2, which were 6881.60, 311.34, and 78.08 mg/kg, respectively. In terms of the fatty acid composition of camellia seed oils, the content of oleic acid increased at first and then decreased, the content of linoleic acid and palmitic acid decreased gradually, while the content of stearic acid increased gradually. A total of 37 volatile compounds were identified in different samples, including 12 aldehydes, 5 ketones, 12 alcohols, 2 acids, 5 esters, and 1 other. With the increase of the ripening degree, the concentration of aldehydes and alcohols increased at first and then decreased, the concentration of ketones and esters decreased gradually, but the concentration of acid compounds had no obvious rule. In addition, the camellia seed oils from various harvest dates were classified and comprehensively evaluated by principal component analysis and grey relation analysis. The results showed that different camellia seed oils could be divided into three groups, and the comprehensive score of camellia seed oils on September 30 was the highest. In general, this work can provide theoretical guidance for the harvest date of Camellia semiserrata.

Genome-wide transcription start site mapping in the facultative intracellular pathogen Brucella melitensis by Capping-seq.

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that -35 and -10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5' ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5' ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella.

The role of NUDT21 in microRNA-binging sites of EZH2 gene increases the of risk preeclampsia.

OBJECTIVES: Preeclampsia (PE) is a major cause of mortality and morbidity among pregnant mothers and their fetuses worldwide. Recent studies have shown that several microRNAs (miRNAs) play crucial role in pathogenesis of PE patients; however, the mechanisms responsible for differences in miRNA function in PE largely remain to be determined. MATERIALS AND METHODS: We studied that NUDT21 expression was markedly increased, whereas EZH2 was decreased in placental samples from patients with PE. We identified NUDT21 as an interaction partner of enhancer of zeste homologue 2 (EZH2). NUDT21 co-localized with EZH2 in the human trophoblast cell line, HTR-8/SVneo and NUDT21 was shown to bind to EZH2 in RNA immunoprecipitation assays. NUDT21 has previously been reported to be involved in alternative polyadenylation; thus, the interaction between NUDT21 and EZH2 may play an important role in the crosstalk between alternative polyadenylation (APA) and miRNA-mediated gene silencing in PE. RESULTS: In the human trophoblast cell line HTR-8/SVneo, loss-of-function assays indicated that knockdown of NUDT21 suppressed cell proliferation, migration and tube formation. Furthermore, functional studies showed that NUDT21 elongated the 3'-UTR of mRNAs thereby exposing more miRNA binding sites (including miR138 and miR363), which enhanced the efficiency of miRNA-mediated gene silencing and promoted EZH2 binding. CONCLUSIONS: This is the first report about the relationship of NUDT21 and EZH2. The data indicate that the aberrant expression of NUDT21 contributes to PE by targeting 3'-UTR of EZH2 mRNA. These findings may provide novel targets for future investigations into therapeutic strategies for PE.

Epigallocatechin gallate inhibits cell growth and regulates miRNA expression in cervical carcinoma cell lines infected with different high-risk human papillomavirus subtypes.

The aim of the present study was to investigate the inhibitory effects of the polyphenol epigallocatechin-3-gallate (EGCG) on the growth of cervical carcinoma cell lines infected with different high-risk human papillomavirus (HPV) subtypes, as well as the associated regulation of microRNA (miR) expression. Cell proliferation was measured using an MTT assay. The effects of 7 different concentrations of EGCG (100, 80, 60, 40, 20, 10 and 0 µg/ml) on HeLa cell proliferation were assessed. HeLa cell growth was significantly inhibited by EGCG in a dose- and time-dependent manner (P<0.05), and the IC50 was 90.74 and 72.74 µg/ml at 24 and 48 h, respectively. The expression of miR-210, miR-29a, miR-203 and miR-125b in HeLa (HPV16/18+), SiHa (HPV16+), CaSki (HPV16+) and C33A (HPV-) cell lines was measured using quantitative polymerase chain reaction analysis. In CA33 cells, miR-203 (all P<0.001) and miR-125b (P<0.01 and <0.0001) were significantly downregulated by EGCG, and miR-210 was significantly upregulated with 40 and 60 µg/ml EGCG (P<0.0001). miR-125b was significantly downregulated (P<0.001 and <0.0001), and miR-210 and miR-29 were significantly upregulated by ≤80 µg/ml EGCG in HeLa cells (all P<0.0001). In CaSki cells, miR-210, miR-29a (all P<0.001) and miR-125b (P<0.01-0.0001) were significantly upregulated by EGCG. In SiHa cells, miR-125b (both P<0.001) and miR-203 (P<0.01 and <0.0001) were significantly upregulated by EGCG. In conclusion, the results of the present study suggest that EGCG suppresses cervical carcinoma cell growth, possibly via regulating the expression of miRs, suggesting their potential as therapeutic targets for the control and prevention of cervical cancer. Additionally, EGCG may be considered a novel anti-cervical cancer drug in the future.

Isolation of Flavonoids From Wild Aquilaria sinensis Leaves by an Improved Preparative High-Speed Counter-Current Chromatography Apparatus.

Four flavonoids including apigenin-7,4'-dimethylether, genkwanin, quercetin, and kaempferol were isolated in a preparative or semi-preparative scale from the leaves of wild Aquilaria sinensis using an improved preparative high-speed counter-current chromatography apparatus. The separations were performed with a two-phase solvent system composed of hexane-ethyl acetate, methanol-water at suitable volume ratios. The obtained fractions were analyzed by HPLC, and the identification of each target compound was carried out by ESI-MS and NMR. The yields of the above four target flavonoids were 4.7, 10.0, 11.0 and 4.4%, respectively. All these four flavonoids exhibited nitrite scavenging activities with the clearance rate of 12.40 ± 0.20%, 5.84 ± 0.03%, 28.10 ± 0.17% and 5.19 ± 0.11%, respectively. Quercetin was originally isolated from the Thymelaeaceae family, while kaempferol was isolated from the Aquilaria genus for the first time. In cytotoxicity test these two flavonoids exhibited moderate inhibitory activities against HepG2 cells with the IC50 values of 12.54 ± 1.37 and 38.63 ± 4.05 µM, respectively.

Interacting Effects Induced by Two Neighboring Pits Considering Relative Position Parameters and Pit Depth.

For pre-corroded aluminum alloy 7075-T6, the interacting effects of two neighboring pits on the stress concentration are comprehensively analyzed by considering various relative position parameters (inclination angle θ and dimensionless spacing parameter λ) and pit depth (d) with the finite element method. According to the severity of the stress concentration, the critical corrosion regions, bearing high susceptibility to fatigue damage, are determined for intersecting and adjacent pits, respectively. A straightforward approach is accordingly proposed to conservatively estimate the combined stress concentration factor induced by two neighboring pits, and a concrete application example is presented. It is found that for intersecting pits, the normalized stress concentration factor Ktnor increases with the increase of θ and λ and always reaches its maximum at θ = 90°, yet for adjacent pits, Ktnor decreases with the increase of λ and the maximum value appears at a slight asymmetric location. The simulations reveal that Ktnor follows a linear and an exponential relationship with the dimensionless depth parameter Rd for intersecting and adjacent cases, respectively.

Transcriptome analysis of the tea oil camellia (Camellia oleifera) reveals candidate drought stress genes.

BACKGROUND: The tea-oil camellia (Camellia oleifera) is the most important oil plant in southern China, and has a strong resistance to drought and barren soil. Understanding the molecular mechanisms of drought tolerance would greatly promote its cultivation and molecular breeding. RESULTS: In total, we obtained 76,585 unigenes with an average length of 810 bp and an N50 of 1,092 bp. We mapped all the unigenes to the NCBI 'nr' (non-redundant), SwissProt, KEGG, and clusters of orthologous groups (COG) databases, where 52,531 (68.6%) unigenes were functionally annotated. According to the annotation, 46,171 (60.8%) unigenes belong to 338 KEGG pathways. We identified a series of unigenes that are related to the synthesis and regulation of abscisic acid (ABA), the activity of protective enzymes, vitamin B6 metabolism, the metabolism of osmolytes, and pathways related to the biosynthesis of secondary metabolites. After exposed to drought for 12 hours, the number of differentially-expressed genes (DEGs) between treated plants and control plants increased in the G4 cultivar, while there was no significant increase in the drought-tolerant C3 cultivar. DEGs associated with drought stress responsive pathways were identified by KEGG pathway enrichment analysis. Moreover, we found 789 DEGs related to transcription factors. Finally, according to the results of qRT-PCR, the expression levels of the 20 unigenes tested were consistent with the results of next-generation sequencing. CONCLUSIONS: In the present study, we identified a large set of cDNA unigenes from C. oleifera annotated using public databases. Further studies of DEGs involved in metabolic pathways related to drought stress and transcription will facilitate the discovery of novel genes involved in resistance to drought stress in this commercially important plant.

Downregulation of human epidermal growth factor receptor 2 by short hairpin RNA increases chemosensitivity of human ovarian cancer cells.

The aim of the current study was to investigate the suppressive effects of pSilencer T7-human epidermal growth factor receptor 2 (HER2)-short hairpin RNA (shRNA) recombinant plasmids on human SKOV3 ovarian cancer cell growth and sensitivity to carboplatin (CBP). Three different pairs of shRNAs (shRNAa, shRNAb and shRNAc), targeting the HER2 gene, were selected and transfected into human SKOV3 cells, respectively. The expression levels of HER2 were then detected by immunohistochemical (IHC), semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. In addition, cell cycle and cell growth were investigated using cell counting kit-8 (CCK-8). The results of the IHC and western blot analyses revealed that shRNAb significantly inhibited HER2 protein expression in SKOV3 cells. shRNAb exhibited an improved effect on HER2 expression compared with shRNAa (P<0.01), while shRNAc did not affect HER2 expression. Nontransfected and nonspecific shRNA groups were used as the negative controls. Knockdown of HER2 expression by shRNA was initiated at 24 h following transfection, achieving an optimum effect at 48 h and lasting for at least 72 h after the treatment. The CCK-8 cell growth assay indicated that the knockdown of HER2 expression in the SKOV3 cell line resulted in significant growth suppression and cell cycle arrest. In addition, inhibition of HER2 significantly increased SKOV3 cell sensitivity to CBP treatment. In conclusion, pSilencer T7-HER2-shRNA significantly inhibited HER2 expression in human ovarian cancer cells in vitro and induced chemotherapeutic sensitivity to CBP.

[Comparison on efficacy between the different timings of methotrexate administration combined with laproscopy in the treatment of salpingocyesis].

OBJECTIVES: To compare pretreatment and intra-operative methotrexate (MTX) administration when combined with laproscopy in the treatment of salpingocyesis. METHODS: All 200 patients with salpingocyesis treated with laproscopy in Jiangwan Hospital from Jan, 2010 to Dec, 2014 were enrolled and divided into group A and B, 100 patients in each. In group A, the patients were administered with MTX 50 mg/kg by intramuscular injection 24-48 h before the surgery. In group B, the patients were administered with MTX 20 mg in 2 ml normal saline to the target tubal seromuscular layer after the ectopic pregnancies were removed. Then, the following characteristics would be compared between the groups including the medical condition during the operation, post-operative body temperature, serum ß-human chorionic gonadotropin (ß-HCG) reduction, the rate of refractory ectopic pregnancy and tubal patency. RESULTS: The bleeding volume during the surgery (P<0.01) was significantly reduced and the operation duration (P<0.05) was significantly shortened in group A. The serum ß-HCG level (P<0.05) was also significantly reduced 4 and 7 days after the surgery in group A. There was significantly difference on the rate of tubal patency (P<0.01) between the groups. There was no refractory ectopic pregnancy in either groups after the surgery. And there were no significant differences on the post-operative temperature, period of serum ß-HCG moving to the normal level between the groups (P>0.05). CONCLUSIONS: Both pretreatment and intra-operative MTX administration showed good efficacy. But the surgery duration shortening, bleeding decreasing during the operation and tubes retaining were seen in group A. For this reason, we believe pro-operative MTX administration would be better for the patients with childbearing requests.

MiR-125b inhibits tumor growth and promotes apoptosis of cervical cancer cells by targeting phosphoinositide 3-kinase catalytic subunit delta.

BACKGROUND: [corrected] microRNAs (miRNAs) are involved in cancer-related processes. The miRNA-125b (miR-125b) has been identified as miRNA over-expressed in a wide variety of cancers. However, the role of miR-125b in the context of cervical carcinoma remains unknown. METHODS: In this study, the effect of miR-125b on the proliferation and apoptosis of human cervical cells was analyzed by MTT assay and Flow cytometry analysis. we identified phosphoinositide 3-kinase catalytic subunit delta (PIK3CD) as a novel miR-125b target. RESULTS: overexpression of miR- 125b in HeLa cervical cancer cells decreased cell proliferation, induced apoptosis and down-regulated expression of PIK3CD. To identify the mechanisms responsible, we investigated the PI3K/Akt pathway and found that PI3K, phospho-Akt, and phospho-mTOR were all down-regulated, while Bid was up-regulated in miR-125b-overexpressing subclones. In vivo, over expression of miR-125b in HeLa cells markedly reduced their ability to form tumors. CONCLUSION: these results suggest that miR-125b suppresses tumor growth activity by targeting the PI3K/ Akt/mTOR signaling pathway, and may provide a target for effective therapies.