Singlet oxygen induces cell wall thickening and stomatal density reducing by transcriptome reprogramming.

Singlet oxygen (1O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants.

Two new photoactive metal-organic compounds for degradation of methylene blue and treatment in ophthalmic local anesthesia.

Two new metal-organic compounds, namely [Cu(CrO4)(4,4'-bipy)(H2O)]·n(H2O) (1) together with [Mn(Cr2O7)(bpp)2]n (2) (4,4'-bipy is 4,4'-bipyridine and bpp is 1,3-bis(4-pyridyl)propane), were hydrothermally generated, which were characterized structurally through a series of characterization techniques. Moreover, compounds 1 and 2 have 2.95 eV and 3.02 eV of narrow optical band gap values, and possess outstanding photocatalytic effects for the methylene blue degradation under irradiation of visible light. The application of above compounds in the ophthalmic local anesthesia was examined and the specific mechanism was tested. First of all, the acetylcholine content in the synaptic cleft was measured with enzyme linked immunosorbent assay (ELISA) assay after treated with the CPs. The acetylcholine receptor relative expression on nerve cells was subsequently measured via real time reverse transcription-polymerase chain reaction (RT-PCR) under the treatment of compounds. In the end, the complexes' toxicity was evaluated by Cell Counting Kit-8 (CCK-8) detection.

[Spatial Characteristics and Potential Ecological Risk Factors of Heavy Metals in Cultivated Land in the Transition Zone of a Mountain Plain].

The purpose of this study was to explore the spatial distribution characteristics and potential ecological risk factors of heavy metal pollution in cultivated soil in the transitional zone of a typical mountain plain. In this study, a county in Sichuan Province, which has the typical transitional topography of the Chengdu Plain-Longmen Mountain transition zone, was taken as the study area. Geostatistics and potential ecological risk index methods were used to analyze the spatial distribution of Cd, Cr, Cu, Pb, and Zn from cultivated land in the study area, and a potential ecological risk assessment was carried out. This was combined with geographical detector analysis to further explore the main influencing factors leading to the spatial difference in potential ecological risk. The results showed that:① The average values of ω(Cd), ω(Cr), ω(Cu), ω(Pb), and ω(Zn) of cultivated land in the study area were 0.39, 123.00, 31.28, 51.04, and 119.66 mg·kg-1, respectively, which were more than 2.60, 1.59, 1.19, 2.00, and 1.54 times the soil background value in the study area. ② The high value areas of Cd and Cr in cultivated land in the study area were mainly distributed in the west of the mountain area, the south of the transition zone, and the west of the plain area; the high value areas of Cu and Zn were mainly distributed in the west of the mountain area; the high value areas of Pb were mainly distributed in the south of the transition zone and the west of the plain area; and the low values of the five heavy metals were mainly distributed in the north of the transition zone. ③ The soil of cultivated land in the study area was dominated by slight potential ecological risk, and the average value of potential ecological risk index of heavy metals was Cd(32.58) > Pb(3.12) > Cu(2.82) > Cr(1.58) > Zn(0.98). Cd was the main factor causing the potential ecological risk of cultivated land in the study area. ④ There were significant differences in the key influencing factors of soil potential ecological risk of cultivated land in different topographic regions of the study area. The distance from the river and the soil type were the key factors affecting the mountain area; the multiple cropping index and the distance from the industry and mine were the key factors affecting the transition zone, and the soil pH value and the amount of chemical fertilizer were the key factors affecting the plain area.

Prediction models for development of hepatocellular carcinoma in chronic hepatitis B patients.

Chronic hepatitis B (CHB)-related hepatocellular carcinoma (HCC) is a major health problem in Asian-Pacific regions. Antiviral therapy reduces, but does not completely prevent, HCC development. Thus, there is a need for accurate risk prediction to assist prognostication and decisions on the need for antiviral therapy and HCC surveillance. A few risk scores have been developed to predict the occurrence of HCC in CHB patients. Initially, the scores were derived from untreated CHB patients. With the development and extensive clinical application of nucleos(t)ide analog(s) (NA), the number of risk scores based on treated CHB patients has increased gradually. The components included in risk scores may be categorized into host factors and hepatitis B virus factors. Hepatitis activities, hepatitis B virus factors, and even liver fibrosis or cirrhosis are relatively controlled by antiviral therapy. Therefore, variables that are more dynamic during antiviral therapy have since been included in risk scores. However, host factors are more difficult to modify. Most existing scores derived from Asian populations have been confirmed to be accurate in predicting HCC development in CHB patients from Asia, while these scores have not offered excellent predictability in Caucasian patients. These findings support that more relevant variables should be considered to provide individualized predictions that are easily applied to CHB patients of different ethnicities. CHB patients should receive different intensities of HCC surveillance according to their risk category.

[Effect of the small needle knife through the Zusanli(ST 36) on behavior and hippocampal expression of NLRP3 and IL-1ß in myalgia comorbid depressed rats].

OBJECTIVE: To observe the effect of the small needle knife through the Zusanli(ST 36) on behavior and hippocampal expression of NLRP3 and IL-1ß in myalgia comorbid depressed rats. METHODS: The rat models of myalgia comorbid depression were prepared by intraperitoneal injection of acute reserpine. Twenty-four SD male rats were randomly divided into control group, model group, small needle knife group and amitriptyline group, 6 rats in each group. The open field behavior and mechanical pain threshold of each group were detected. The thermal pain threshold was detected by intelligent hot plate test. The expression of NLRP3 and IL-1ß in hippocampus of rats was detected by Western blotting. RESULTS: Compared with the model group, the mechanical pain threshold of the foot was significantly improved in the small needle knife group (P<0.01). Compared with the amitriptyline group, the small needle knife stimulation of Zusanli(ST 36) can significantly increase the thermal pain threshold in rats(P<0.05); in the comparison of the horizontal movement distance and the number of crossings in the open field behavioral rats, the total distance of the open field activity of the small needle knife group was significantly increased(P<0.05). Compared with the model group, the number of crossings in the small needle knife group had no statistically significant difference (P>0.05). The expressions of NLRP3 and IL-1ß in the hippocampus of the model group were significantly increased(P<0.05), and the expression of IL-1ß in the small needle knife group was significantly decreased (P<0.05). The stimulation of small needle knife at Zusanli(ST 36) could inhibit the expression of NLRP3 in hippocampus of rats. However, there was no statistically significant difference compared with the model group (P>0.05). CONCLUSIONS: Small needle knife can improve the pathological state of myalgia comorbid depression caused by reserpine in rats. The mechanism may be related to the inhibition of NLRP3 inflammasome and IL-1ß expression in central hippocampus.

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system.

AIM: To investigate the biological function of F protein by yeast two-hybrid system. METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (alpha type) containing liver cDNA library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection and screening. After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-alpha-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.

[Screening and cloning target genes transactivated by hepatitis C virus F protein using suppression subtractive hybridization technique].

OBJECTIVES: To identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique. METHODS: Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR. RESULTS: The subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown. CONCLUSIONS: The obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system.

AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes. METHODS: We constructed p7 protein bait plasmid by cloning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (alpha type) containing liver cDNA library plasmid, pACT2 in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics. RESULTS: Fifty colonies were selected and sequenced. Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens. CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

[Impact of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2].

OBJECTIVE: To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2. METHODS: Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells. RESULTS: PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells. CONCLUSION: RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

[Application of continuous epidural analgesia with ropivacaine and fentanyl during delivery].

OBJECTIVE: To evaluate the safety and efficiency of ropivacaine and fentanyl for continuous epidural analgesia during delivery. METHODS: Altogether 98 full-term primigravidas with vertex presentation were selected for this study. Epidural catheter was placed during delivery with the cervical dilation of 3 cm, and the mixture of 0.2% ropivacaine and 2 microgram/ml fentanyl at the initial dose of 5 ml was given for continuous epidural analgesia. Drug infusion was discontinued when the second stage of delivery started. Another 98 primigravidas of similar conditions without analgesia served as the control group. RESULTS: Analgesic group showed obvious pain-relieving effect (P<0.001) during the delivery, in which the active phase was significantly shortened (P<0.05). No obvious adverse effect arose in the mother and fetuses from the administration of analgesia. CONCLUSION: Continuous epidural analgesia by pumping ropivacaine and fentanyl is effective and convenient for pain relief during delivery, and can be beneficial to the smooth progress of delivery.