The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner.

Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

Molecular Characterization of Predominant Serotypes, Drug Resistance, and Virulence Genes of Streptococcus pneumoniae Isolates From East China.

Streptococcus pneumoniae is a common diplococcus pathogen found worldwide. The characterization of predominant serotypes, drug resistance, and virulence genes of S. pneumoniae isolates prevailing in different areas and countries is clinically important for choice of antibiotics and improvement of vaccines. In this study, pneumonia (78.7%) and meningitis (37.0%) were the predominant diseases observed in the 282 (children) and 27 (adults) S. pneumoniae-infected patients (p < 0.05) from seven hospitals in different areas of East China. Of the 309 pneumococcal isolates, 90.3% were classified by PCR into 15 serotypes, with serotypes 19F (27.2%) and the 6A/B (19.1%) being most predominant (p < 0.05). Importantly, serotypes 15A and 15B/C combined for a total of 10.4% of the isolates, but these serotypes are not included in the 13-valent pneumococcal capsule conjugate vaccine used in China. Antimicrobial susceptibility analysis by the E-test showed that >95% of the 309 pneumococcal isolates were susceptible to moxifloxacin and levofloxacin, as well as 18.4, 85.8, and 81.6% of the isolates displayed susceptibility to penicillin, cefotaxime, and imipenem, respectively. A significant correlation between the prevalence of predominant serotypes and their penicillin resistance was observed (p < 0.05). In particular, >95% of all the pneumococcal isolates showed resistance to erythromycin and azithromycin. Of the nine detected virulence genes, the lytA, ply, hysA, and nanA were the most common with 95-100% positive rates in the 309 pneumococcal isolates, while the pavA and psaA genes displayed a significant correlation with pneumococcal bacteremia and meningitis (p < 0.05). Overall, our data suggested that the predominant serotypes, drug resistance, and virulence genes of the S. pneumoniae isolates prevailing in East China are distinct from those observed in other areas of China and adjacent countries.

Thrombophilic-type placental pathologies and skeletal growth delay following maternal administration of angiostatin4.5 in mice.

During placentation, the concentration of fibrinous deposits on the surfaces of maternal vasculature plays a role in villous development and has been strongly implicated in the pathophysiology of human fetal growth restriction (FGR). Fibrinous deposits are conspicuous sites of platelet aggregation where there is local activation of the hemostatic cascade. During activation of the hemostatic cascade, a number of pro- and antiangiogenic agents may be generated at the cell surface, and an imbalance in these factors may contribute to the placental pathology characteristic of FGR. We tested the hypothesis that angiostatin(4.5) (AS(4.5)), a cleavage fragment of plasminogen liberated at the cell surface, is capable of causing FGR in mice. Increased maternal levels of AS(4.5) in vivo result in reproducible placental pathology, including an altered vascular compartment (both in decidual and labyrinthine layers) and increased apoptosis throughout the placenta. In addition, there is significant skeletal growth delay and conspicuous edema in fetuses from mothers that received AS(4.5). Maternally generated AS(4.5), therefore, can access maternal placental vasculature and have a severe effect on placental architecture and inhibit fetal development in vivo. These findings strongly support the hypothesis that maternal AS(4.5) levels can influence placental development, possibly by directly influencing trophoblast turnover in the placenta, and contribute to fetal growth delay in mice.

Assessment of heterogeneity in collective endothelial cell behavior with multicolor clonal cell tracking to predict arteriovenous remodeling.

Arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single-cell level, little is known about how collective cell migration contributes to the organization of the balanced distribution between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from early vasculogenesis throughout arteriovenous remodeling. We reveal that the genesis of arteries and veins relies on the coordination of 10 types of collective cell dynamics. Within these behavioral categories, we identify a heterogeneity of collective cell motion specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that cell-intrinsic Notch signaling and cell-extrinsic blood flow act as regulators in maintaining the heterogeneity of collective endothelial cell behavior, which, in turn, instructs the future territory of arteriovenous remodeling.

Bio-resolution of glycidyl (o, m, p)-methylphenyl ethers by Bacillus megaterium.

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84-99% enantiomeric excess and with 21-73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.

Maternal administration of anti-angiogenic agents, TNP-470 and Angiostatin4.5, induces fetal microphthalmia.

PURPOSE: Agents specifically targeting the vasculature as a mode of therapy are finding increasing use in the clinic, primarily in the treatment of colon cancer (Avastin) and age-related macular degeneration (Lucentis). We have previously shown that maternal administration of angiogenic inhibitors (TNP-470 [O-[chloroacetyl-carbamoyl]fumagillol, initially called AGM-1470], the first angiogenic inhibitor to undergo clinical trials, and Angiostatin(4.5), currently in phase I-III clinical trials) cause fetal growth restriction and/or placental abnormalities. During a rapid growth phase of ocular development in the mouse (embryonic days 12 to 19 [E12-E19]), the placenta mediates the metabolic requirements of the fetus and consequently may impact upon the growth of the highly oxygen sensitive fetal eye. METHODS: We injected pregnant dams (between E10.5 - E18.5) with anti-angiogenic agents, which caused either a placental insufficiency type of IUGR (intrauterine growth restriction; i.e., TNP-470) or frank placental pathology (Angiostatin(4.5) [AS(4.5)]), and assessed changes in absolute ocular dimensions, tissue types, and vascular profiles using stereological techniques. RESULTS: The experiments showed that ocular volumes were significantly reduced in fetal mice where dams were treated with either TNP-470 or AS(4.5). Furthermore, TNP-470 specifically caused a reduction in hyaloid blood vessel length and volume, the only intraocular vascular circulation in fetal mice. CONCLUSIONS: These experiments support the hypothesis that the angiogenic inhibitors (specifically TNP-470 and AS(4.5)) induce microphthalmia either indirectly by their known effects on placental morphology (and/or function) or directly via altering microvascular growth in the fetus. These results also warrant further investigation of a new experimental paradigm linking placental pathology-related fetal growth restriction and microphthalmia.

A long-wavelength, fluorogenic probe for epoxide hydrolase: 7-(2-(oxiran-2-yl)ethoxy) resorufin.

Epoxide hydrolase plays an important role in the detoxification of genotoxic compounds and in the control of physiological signaling molecules. Altered levels of epoxide hydrolase activity are associated with many diseases, such as emphysema, lung cancer, ovarian cancer, and laryngeal carcinoma. We designed and synthesized a resorufin-based fluorogenic probe, 7-(2-(oxiran-2-yl)ethoxy) resorufin, which was hydrolyzed by microsomal epoxide hydrolase to form the corresponding diol, which upon further treatment with sodium periodate released the strongly fluorescent resorufin. The probe exhibits good biological compatibility and photophysical properties, such as long wavelength excitation (571 nm) and emission (585 nm) and a wide working pH range (from 6.0 to 10.0), and thus facilitates the determination of the activity of microsomal epoxide hydrolase.

Differential binding of plasminogen, plasmin, and angiostatin4.5 to cell surface beta-actin: implications for cancer-mediated angiogenesis.

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and approximately 85% of kringle 5. In culture, cancer cell surface globular beta-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface beta-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized beta-actin similarly, with a Kd of approximately 140 nmol/L. The binding is inhibited by epsilon-aminocaproic acid (epsilonACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from beta-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 micromol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface beta-actin, and the truncated kringle 5 in AS4.5 results in its release from beta-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.

Self-assembly of metal/semiconductor heterostructures via ligand engineering: unravelling the synergistic dual roles of metal nanocrystals toward plasmonic photoredox catalysis.

Metal nanocrystals (NCs) have been recognized as an important class of nanomaterials by virtue of their unique surface plasmon resonance (SPR) effect and pivotal roles as electron traps in photocatalysis. Nevertheless, it is still challenging to unambiguously unravel and simultaneously harness the dual synergistic roles of metal NCs in a single photocatalytic system for solar-to-chemical energy conversion. Herein, an efficient ligand-triggered electrostatic self-assembly strategy was developed to achieve the spontaneous and monodispersed attachment of Au NCs onto 1D WO3 nanorods (NRs) via pronounced electrostatic attractive interaction, in which tailor-made positively charged Au NCs were closely integrated with negatively charged WO3 NRs. The intimate integration of Au NCs with WO3 NRs at the nanoscale could significantly benefit the extraction, separation, and migration of plasmon-induced energetic hot carriers over Au NCs and promote the separation of photogenerated charge carriers over the WO3 substrate. Such a cooperative synergy stemming from SPR and the electron-withdrawal effects of the Au NCs resulted in distinctly enhanced photoredox catalytic performances for plasmonic photocatalysis under both simulated solar and visible light irradiation. Our study highlights the significance of utilizing a rational interface design between metal NCs and semiconductors for excavating the multifarious roles of metal NCs in substantial solar energy conversion.

In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna.

PURPOSE: Angiostatin4.5 (AS4.5), the endogenous human angiostatin, is derived from plasminogen in a two-step process. A plasminogen activator converts plasminogen to plasmin, then plasmin undergoes autoproteolysis to AS4.5. A free sulfhydryl donor can mediate plasmin autoproteolysis. To translate this process to human cancer therapy, we conducted a phase I trial of administration of a tissue plasminogen activator (tPA) with a free sulfhydryl donor (mesna). PATIENTS AND METHODS: Fifteen patients with advanced solid tumors were treated. The dose of tPA was escalated (cohorts; 1, 2, 3, 5, and 7.5 mg/h for 6 hours). Mesna was administered as a 240 mg/m2 bolus followed by an infusion of 50 mg/h, concurrent with tPA. Both tPA and mesna were administered 3 consecutive days every 14 days. RESULTS: No dose-limiting toxicity was observed. Two AS4.5 isoforms were generated, Lys-AS4.5 and Glu-AS4.5. Mean baseline Lys-AS4.5 level was 20.4 nmol/L (SE, 2.9). In the 5 mg/h tPA cohort, Lys-AS4.5 levels increased by an average of 143% or 24 nmol/L (SE, 4.9) above baseline. Glu-AS4.5 (M(r) approximately 62,000) was also generated (additional 77 amino acids at amino terminus compared with Lys-AS4.5). Glu-AS4.5 level at baseline was undetectable in four of five patients in the 5 mg/h tPA cohort, but at end of infusion, was approximately 67 nmol/L (SE, 20). Two patients in the 5 mg/h tPA cohort experienced decreases in tumor markers with treatment, although no clinical objective responses were observed. CONCLUSION: This study shows that in vivo generation of AS4.5 is safe in humans and may provide a practical approach to achieve antiangiogenic therapy.

Cell surface-dependent generation of angiostatin4.5.

Angiostatin4.5 (AS4.5) is a naturally occurring human angiostatin isoform, consisting of plasminogen kringles 1-4 plus 85% of kringle 5 (amino acids Lys78 to Arg529). Prior studies indicate that plasminogen is converted to AS4.5 in a two-step reaction. First, plasminogen is activated to plasmin. Then plasmin undergoes autoproteolysis within the inner loop of kringle 5, which can be induced by a free sulfhydryl donor or an alkaline pH. We now demonstrate that plasminogen can be converted to AS4.5 in a cell membrane-dependent reaction. Actin was shown previously to be a surface receptor for plasmin(ogen). We now show that beta-actin is present on the extracellular membranes of cancer cells (PC-3, HT1080, and MDA-MB231), and beta-actin can mediate plasmin binding to the cell surface and autoproteolysis to AS4.5. In the presence of beta-actin, no small molecule-free sulfhydryl donor is needed for generation of AS4.5. Antibodies to actin reduced membrane-dependent generation of AS4.5 by 70%. In a cell-free system, addition of actin to in vitro-generated plasmin resulted in stoichiometric conversion to AS4.5. Annexin II and alpha-enolase have been reported to be plasminogen receptors, but we did not demonstrate a role for these proteins in conversion of plasminogen to AS4.5. Our data indicate that membrane-associated beta-actin, documented previously as a plasminogen receptor, is a key cell membrane receptor capable of mediating conversion of plasmin to AS4.5. This conversion may serve an important role in regulating tumor angiogenesis, invasion, and metastasis, and surface beta-actin may also serve as a prognostic marker to predict tumor behavior.

Cloning and characterization of human ubiquitin binding enzyme 2 cDNA.

OBJECTIVE: To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern. METHODS: According to the sequence of human EST, which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple-tissue Northern blot. RESULTS: Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows that they are expressed exclusively in adult human heart, placenta, and pancreas but no transcripts can be detected in brain, lung, liver, skeletal muscle or kidney. CONCLUSIONS: The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.

[Decomposition and phosphorus dynamics of the litters in standing and litterbag of the Hangzhou Bay coastal wetland ].

Wetlands litter decomposition affects wetlands nutrient cycling. The decomposition progress of standing litter was monitored and the litterbag simulation experiment was carried out in order to analyze dynamics of litter decomposition and phosphorus release in Phragmites australis (PA), Spartina alterniflora (SA) and Scirpus mariqueter (SM) marshes of Hangzhou Bay coastal wetland. Results show that the dry mass of standing litter and P concentration decrease gradually and the litter drops to the sediment surface after 180 d. There are distinctive stages of the plant litter decomposition in litterbag simulation experiments. The loss rate is faster during 0- 15 d than that of later days. The loss rate in root decomposition of three plants are SM > PA > SA, while the trend is opposite for that of aboveground tissues. The time needed for 95% of dry mass decomposition in the plant tissues is between 1. 2- 8. 3 a. The P concentration in litters decreases rapidly in the initial stage and then increases slowly while the net P pools decreases all the time. Pearson's correlation coefficient shows that there is no significant correlation between the litter decomposition rate and C/N ratio. However, the litter C/P ratio affects greatly on plant decomposition rate. Environmental factors in the atmospheric temperature also have an impact on the decomposition rate of leaves. The different decomposition progresses between standing litter and litterbag are caused by environmental factors.

[Dynamics of carbon, nitrogen and phosphorus storage of three dominant marsh plants in Hangzhou Bay coastal wetland].

Salt marshes perform important ecosystem functions in carbon, nitrogen and phosphorus recycling. The plant biomass, content and pools of C, N and P were measured seasonally in three marsh species Phragmites australis, Spartina alterniflora and Scirpus mariquezer in Hangzhou Bay coastal wetland for the dynamics of C, N and P storage. The results showed that seasonal variation of aboveground biomass displayed a unimodal curve. The seasonal variability of plant OC content in the aboveground part of the plants was not significant, while the TN and TP content decreased significantly from spring to winter. The seasonal variability of plant C, N and P pools was significant. And there was a significant relationship between plant C/N/P pools and biomass. The pools among plant species were significantly different. S. mariqueter had the lowest C/N/P pools. TN pool in the aboveground part of P. australis was higher than that of S. aterniflora, but its TP pool was lower than that of S. alterniflora, and there was no significant difference for OC pools between P. australis and S. alterniflora. C fixation of the three marsh species was 380%, 376% and 55.5% of the average C fixation of terrestrial vegetations in China, and 463%, 458% and 67.7% of the average C fixation of terrestrial vegetations of the world. Considering the purification capacity of N and P, July would be the best harvest time of the study area for three plants. And the harvest of S. alterniflora could remove the biggest amount of P, since P was a limiting nutrient for phytoplankton growth. In conclusion, the marsh plants had strong C fixation and N/P purification ability.

[Community composition, seasonal dynamics and interspecific correlation of waterbirds in the Qiantangjiang River estuary and Hangzhou Bay].

Waterbird surveys were conducted regularly in the Qiantangjiang River estuary and Hangzhou Bay from July 2007 to November 2011. A total of 128 species (nine orders and 18 families) were recorded, including 119 migrants which accounted for 93% of the total species; eleven species were listed as National Protected Species. Inter-specific correlation analysis for 13 shorebird populations and nine duck populations recorded over time found that 21 pairs of shorebirds and 23 pairs of ducks were correlated. By looking at seasonal dynamics and migration patterns we were able to divide the migration process into six stages: (1) late July to late September was the migration peak of shorebirds, which were dominated by Limosa limosa, Calidris ruficollis and Charadrius mongolus. (2) Early October to mid-December was the migration peak of wintering migrants of shorebirds and ducks, which were the first two large groups in our study areas. (3) Late December to mid-February was the wintering period of migration waterbirds. (4) Late February to late March was the peak migration of ducks and the winter migrants of shorebirds dominated by Calidris alpina. (5) Early April to mid-May was the migration peak of passage migrants such as, Calidris ruficollis, Calidris acuminate and Limosa limosa but the population size of shorebird winter migrants dominated by Calidris alpine was still larger than the former. (6) Late May to mid-July was the breeding season of all egrets, summer migrants of gulls and several species of shorebirds. Our surveys show that interaction among species is possibly an important determinant of community composition of shorebirds and wintering ducks during the migration season. It may be the geographical position and community composition of migrant shorebirds across Hangzhou Bay that mean during the northward migration there are far more shorebirds than during the southward migration.

[Soil organic carbon content and its distribution pattern in Hangzhou Bay coastal wetlands].

In this paper, the soil organic carbon (SOC) content and its distribution pattern in the natural intertidal zones and reclaimed wetlands of Hangzhou Bay were studied, aimed to explore the effects of vegetation succession, exotic species invasion, and reclamation on the SOC in costal wetlands of the Bay. In intertidal zones, the surface SOC content ranged from 4.41 to 8.58 g x kg(-1), with an average of 6.45 g x kg(-1), and differed significantly under different vegetations, with a tendency of under Phragmites australis (8.56 +/- 0.04 g x kg(-1)) > Spartina alterniflora (7.31 +/- 0.08 g x kg(-1)) > Scirpus mariqueter (5.48 +/- 0.29 g x kg(-1)) > mudflats (4.47 +/- 0.09 g x kg(-1)); in reclaimed wetlands, the surface SOC content was 7.46 +/- 0.25 g x kg(-1) in the 1960s, 1.96 +/- 0.46 g x kg(-1) in the 1980s, and 5.12 +/- 0.16 g x kg(-1) in 2003, showing a trend of increased after an initial decrease with increasing reclamation year. The SOC in the profiles all showed a decreasing trend from the surface to the bottom. The SOC in intertidal zones and reclaimed wetlands was significantly negatively correlated with soil pH, and positively correlated with soil total nitrogen (TN), suggesting a large reserve of organic nitrogen in TN. The correlation between SOC and soil C/N ratio was not obvious in intertidal zones, but significantly positive in reclaimed wetlands, indicating that reclamation affected soil C/N ratio to a certain extent. This study showed that in the intertidal zones, soil carbon sequestration capacity increased gradually with plant community succession. However, the invasion of exotic species Spartina alternflora might decrease the capacity of carbon sequestration in intertidal zones. It was also found that the changes of soil moisture content, particle composition, vegetation coverage, and reclamation history were the main factors affecting the SOC distribution in reclaimed wetlands.

[Seasonal changes of fish species composition and diversity in mudflat wetlands of Hangzhou Bay].

In order to understand the spatiotemporal variation of fish species composition and biodiversity in the mudflat wetlands of Hangzhou Bay, thirty six surveys were conducted in the mudflat area, inning area, and aquaculture area in the south bank of the Bay in. March (early spring), May (spring), July (summer), and October (autumn), 2009. A total of 41 species belonging to 9 orders and 16 families were observed, among which, Cyprinid had the largest species number (14 species, 33.3% of the total), followed by Gobiidae (8 species, 19.1%). According to the lifestyle of fish, these 41 species could be divided into five ecological types, i.e., freshwater type (21 species), brackish-water type (16 species), inshore type (2 species), anadromous type (Coilia ectenes), and catadromios type (Anguilla japonica). The fish abundance was the highest (54. 5 fish per net) in summer, followed by in spring and autumn, and the lowest (17.7 fish per net) in early spring. In the three habitats, mudflat area and inning area had the similar seasonal change of fish abundance, i.e., the lowest in early spring, the highest in summer, and then decreased in autumn. Only two or three species were the dominant species in different seasons. In mudflat area, the dominant species were Mugil cephalus and Liza carinatus; while in inning and aquaculture areas, the dominant species were Carassius auratus, Hemiculter leucisculus, and Pseudorasbora parva. The values of Margalef's richness index (D), Pielou's evenness index (J), and Shannon index (H) were lower in March than in other months, but had no significant differences among May, July, and October (P > 0.05). The H value ranged in 0. 27-2. 13, being the lowest in March and higher in May and October (1.66 and 1.63, respectively). Overall, the fish abundance and biodiversity in the mudflat wetlands of Hangzhou Bay had apparent seasonal changes.