PDIA3 defines a novel subset of adipose macrophages to exacerbate the development of obesity and metabolic disorders.

Adipose tissue macrophages (ATMs) play important roles in maintaining adipose tissue homeostasis and orchestrating metabolic inflammation. Given the extensive functional heterogeneity and phenotypic plasticity of ATMs, identification of the authentically pathogenic ATM subpopulation under obese setting is thus necessitated. Herein, we performed single-nucleus RNA sequencing (snRNA-seq) and unraveled a unique maladaptive ATM subpopulation defined as ATF4hiPDIA3hiACSL4hiCCL2hi inflammatory and metabolically activated macrophages (iMAMs), in which PDIA3 is required for the maintenance of their migratory and pro-inflammatory properties. Mechanistically, ATF4 serves as a metabolic stress sensor to transcribe PDIA3, which then imposes a redox control on RhoA activity and strengthens the pro-inflammatory and migratory properties of iMAMs through RhoA-YAP signaling. Administration of Pdia3 small interfering RNA (siRNA)-loaded liposomes effectively repressed adipose inflammation and high-fat diet (HFD)-induced obesity. Together, our data support that strategies aimed at targeting iMAMs by suppressing PDIA3 expression or activity could be a viable approach against obesity and metabolic disorders in clinical settings.

New Interpretation of Neonatal Outcomes by Phenotypically Classified Preterm Syndrome: A Retrospective Cohort Study.

OBJECTIVE: The global aim to lower preterm birth rates has been hampered by the insufficient and incomplete understanding of its etiology, classification, and diagnosis. This study was designed to evaluate the association of phenotypically classified preterm syndromes with neonatal outcomes; to what extent would these outcomes be modified after the obstetric interventions, including use of glucocorticoid, magnesium sulfate, and progesterone. METHODS: This was a retrospective cohort study conducted at Tongji Hospital (composed of Main Branch, Optical Valley Branch and Sino-French New City Branch) in Wuhan. A total of 900 pregnant women and 1064 neonates were retrospectively enrolled. The outcomes were the distribution of different phenotypes among parturition signs and pathway to delivery, the association of phenotypically classified clusters with short-term unfavorable neonatal outcomes, and to what extent these outcomes could be modified by obstetric interventions. RESULTS: Eight clusters were identified using two-step cluster analysis, including premature rupture of fetal membranes (PPROM) phenotype, abnormal amniotic fluid (AF) phenotype, placenta previa phenotype, mixed condition phenotype, fetal distress phenotype, preeclampsia-eclampsia & hemolysis, elevated liver enzymes, and low platelets syndrome (PE-E&HELLP) phenotype, multiple fetus phenotype, and no main condition phenotype. Except for no main condition phenotype, the other phenotypes were associated with one or more complications, which conforms to the clinical practice. Compared with no main condition phenotype, some phenotypes were significantly associated with short-term adverse neonatal outcomes. Abnormal AF phenotype, mixed condition phenotype, PE-E&HELLP phenotype, and multiple fetus phenotype were risk factors for neonatal small-for gestation age (SGA); placenta previa phenotype was not associated with adverse outcomes except low APGAR score being 0-7 at one min; mixed condition phenotype was associated with low APGAR scores, SGA, mechanical ventilation, and grade HI-W intraventricular hemorrhage (IVH); fetal distress phenotype was frequently associated with neonatal SGA and mechanical ventilation; PE-E&HELLP phenotype was correlated with low APGAR score being 0-7 at one min, SGA and neonatal intensive care unit (NICU) admission; multiple fetus phenotype was not a risk factor for the outcomes included except for SGA. Not all neonates benefited from obstetric interventions included in this study. CONCLUSION: Our research disclosed the independent risk of different preterm phenotypes for adverse pregnancy outcomes. This study is devoted to putting forward the paradigm of classifying preterm birth phenotypically, with the ultimate purpose of defining preterm phenotypes based on multi-center studies and diving into the underlying mechanisms.

[Metagenomic Analysis of Resistance Genes in Membrane Cleaning Sludge].

Wastewater treatment plants (WWTPs) are considered important reservoirs of antibiotic resistance genes (ARGs) and function as the main sources of ARGs in the environment. Membrane bioreactors (MBRs) have been recognized as effective tools for removing ARGs in WWTPs.There are a large number of pathogens and resistance genes in colloids, particulate matter, suspended matter, and microbial metabolites in intercepted wastewater by MBR. However, the distribution characteristics of resistance genes in membrane cleaning sludge remains unclear. In this study, resistance genes of membrane cleaning sludge were analyzed using a metagenomic technique. The results showed that there were 39 phyla in the membrane cleaning sludge. Proteobacteria, Nitrospirae, and Actinobacteria were the dominant phyla. The dominant genera were Nitrospira, Pseudomonas, and Bradyrhizobium. The pathogens accounted for 10.54% of all bacteria in the sample, among which Pseudomonas had the highest abundance, accounting for 3.94%. A total of 17 types of antibiotic resistance genes and 16 types of metal resistance genes (MRGs) (15 types of single metal resistance genes and 1 types of multi-heavy metal resistance gene) were identified. Multidrug resistance genes had the highest abundance, accounting for 49.08%. Multi-heavy metal resistance genes were the most abundant, accounting for 34.58%. The copper resistance genes were the most abundant of the single metal resistance genes, accounting for 19.99%. The most important functional pathway of microbial community in the membrane cleaning sludge was metabolic related, and many genes identified were related to human diseases. The numbers of genes related to bacterial resistance and bacterial infectious diseases were the largest, accounting for 34.50% and 16.62%, respectively. These results indicate that there were abundant ARGs, MRGs, and pathogens in the membrane cleaning sludge, which has potential environmental health risks. It is necessary to strengthen the control of ARGs, MRGs, and pathogens in membrane cleaning sludge to provide guidance for selecting appropriate technologies for effectively removing ARGs, MRGs, and pathogens.

Application of Nano-Hydroxyapatite Derived from Oyster Shell in Fabricating Superhydrophobic Sponge for Efficient Oil/Water Separation.

The exploration of nonhazardous nanoparticles to fabricate a template-driven superhydrophobic surface is of great ecological importance for oil/water separation in practice. In this work, nano-hydroxyapatite (nano-HAp) with good biocompatibility was easily developed from discarded oyster shells and well incorporated with polydimethylsiloxane (PDMS) to create a superhydrophobic surface on a polyurethane (PU) sponge using a facile solution-immersion method. The obtained nano-HAp coated PU (nano-HAp/PU) sponge exhibited both excellent oil/water selectivity with water contact angles of over 150° and higher absorption capacity for various organic solvents and oils than the original PU sponge, which can be assigned to the nano-HAp coating surface with rough microstructures. Moreover, the superhydrophobic nano-HAp/PU sponge was found to be mechanically stable with no obvious decrease of oil recovery capacity from water in 10 cycles. This work presented that the oyster shell could be a promising alternative to superhydrophobic coatings, which was not only beneficial to oil-containing wastewater treatment, but also favorable for sustainable aquaculture.

A Participant-assigned Interventional Research of Precesarean Internal Iliac Artery Balloon Catheterization for Managing Intraoperative Hemorrhage of Placenta Previa and Placenta Accreta Spectrum Disorders After Cesarean Section.

Placenta accreta spectrum disorder (PASD) and placenta previa (PP) are two of the most hideous obstetric complications which are usually associated with a history of cesarean section (CS). Moreover, women with PASD, PP and/or a cesarean scarred uterus are more likely to have adverse pregnancy outcomes, including blood transfusion, hysterectomy, pelvic organs damage, postpartum hemorrhage, disseminated intravascular coagulation, multi-organ dysfunction syndrome and even maternal or fetal death. This study aimed to investigate the efficacy of precesarean internal iliac artery balloon catheterization (BC) for managing severe hemorrhage caused by PASD and PP with a history of CS. This participant-assigned interventional study was conducted in Tongji Hospital. We recruited 128 women with suspected PASD, PP and a history of CS. Women in the BC group accepted precesarean BC of bilateral internal iliac arteries before the scheduled cesarean delivery. Women in the control group underwent a conventional cesarean delivery. Intraoperative hemorrhage, transfusion volume, radiation dose, exposure time, complications and neonatal outcomes were discussed. There were significant differences in calculated blood loss (CBL) between BC group and control group (1015.0±144.9 vs. 1467.0±171.0 mL, P=0.04). Precesarean BC could reduce intraoperative red blood cell (RBC) transfusion as compared with control group (799.5±136.1 vs. 1286.0±161.6 mL, P=0.02) and lessen the rate of using blood products (57.1% vs. 76.4%, P=0.02). The incidence of hysterectomy was also lower in BC group than in control group. Postpartum outcomes showed no significant differences between the two groups, except that postoperation hospitalization was longer in BC group than in control group (6.7±0.4 vs. 5.8±0.2 days, P=0.03). Precesarean BC of internal iliac artery is an effective method for managing severe hemorrhage caused by PASD and PP with a cesarean scarred uterus, as it could reduce intraoperative blood loss, lessen intraoperative RBC transfusions and potentially decrease hysterectomies.

Phosphorus Co-Existing in Water: A New Mechanism to Boost Boron Removal by Calcined Oyster Shell Powder.

The removal of boron (B) from water by co-precipitation with hydroxyapatite (HAP) has been extensively studied due to its low cost, ease of use and high efficiency. However, there is no explicit mechanism to express how resolved B was trapped by HAP. Thus, in this work, the process of removing B from water was studied using a low-cost calcium (Ca) precipitation agent derived from used waste oyster shells. The results showed that the removal rate of B in the simulated wastewater by calcined oyster shell (COS) in the presence of phosphorus (P) is up to more than 90%, as opposed to virtually no removal without phosphate. For B removal, the treated water needs to be an alkaline solution with a high pH above 12, where B is removed as [CaB(OH)4]+ but is not molecular. Finally, the synergistic mechanism of co-precipitation between HAP and dissolved B, occlusion co-precipitation, was explained in detail. The proposed method discovered the relationship between Ca, P and B, and was aimed at removing B without secondary pollution through co-precipitation.

[Stimulus and reward information encoded by population neurons in the primate prefrontal cortex and striatum].

It has been reported that single-unit activity in the prefrontal cortex (PFC) and striatum represented visual stimulus and reward information. But how to encode these pieces of information is quite complex from the view of single-neuron activity. Different neurons represented stimulus or reward information in different task epochs with increasing or decreasing their activities relative to their baseline firing rates. The present paper was aimed to study whether population neurons in the two brain areas could stably encode task-relevant parameters in a whole trial period. We recorded single-unit activities in the lateral PFC (LPFC) and striatum while the monkey was performing a stimulus- reward prediction task, and analyzed the neuronal activities by the method of a multi-variable regression model and the linear support vector machine. The results showed that, although proportions of task-related neurons in the two areas varied largely in the whole trial period, LPFC population neurons encoded reward and stimulus information stably and reliably. Population neurons in the striatum encoded only reward information, not stimulus information. A group of neurons in the two areas represented combined information of stimulus and reward. Further analysis showed that LPFC neurons encoded reward information for a group of relevant stimuli, while striatal neurons encoded reward information for a specific stimulus. These results suggest that both LPFC and striatal population neurons are able to stably represent task-relevant information, but from different aspects of the task. The different strategies to encode information in the LPFC and striatum suggest their different contributions in reward-based decision making.

Easy Synthesis of BiVO4 for Photocatalytic Overall Water Splitting.

Developing a photocatalyst system to generate hydrogen from water is a topic of great interest for fundamental and practical importance. In this study, we develop a new Z-scheme photocatalytic system for overall water splitting that consists of Rh/K4Nb6O8 for H2 evolution, Pt/BiVO4 for O2 evolution, and I-/IO3 - for an electron mediator under UV light irradiation. The oxygen evolution photocatalyst BiVO4 was prepared by the microwave-assisted hydrothermal method. The method is fast and simple, as compared to conventional hydrothermal synthesis. The catalysts were characterized by powder X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and UV-visible spectroscopy. The photocatalytic water splitting is investigated in (i) aqueous AgNO3 as sacrificial electron scavengers and (ii) a Z-scheme photocatalytic water splitting system. The BiVO4 photocatalysts prepared by the microwave-assisted hydrothermal method not only showed a very high oxygen evolution rate (2622 µmol g-1 h-1) of water splitting reaction in an aqueous AgNO3 solution but also achieved a high H2 evolution rate (340 µmol g-1 h-1) and O2 evolution rate (172 µmol g-1 h-1) in a Z-scheme overall water splitting system.

Experience of Clinical Management for Pregnant Women and Newborns with Novel Coronavirus Pneumonia in Tongji Hospital, China.

Based on the New Diagnosis and Treatment Scheme for Novel Coronavirus Infected Pneumonia (Trial Edition 5), combined with our current clinical treatment experience, we recently proposed a revision of the first edition of "Guidance for maternal and fetal management during pneumonia epidemics of novel coronavirus infection in the Wuhan Tongji Hospital". This article focused on the issues of greatest concern of pregnant women including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnostic criteria, inspection precautions, drug treatment options, indications and methods of termination of pregnancy, postpartum fever, breastfeeding considerations, mode of mother-to-child transmission, neonatal isolation and advice on neonatal nursing, to provide valuable experience for better management of SARS-CoV-2 infection in pregnant women and newborns.

Circ_0063517 acts as ceRNA, targeting the miR-31-5p-ETBR axis to regulate angiogenesis of vascular endothelial cells in preeclampsia.

AIMS: Accumulated evidence indicates that the dysregulation of circular RNAs (circRNAs) plays pivotal roles in many human diseases including preeclampsia (PE). Circ_0063517 has been verified to be down-regulated in PE. But the role of circ_0063517 in PE is still unclear. This research aims to probe into the effect of circ_0063517 on angiogenesis in PE development. MAIN METHODS: The expression of circ_0063517, endothelin receptor type B (ETBR) and miR-31-5p was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MTT assay, colony formation assay, scratch assay, transwell assay, and tube formation assay were performed to detect proliferation, migration, and angiogenesis, respectively. Dual luciferase reporter system and RNA immunoprecipitation (RIP) assay were carried out to determine the interaction between miR-31-5p and circ_0063517(or ETBR). ETBR, VEGFRA, and VEGFR2 levels were detected by western blot analysis. The effect of circ_0063517 and ETBR on angiogenesis was evaluated in N-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced PE in vivo. KEY FINDINGS: The levels of circ_0063517 and ETBR were down-regulated in the placenta tissue of PE patients. Conversely, the level of miR-31-5p was up-regulated in PE. Overexpression of circ_0063517 or knockdown of miR-31-5p facilitated growth, migration, and angiogenesis of vascular endothelial cells. Circ_0063517 knockdown-induced repression of the expression of ETBR, VEGFA, and VEGFR2 was partly counteracted by ETBR overexpression. Mechanistically, circ_0063517 sponged miR-31-5p to regulate ETBR expression. Finally, circ_0063517 promoted angiogenesis via enhancing ETBR expression in PE in vivo. SIGNIFICANCE: Our findings suggest that circ_0063517-miR-31-5p-ETBR axis regulates angiogenesis during the pathological process of PE.

HIF-1α regulates angiogenesis via Notch1/STAT3/ETBR pathway in trophoblastic cells.

Background: Preeclampsia is a pregnancy-related complication and the major cause to maternal and fetal mortality. Despite extensive studies, the pathogenesis of this disease still remains unknown. Here we explored the roles of HIF-1α and Notch1/ETBR in preeclampsia.Methods: Immunohistochemistry, RT-qPCR and western blot were used to measure levels of Notch1 and ETBR in placentas of preeclampsia patients. Transwell invasion assay and in vitro Matrigel assay were used to test the functions of Notch1, HIF-1α and ETBR in invasion and angiogenesis of trophoblast cells. In addition, we used reduced uterine perfusion pressure (RUPP) rat model to study preeclampsia in vivo.Results: We found that Notch1 and ETBR were down-regulated in the placenta of patients with preeclampsia. Hypoxia promoted invasion and angiogenesis of trophoblast cells, and up-regulated expressions of HIF-1α, Notch1/ETBR. Overexpression of Notch1 facilitated invasion and angiogenesis of trophoblast cells while HIF-1α inhibitor suppressed. Furthermore, Notch1 or ETBR could promote angiogenesis of trophoblast cells in RUPP rats.Conclusions: Our study reveals that HIF-1α and Notch1/ETBR play important roles in preeclampsia. Hypoxia-induced HIF-1αregulated Notch1/ETBR signaling, thereby modulating invasion and angiogenesis of trophoblast cells. These results shed light on molecular mechanisms of preeclampsia and provide potential targets for preeclampsia therapy.

Risk Factors and Pregnancy Outcomes: Complete versus Incomplete Placenta Previa in Mid-pregnancy.

This prospective study was conducted to compare risk factors and pregnancy outcomes between women with complete placenta previa and those with incomplete placenta previa diagnosed in mid-pregnancy. The study was carried out from April 2014 to December 2015, during which 70 patients with complete previa and 113 with incomplete previa between 20+0 weeks and 25+6 weeks of gestation were included. Maternal demographics and pregnancy outcomes were compared between the two groups. Comparisons between categorical variables were tested by chi-squared test and those between continuous variables by Student t test. Resolution of previa occurred in 87.43% of the studied women. The mean gestational age at resolution was 32.1±4.4 weeks. Incidence of maternal age ≥35 years and incidence of prior uterine operation ≥3 were high in women with complete previa (28.6% vs 8.8%, P=0.003; 28.6% vs. 8.8%, P=0.003). Resolution of previa occurred less often in complete previa group (74.3% vs. 95.6%, P=0.001). Women with complete previa admitted earlier (37.3±2.0 weeks 38.1±1.4 weeks, P=0.011) and delivered earlier (37.7±1.2 weeks vs. 38.3±1.4 weeks, P=0.025). Maternal age ≥35 years and prior uterine operation ≥3 increase the risk of complete previa in mid-pregnancy. Placenta previa is more likely to persist in women with complete previa than those with incomplete previa diagnosed in midpregnancy. What is more, women with complete previa in mid-pregnancy delivers earlier.

Relationship between placenta location and resolution of second trimester placenta previa.

This prospective study was conducted to assess the rate of resolution of second trimester placenta previa in women with anterior placenta and posterior placenta, and that in women with and without previous cesarean section. In this study, placenta previa was defined as a placenta lying within 20 mm of the internal cervical os or overlapping it. We recruited 183 women diagnosed with previa between 20+0 weeks and 25+6 weeks. They were grouped according to their placenta location (anterior or posterior) and history of cesarean section. Comparative analysis was performed on demographic data, resolution rate of previa and pregnancy outcomes between anterior group and posterior group, and on those between cesarean section group and non-cesarean section group. Women with an anterior placenta tended to be advanced in parity (P=0.040) and have increased number of dilatation and curettage (P=0.044). The women in cesarean section group were significantly older (P=0.000) and had more parity (P=0.000), gravidity (P=0.000), and dilatation and curettage (P=0.048) than in non-cesarean section group. Resolution of previa at delivery occurred in 87.43% women in this study. Women with a posterior placenta had a higher rate of resolution (P=0.030), while history of cesarean section made no difference. Gestational age at resolution was earlier in posterior group (P=0.002) and non-cesarean section group (P=0.008) than in anterior group and cesarean section group correspondingly. Placenta location and prior cesarean section did not influence obstetric outcomes and neonatal outcomes. This study indicates that it is more likely to have subsequent resolution of the previa when the placenta is posteriorly located for women who are diagnosed with placenta previa in the second trimester.

Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification.

A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0 µg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0 µg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13 min. In contrast, the DNA from 2000 CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1 min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3 min, which was statistically identical (P<0.05) to the Tp value of 19.9 min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1 min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica.

Synthetic chalcones with potent antioxidant ability on H2O2-induced apoptosis in PC12 cells.

Chalcone derivatives (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one and (E)-3-(4-hydroxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (Compounds 1 and 2) have been demonstrated to be potent anti-inflammatory agents in our previous study. In light of the relationship of intracellular mechanisms between anti-inflammatories and antioxidants, we further designed and synthesized a series of chalcone derivatives based on 1 and 2, to explore their antioxidant efficacy. The majority of the derivatives exhibited strong protective effects on PC12 (PC12 rat pheochromocytoma) cells exposed to H2O2, and all compounds were nontoxic. A preliminary structure-activity relationship was proposed. Compounds 1 and 1d ((E)-2-methoxy-4-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl) phenyl acrylate) exerted the action in a good dose-dependent manner. Quantitative RT-PCR (qRT-PCR) and western blot analysis showed that 1 and 1d significantly improve the expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant genes g-Glutamylcysteine Ligase Catalytic Subunit (GCLC) and heme oxygenase-1 (HO-1) and their corresponding proteins (γ-glutamyl cysteine synthase (γ-GCS) and HO-1) in PC12 cells. Inhibition of GCLC and HO-1 by specific inhibitors, L-buthionine-S-sulfoximine (BSO) and zinc protoporphyrin (ZnPP), respectively, partially reduce the protective effect of 1 and 1d. These data present a series of novel chalcone analogs, especially compounds 1 and 1d, as candidates for treating oxidative stress-related disease by activating the Nrf2-antioxidant responsive element (ARE) pathway.

Effect of baicalein on the expression of VIP in extravillous cytotrophoblasts infected with human cytomegalovirus in vitro.

This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCID50 was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by virus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in infected HPT-8 cells was decreased (P<0.05), and the levels of VIP mRNA and protein, and the concentration were raised to the normal (P>0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.

[Effects of TSP2-8 and CUB1+2 domains on secretion direction of von Willebrand factor-cleaving protease].

This study was aimed to explore if the intracellular transportation direction of von Willebrand factor-cleaving protease (ADAMTS13, vWF-CP) after synthesis is determined by the carboxyl terminal TSP2-8CUB1+2 domains of ADAMTS13 and to decipher the relationship between the structure and function of ADAMTS13. The recombinant plasmids pcDNA3.1-ADAMTS13 and pcDNA3.1-delTSP2-8CUB1+2 ADAMTS13 were introduced into Madin-Darby canine kidney cells (MDCK) by lipofectamine-mediated DNA transfection. Positive cell clones gained after antibiotic-screening were grown on 6-well transwell filter units with a zeolite membrane in the middle layer. The conditioned culture media in both apical and basolateral wells were collected when cells reached confluency and the tight cell monolayer formed. ADAMTS13 proteases in the conditioned media were determined by Western blot, and the direction of ADAMTS13 secretion in polarized cells was comparatively analyzed. The results showed that Madin-Darby canine kidney cells stably expressing wild-type ADAMTS13 were grown on 6-well transwell filter units, then ADAMTS13 protease was only determined in the apical area of the transwell filter units by Western blot, but the recombinant ADAMTS13 protease was determined both in the apical and basolateral area of cells in the group of expressing TSP2-8CUB-1+2 domain-deleted ADAMTS13. It is concluded that the metalloprotease ADAMTS13 is sorted apically in polarized cells, and the carboxyl-terminal TSP2-8 and CUB1+2 domains of ADAMTS13 are important for the direction of ADAMTS13 protease transportation in the cells after being synthesized.

Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio).

An aminopeptidase from zebrafish (Danio rerio) was purified 1247-fold to homogeneity with 35.4% recovery by column chromatography successively on DEAE-sephacel, hydroxyapatite, and phenyl-sepharose. The molecular mass of the enzyme was estimated at 98 kDa by SDS-PAGE and gel filtration. Optimum temperature and pH of the enzyme were 45°C and 7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with k(cat)/K(m) of 4.2×10(6)M(-1)s(-1) and an activation energy of 68.9 kJ M(-1) [corrected], respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn(2+) seemed to be its metal cofactor(s). Some l-amino acids significantly inhibited its activity, and l-cysteine was a non-competitive inhibitor with a K(i) of 0.27 mM. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive (gi: 255683530) (EC 3.4.11.14), suggesting that the present enzyme is a puromycin-sensitive aminopeptidase of zebrafish.

[Dynamics of polycyclic aromatic hydrocarbons (PAHs) in the paddy-soil system during the crop rotation process].

The concentrations of polycyclic aromatic hydrocarbons (PAHs) in the paddy root-soil system were determined to study the dynamic and the influencing factors during crop rotation period. It showed that the dynamic of PAHs in paddy roots was most correlative with the factor of root surface area, but less correlated with PAHs in air and particles, which indicates that the physiological characters rather than the environment media are the main factors influencing the PAHs accumulation in paddy roots. According to the EPA risk standard about BaP and sigma PAHs, the PAHs accumulation in the paddy seeds won't decrease the food security to human being. The PAHs concentrations in paddy soil showed a declined trend during the period of paddy growth, which was affected not only by the processes of water elution and microbe degradation, but also depended on the absorption rate of paddy roots. When the crop rotation begins and paddy planting rolls into the next growing period, the PAHs in the paddy soil will again increase into a higher level which is correlated with the TOC content in the soil.