Improvement of soybean transformation via Agrobacterium tumefaciens methods involving α-aminooxyacetic acid and sonication treatments enlightened by gene expression profile analysis.

KEY MESSAGE: Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, ß-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.

Lonicerin alleviates ovalbumin-induced asthma of mice via inhibiting EZH2/NF-κB signaling pathway.

Asthma is the most common chronic disease in the respiratory system of children caused by abnormal immunity that responses to common antigens. Lonicerin exerts anti-inflammatory activity in other inflammatory models through targeting enhancer of zeste homolog 2 (EZH2) that is related to asthma. We sought to explore the role and mechanism of lonicerin in regulating allergic airway inflammation. Mice were intraperitoneally injected 10 µg ovalbumin (OVA) on postnatal day 5 (P5) and P10, and then inhaled 3% aerosolized OVA for 10 min every day on P18-20, to establish asthmatic mice model. Lonicerin (10 or 30 mg/kg) was given to mice by intragastric administration on P16-P20. Notably, the administration of lonicerin amended infiltration of inflammatory cells and mucus hypersecretion. OVA-specific IgE level, inflammatory cell count and inflammatory cytokines in asthmatic mice were reduced after lonicerin treatment. Moreover, it suppressed the activity of EZH2 and activation of nuclear factor-kappa B (NF-ĸB) as evidenced by decreasing tri-methylation of histone H3 at lysine 27 and reducing nuclear translocation of NF-κB p65. In a word, Lonicerin may attenuate asthma by inhibiting EZH2/NF-κB signaling pathway.

Clinical value of blood related indexes in the diagnosis of bacterial infectious pneumonia in children.

BACKGROUND: This study sought to explore the value of hematological indexes [i.e., the neutrophil count/lymphocyte count ratio (NLR), the platelet count/lymphocyte count ratio (PLR), and red cell distribution width (RDW) index] in the diagnosis of bacterial infectious pneumonia in children. METHODS: Fifty cases of mycoplasma infectious pneumonia, 50 cases of bacterial infectious pneumonia and 50 healthy children were enrolled in this study. The differences between the NLR, PLR, and RDW index values in each group were compared using the Mann-Whitney test. The correlation coefficients of the NLR, PLR, and RDW index with the interleukin-6 (IL-6) and procalcitonin (PCT) were analyzed using the Spearman's rank test. The specificity and sensitivity of the NLR, PLR, and RDW index in the diagnosis of bacterial pneumonia in children were evaluated by receiver operating characteristic (ROC) curves. RESULTS: The NLR value of the children with bacterial infectious pneumonia was significantly higher than that of the children with mycoplasma infectious pneumonia (P<0.05) and healthy children (P<0.05), while the PLR value of the children with bacterial infectious pneumonia was significantly lower than that of the children with mycoplasma infectious pneumonia (P<0.05) and healthy children (P<0.05). There was no significant difference in the RDW index values of the healthy control children and the children with mycoplasma infectious pneumonia and bacterial infectious pneumonia (P>0.05). There was a positive correlation between NLR and serum IL-6 (R=0.203; P=0.041), and a negative correlation between PLR and serum PCT (R=-0.291; P=0.037). In addition, there was no significant correlation between the RDW index and serum IL-6, and the RDW index and serum PCT in children with bacterial infectious pneumonia. When the 3 indicators were each used to differentiate between healthy children and children with bacterial pneumonia, the area under the PLR curve was the largest for the ROC curve [0.898, 95% confidence interval (CI): 0.815-0.953]. In the differential diagnosis of mycoplasma pneumonia and bacterial pneumonia, the area under the PLR curve was also the largest (0.803, 95% CI: 0.577-0.780). CONCLUSIONS: The PLR has clinical value in the diagnosis of bacterial infectious pneumonia in children.

Identification of 10 differently expressed lncRNAs as prognostic biomarkers for prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) is one of the most frequently diagnosed cancer in males. Previous studies had demonstrated long non-coding RNAs (lncRNAs) played crucial roles in human cancers. In present study, we reported ten disease-free survival time related lncRNAs in PRAD, including RP11-468E2.5, GS1-393G12.13, CTD-2228K2.7, RP11-783K16.13, RP11-631N16.4, CTC-435M10.12, RP11-1109F11.5, RP11-228B15.4, RP11-496I9.1, and RP11-95O2.5. Higher expression of these lncRNAs significantly correlates to shorter DFS time in patients with PRAD. We next constructed lncRNAs regulating PPI networks in PRAD. Bioinformatics analysis revealed these DFS-related lncRNAs were associated with the regulation of cell cycle, glucose metabolic process, histone modification, and RNA splicing. AR and SPOP were identified to be involved in regulating these lncRNAs expression in PRAD. The prognostic value and molecular functions of these lnRNAs in human diseases remained largely unknown. We thought this study for the first time demonstrated that they could act as novel potential biomarkers for PRAD.

Research Progress on Flavor Compounds and Microorganisms of Maotai Flavor Baijiu.

Maotai-flavor liquor is one of the three basic traditional Chinese baijiu and is also the most famous baijiu in the world. Guizhou Maotai baijiu is the representative of Maotai-flavor liquor, which has a long history of culture and is prepared using unique brewing methods. However, the main flavor of Maotai-flavor liquor as well as the mechanism by which its aroma is produced is unclear. In this review, the Da-qu production and fermentation processes for Maotai-flavor liquor are briefly described along with the flavoring constituents of Maotai-flavor liquor that have been recently reported. In addition, the volatile compounds and the aroma derived from Maotai-flavor liquor are discussed. Finally, the microorganisms for the high-temperature Daqu and fermentation processes of Maotai-flavor liquor are discussed. PRACTICAL APPLICATION: Maotai is one of the most famous baijiu in China and the most valuable in the market. However, it is unclear what is the key flavor of Maotai and what microbial metabolism is produced. So, if we can figure out the key flavor substances of Maotai baijiu, we can use the various technology to explore the microbes that produce this flavor to understand the mechanism of the production of Maotai. This will not only achieve breakthroughs in academic value, but also bring higher value to Maotai. On this basis, we can brew Maotai baijiu with better quality according to the fermentation mechanism of Maotai.

[Seasonal variation of microflora in Sinonovacula constricta and its aquacultural pond based on high-throughput sequencing]. / åŸºäºŽé«˜é€šé‡æµ‹åºçš„ç¼¢è›åŠå ¶å »æ®–æ± å¡˜èŒç¾¤ç»“æž„çš„å­£èŠ‚å˜åŒ–.

In order to explore the seasonal changes of the bacterial community structure and the interaction of environmental factors in Sinonovacula constricta ponds, we used high throughput sequencing technology to examine the bacteria of water, sediment, and viscera. The results showed that microflora structure of water samples in winter was significantly different from that in spring, summer and autumn, while there was no significant difference in bacterial community structure of sediment and viscera in different seasons. There was no significant difference of Shannon diversity index in water across different seasons. The Shannon diversity index of sediment and viscera was the lowest in summer and the highest in winter. At the phylum level, Cyanobacteria, Proteobacteria and Tenericutes were the most predominant bacteria in water, sediment, and viscera, respectively. At the genus level, NS3a_marine_group was predominant in winter water, and Synechococcus in the other three seasons. By contrast, dominant bacteria in sediments were norank_f_Anaerolineacea and Nitrospira, and Mycoplasma and Arcobacter were the most abundant bacterial genera in viscera. Synechococcus had a positive correlation with water temperature, COD, PO4--P, NH4+-N, pH, and transparency. The norank_f_Anaerolineacea was positively correlated with water temperature, COD, and TP. Mycoplasma was positively correlated with water temperature, PO4--P, NH4+-N, pH, and transparency. Our results suggest that there were significant differences in the composition and diversity of microflora of S. constricta and ponds in different seasons. Bacteria in water was obviously affected by various environmental factors, especially water temperature and the concentrations of nitrogen and phosphorus.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Human mesenchymal stem cells improve the neurodegeneration of femoral nerve in a diabetic foot ulceration rats.

Neuropathy is observed in 50% of diabetic patients with diabetic foot. This study attempted to explore the potential role of human mesenchymal stem cells-umbilical cord blood (hMSCs-UC) in femoral nerve (FN) neuropathy. The model rats were established by one time administration of streptozotocin and empyrosis on the dorsal hind foot. At 3d, 7d, 14d after treatment with hMSCs-UC or saline through left femoral artery, the serum NGF was examined by ELISA; NF-200 expression in FN was evaluated by immunohistochemistry; the diameter and roundness of FN, the ratio of capillary and muscular fiber of gastrocnemius were calculated under light microscope; and neuronal degenerations, such as demyelization, axonal atrophy, and loose arrangement of nerve fibers, were observed by electronic microscope. The results showed that, in hMSCs-UC-treated model rats, serum NGF was increased with higher positive rate of NF-200. Although the difference in FN diameters was not established among groups, improvement of roundness of FN was confirmed with increase in the numbers of capillary in FN-innervated gastrocnemius; additionally, degenerative neuropathy was significantly improved. Importantly, the functional study of electroneurogram (ENG) showed that, slowed conduction of FN in model rats was significantly restored by hMSCs-CU treatment. These data suggested that hMSCs-UC-treatment partially reverse the neuronal degeneration and nerve function of FN, which might be contributed by the upregulation of NGF with dramatic angiogenesis in FN-innervated gastrocnemius, consequently reversing neuronal structure and function, preventing or curing foot ulceration.

Genetic polymorphisms in the PDZK1 gene and susceptibility to gout in male Han Chinese: a case-control study.

PDZK1 acts as a scaffolding protein for a large variety of transporter and regulatory proteins, and has been identified in the kidney. The PDZK1 locus has been determined to be associated with the serum urate concentration. However, the evidence supporting this protein's association with gout is equivocal. In the current study, we investigated the association between two single nucleotide polymorphisms (SNPs) (rs12129861 and rs1967017) in the PDZK1 gene with gout in a male Chinese Han population. A total of 824 subjects were enrolled in this case-control study (400 gout cases and 424 controls). PDZK1 genotyping was carried out by polymerase chain reaction (PCR) and ligase detection reaction (LDR) assays methods. The relationships were evaluated using the pooled odds ratios (ORs) and their 95 % confidence intervals (CI). The results of our case-control study demonstrated that the gout and control groups exhibited significant differences in the distribution of genotypes at rs12129861 (OR = 0.727, P = 0.015) and rs1967017 (OR = 0.705, P = 0.016), suggesting that PDZK1 genetic polymorphisms were associated with increased risks of gout in male Han Chinese. However, there were no differences in the distribution of genotypes at rs12129861 (odds ratio (OR) = 0.744, P > 0.05) and rs1967017 (OR = 0.706, P > 0.05) in patients with gout with kidney stones and without kidney stones.

Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats.

The aim of this study is to explore the localization of human mesenchymal stem cells from umbilical cord matrix (hMSCs-UC) and the role of these cells in the repair of foot ulcerate tissue in diabetic foot ulcers in rats. A diabetic rat model was established by administering Streptozotocin. Diabetic foot ulceration was defined as non-healing or delayed-healing of empyrosis on the dorsal hind foot after 14 weeks. hMSCs-UC were delivered through the left femoral artery. We evaluated the localization of hMSCs-UC and their role in tissue repair in diabetic foot ulcers by histological analysis, PCR, and immunohistochemical staining. A model for diabetes was established in 54 out of 60 rats (90% success rate) and 27 of these rats were treated with hMSCs-UC. The area of ulceration was significantly and progressively reduced at 7 and 14 days following treatment with hMSCs-UC. This gross observation was strongly supported by the histological changes, including newly developed blood vessels and proliferation of inflammatory cells at 3 days post-treatment, significant increase in granulation tissue at 7 days post-treatment and squamous epithelium or stratified squamous epithelium at 14 days post-treatment. Importantly, human leukocyte antigen type-I (HLA-1) was confirmed in ulcerated tissue by RT-PCR. The expression of cytokeratin 19 was significantly increased in diabetic model rats, with no detectable change in cytokeratin 10. Additionally, both collagens I and III increased in model rats treated with hMSCs-UC, but the ratio of collagen I/III was less significant in treated rats compared with control rats. These results suggest that hMSCs-UC specifically localize to the target ulcerated tissue and may promote the epithelialization of ulcerated tissue by stimulating the release of cytokeratin 19 from keratinocytes and extracellular matrix formation.

[Adaptability of cucumber plug seedlings to daytime temperature].

Cucumber plug seedlings were cultured in a phytotron to study their responses to different daytime temperature. The daytime temperature was controlled at 30 degrees C, 27 degrees C, 24 degrees C, 21 degrees C, 18 degrees C and 15 degrees C, and the night temperature was at 15 degrees C, with the thickness and length of hypocotyl, length and width of the first and the second leaves, dry matter accumulation of above- and underground parts, leaf moisture content, and leaf chlorophyll fluorescence parameters measured. Principal component analysis and clustering analysis were made to analyze the seedling quality under different temperature treatment. There existed significant differences in the test growth indices among different treatments, with the seedling quality decreased at the daytime temperature 24 degrees C > 21 degrees C > 27 degrees C > 30 degrees C > 18 degrees C > 15 degrees C. All the daytime/nighttime temperature treatments could be classified into three groups, i.e., optimum temperature (24 degrees C/15 degrees C), appropriate temperature (21 degrees C/15 degrees C), and inappropriate temperature, and the inappropriate temperature could be further subdivided into two groups, i.e., high-temperature inhibition (27 degrees C/15 degrees C, 30 degrees C/15 degrees C) and low-temperature inhibition (15 degrees C/15 degrees C, 18 degrees C/15 degrees C).

[Cloning and expression pattern analysis of a lipopolysaccharide -and beta-1,3-glucan-binding protein in Portunus trituberculatus].

The lipopolysaccharide -and beta-1,3-glucan-binding protein (LGBP) is a pattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified. The expression of P. trituberculatus in various tissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic process against bacterial infection.