CaERF1- mediated ABA signal positively regulates camptothecin biosynthesis by activating the iridoid pathway in Camptotheca acuminata.

Camptotheca acuminata is one of the primary sources of camptothecin (CPT), which is widely used in the treatment of human malignancies because of its inhibitory activity against DNA topoisomerase I. Although several transcription factors have been identified for regulating CPT biosynthesis in other species, such as Ophiorrhiza pumila, the specific regulatory components controlling CPT biosynthesis in C. acuminata have yet to be definitively determined. In this study, CaERF1, an DREB subfamily of the APETALA2/ethylene response factors (AP2ERFs), was identified in C. acuminata. The transient overexpression and silencing of CaERF1 in C. acuminata leaves confirmed that it positively regulates the accumulation of CPT by inducing the expression of CaCYC1 and CaG8O in the iridoid pathway. Results of transient transcriptional activity assay and yeast one-hybrid assays have showed that CaERF1 transcriptionally activates the expression of CaCYC1 and CaG8O by binding to RAA and CEI elements in the promoter regions of these two genes. Furthermore, the expression of CaCYC1 and CaG8O in CaERF1-silenced leaves was less sensitive to ABA treatment, indicating that CaERF1 is a crucial component involved in ABA-regulated CPT biosynthesis in C. acuminata.

FlhF affects the subcellular clustering of WspR through HsbR in Pseudomonas aeruginosa.

In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of c-di-GMP induces biofilm formation and represses motility. WspR, a hybrid response regulator DGC, produces c-di-GMP when it is phosphorylated. FlhF, a signal recognition particle-type GTPase, is initially localized to the cell poles and is indispensable for polar flagellar localization in Pseudomonas aeruginosa. In this study, we report that deletion of flhF affected biofilm formation and the c-di-GMP level in P. aeruginosa. Phenotypic analysis of a flhF knockout mutant revealed increased biofilm formation, wrinkled colonies on Congo red agar, and an elevated c-di-GMP level compared to the wild-type strain, PAO1. Yeast and bacterial two-hybrid systems showed that FlhF binds to the response regulator HsbR, and HsbR binds to WspR. Deletion of hsbR or wspR in the ΔflhF background abolished the phenotype of ΔflhF. In addition, confocal microscopy demonstrated that WspR-GFP was distributed throughout the cytoplasm and formed a visible cluster at one cell pole in PAO1 and ΔhsbR, but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in ΔflhF. These findings suggest that FlhF influences the subcellular cluster and localization of WspR and negatively modulates WspR DGC activity in a manner dependent on HsbR. Together, our findings demonstrate a novel mechanism for FlhF modulating the lifestyle transition between motility and biofilm via HsbR to regulate the DGC activity of WspR.IMPORTANCECyclic di-GMP (c-di-GMP) is a second messenger that controls flagellum biosynthesis, adhesion, virulence, motility, exopolysaccharide production, and biofilm formation in bacteria. Recent research has shown that distinct diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) produce highly specific outputs. Some DGCs and PDEs contribute to the total global c-di-GMP concentration, but others only affect local c-di-GMP in a microenvironment. However, the underlying mechanisms are unclear. Here, we report that FlhF affects the localization and DGC activity of WspR via HsbR and is implicated in local c-di-GMP signaling in Pseudomonas aeruginosa. This study establishes the link between the c-di-GMP signaling system and the flagellar localization and provides insight for understanding the complex regulatory network of c-di-GMP signaling.

An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

Matrix Metalloproteinases (MMP-2) and Tissue Inhibitors of Metalloproteinases (TIMP-2) in Patients with Inguinal Hernias.

BACKGROUND: The development of inguinal hernia might be related with collagen metabolism, which was regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to evaluate the mRNA and protein expression levels of MMP-2 and TIMP-2 in anterior rectus sheath fascia to investigate the function of them in inguinal hernia formation. METHODS: The study enrolled 48 primary inguinal hernia patients: 32 participants had indirect inguinal hernia and 16 patients suffered direct inguinal hernia. Specimens were taken from the anterior rectus sheath fascia. The amounts of MMP-2 mRNA and TIMP-2 mRNA were evaluated by real-time fluorescence quantitative polymerase chain reaction (RT-PCR), and immunohistochemistry was performed to assess the protein expression of them. RESULTS: The mRNA and protein expression levels of MMP-2 in direct group were significantly higher than those of control group (P < 0.05) and indirect group (P < 0.05), while the expression levels of TIMP-2 in direct group were significantly lower than those of control group (P < 0.05) and indirect group (P < 0.05). The ratio of MMP-2 mRNA/TIMP-2 mRNA in direct group was significantly higher than that of control group (P < 0.05) and indirect group (P < 0.05), and the ratio of indirect group was significantly higher than that of control group (P < 0.05). According to receiver operating characteristic (ROC) curve, MMP-2/TIMP-2 can diagnose direct hernia from controls with area under the curve (AUC) of 0.950 and indirect hernia with AUC of 0.730 effectively. CONCLUSIONS: Elevated level of MMP-2 and decreased level of TIMP-2 may play a role in direct inguinal hernia development. The ratio of MMP-2/TIMP-2 may be useful in identification of direct hernia.

Prospective Analysis of Epigastric, Umbilical, and Small Incisional Hernia Repair Using the Modified Kugel Oval Patch.

The study was performed to determine the complication and recurrence rate using the modified Kugel oval patch for small ventral hernia repair. Another aim of the study was to find risk factors for recurrence in patients with this repair. Seventy-three patients underwent ventral hernia repair with the modified Kugel oval patch between April 2013 and February 2015. Prospective follow-up data (18 months postoperative) were collected and evaluated. A total of 58 primary (18 epigastric/40 umbilical) and 15 incisional ventral hernias were repaired with the modified Kugel oval patch (7.6/10.2 cm/8 × 12 cm). We found three patients suffered superficial wound infection and two participants got hernia recurrence. Placement of mesh was found to be a significant risk factor for hernia recurrence. Small ventral hernia repair using the modified Kugel oval patch is a novel and useful approach and we recommend placing the patch preperitoneally.

Potent and selective inhibition of matrix metalloproteinases by lanthanide trichloride.

Matrix metalloproteinases (MMPs) are a family of Zn-containing and Ca-dependent proteases with vital roles in extracellular matrix remodeling. Deregulation of MMPs occurs in many pathological conditions such as cardiovascular diseases, inflammation, and cancer. The therapeutic potential of MMP inhibitors has been demonstrated in diseases such as arthritis and cancer. Here we demonstrated that the 3-valent lanthanide compounds LaCl3, TbCl3, GdCl3, YbCl3, and EuCl3 inhibit MMPs such as MMP-2, MMP-13, and MMP-14 (MT1-MMP). The inhibition is more potent and selective toward MT1-MMP compared to the other MMPs. EuCl3 was further selected to study the enzyme kinetics of the MT1-MMP inhibition. The results showed that the inhibition is a mixed type with anti-competition and non-competitive types, which indicated that inhibition was achieved by the compound bound to the non-active center of MT1-MMP and changing the enzyme conformation. The interaction between EuCl3 and MT1-MMP was further studied by UV-visible (UV-vis) light absorption. EuCl3 caused a slight blue shift of the maximum absorption wavelength of MT1-MMP, indicating the interaction reduced protein hydrophobicity. Moreover, EuCl3 exerted substantial inhibitory effects on the migration of HT-1080 cells. Thus, EuCl3 may play a role in modulating tumor cell behavior by inhibiting MMPs activities especially the MT1-MMP activity. These findings provide initial insight into the biological activity and potential therapeutic value of EuCl3.

Inactivation mechanism of Vibrio parahaemolyticus via supercritical carbon dioxide treatment.

The effects of supercritical carbon dioxide (SC-CO2) treatments on Vibrio parahaemolyticus cells were determined using viable plate count method at different treatment times (10 and 40min), pressures (10-25MPa), and temperature (40°C). Using the changes in the physical (absorbance, transmission electron microscope and contents of fatty acids) and chemical indexes (pH value, activity of Na+K+-ATPase, SDS-PAGE) were for further understand the mechanisms of bacterial inactivation under SC-CO2. The result showed that 25MPa treatment for 40min in 40°C could significantly (P<0.05) enhance inactivation of V. parahaemolyticus. The pH value and activity of Na+K+-ATPase of SC-CO2 treated groups significantly (P<0.05) decreased compared with blank group. Damage to the cell membrane and cytoplasmic components can be observed on transmission electron microscope images. Results of SDS-PAGE and UV-absorbing substances also showed that the leakage of proteins and cytoplasmic materials increased with the SC-CO2 treatment time and pressure. Therefore, our results indicate that SC-CO2 can be applied to inactivate V. parahaemolyticus by causing a low pH, as well as severe damage to key substances and structures.

Development and validation of a subcritical 1,1,1,2-tetrafluoroethane extraction technique: Gas chromatography-mass spectrometry method for the simultaneous determination of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and polybrominated biphenyl ethers in aquatic products.

A simple, rapid, and green method was developed for the simultaneous analysis of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and polybrominated biphenyl ethers in aquatic products using subcritical 1,1,1,2-tetrafluoroethane extraction coupled with gas chromatography and mass spectrometry. Effects of the extraction temperature, pressure, and cosolvent volume on the extraction efficiency were investigated by extracting spiked oyster samples. The results show that the maximum extraction efficiency was obtained at 40°C, 12 MPa, and a cosolvent (dichloromethane) volume of 5.0 mL. Under these conditions, the calibration curves had good linearity with square of the correlation larger than 0.998 in the concentration range of 5-800 ng/mL; limit of detection and limit of quantitation were 0.16-2.83 and 0.55-9.43 ng/g, respectively. At spiked levels of 10, 30, and 50 ng/g, the average recoveries were 70.4-80.4% for polycyclic aromatic hydrocarbons, 74.0-83.6% for polychlorinated biphenyls, and 66.9-78.0% for polybrominated biphenyl ethers, with average relative standard deviations of less than 16.3%. The established method has no significant differences in recovery compared to traditional methods and is suitable for the analysis of real samples.

Application of response surface methodology to optimise microbial inactivation of shrimp and conch by supercritical carbon dioxide.

BACKGROUND: Supercritical carbon dioxide (SC-CO2 ) has been shown to have a good pasteurising effect on food. However, very few research papers have investigated the possibility to exploit this treatment for solid foods, particularly for seafood. Considering the microbial safety of raw seafood consumption, the study aimed to explore the feasibility of microbial inactivation of shrimp (Metapenaeus ensis) and conch (Rapana venosa) by SC-CO2 treatment. RESULTS: Response surface methodology (RSM) models were established to predict and analyse the SC-CO2 process. A 3.69-log reduction in the total aerobic plate count (TPC) of shrimp was observed by SC-CO2 treatment at 53°C, 15 MPa for 40 min, and the logarithmic reduction in TPC of conch was 3.31 at 55°C, 14 MPa for 42 min. Sensory scores of the products achieved approximately 8 (desirable). The optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55°C, 15 MPa and 40 min. CONCLUSION: SC-CO2 exerted a strong bactericidal effect on the TPC of shrimp and conch, and the products maintained good organoleptic properties. This study verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment.

Optimization of subcritical fluid extraction of carotenoids and chlorophyll a from Laminaria japonica Aresch by response surface methodology.

BACKGROUND: The carotenoids and chlorophyll a of Laminaria japonica Aresch were extracted using ethanol-modified subcritical 1,1,1,2-tetrafluoroethane (R134a). In the present study, the effects of pressure (5-17 MPa), temperature (303-333 K) and the amount of cosolvent(2-6% R134a, w/w)were investigated. Response surface methodology (RSM) combined with a Box-Behnken design was applied to evaluate the significance of the three independent variables on each response. A desirability function was conducted to simultaneously optimize the multiple responses. RESULTS: The optimum extraction conditions were as follows: extraction temperature 324.13 K, extraction pressure 17 MPa and a cosolvent amount of 4.73%. Under these conditions, the yields of carotenoids and chlorophyll a were predicted to be 0.239 and 2.326 g kg(-1), respectively. CONCLUSION: It has been proved that subcritical R134a is a potential solvent, which can be an alternative to supercritical CO2 for extraction of natural ingredients under mild conditions.

Development of a subcritical fluid extraction and GC-MS validation method for polychlorinated biphenyls (PCBs) in marine samples.

This paper describes a new procedure for extracting polychlorinated biphenyls (PCBs) from marine samples using subcritical 1,1,1,2-tetrafluoroethane (R134a). The extraction procedure was optimized at temperatures varying from 20 to 70°C and pressures ranging from 3 to 15 MPa. The volume of the co-solvent was then optimized using 1,1,1,2-tetrafluoroethane (R134a) as the subcritical phase. PCBs were characterized by GC-MS using the optimized conditions of 3 MPa, 30°C, and a co-solvent volume of 6 mL. The average yields of PCBs from subcritical fluid extraction of spiked oyster samples were measured and found to be greater than 90%, with relative standard deviations (RSD) of less than 10%. Detection limits of this method were in the range of 0.045-0.108 ng/g of dry mass. The method was compared to Soxhlet extraction and then applied for monitoring PCBs in oysters from Qingdao, Shandong, China.

Optimisation for subcritical fluid extraction of 17-methyltestosterone with 1,1,1,2-tetrafluoroethane for HPLC analysis.

A simple, rapid and sensitive method was developed for determination of 17α-methyltestosterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) extraction and high performance liquid chromatography (HPLC). Response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure 5 MPa, temperature 31°C, and co-solvent volume 3.35ml. The analysis was carried out on XDB-C(18) column (4.6 mm × 250 mm, 5 µm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 0.8 ml/min, temperature 30°C and wavelength 245 nm. Good linearity of detection was obtained for 17α-methyltestosterone between concentrations of 50-250 ng/ml, r(2)=0.999. The method was validated using samples fortified with 17α-methyltestosterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.

Development and validation of a subcritical fluid extraction and high performance liquid chromatography assay for medroxyprogesterone in aquatic products.

A simple, rapid and sensitive method was developed for the determination of medroxyprogesterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) and high performance liquid chromatography (HPLC). A response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure, 3 MPa; temperature, 25 °C; and co-solvent volume, 6 ml. The analysis was carried out on Zorbax SB-C18 column (4.6 mm × 150 mm, 5 µm) with the mobile phase acetonitrile-water (55:45, v/v), flow rate 1.0 ml/min, temperature 30 °C and wavelength 240 nm. Good linearity of detection was obtained for medroxyprogesterone between concentrations of 50-250 ng/ml, r²=0.999. The method was validated using samples fortified with medroxyprogesterone at levels of 10, 30 and 50 ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.