We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by co-injection of two transgenes, a “reverse” tetracycline-controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline-responsive promoter (TetO). Here, Tnnt2-rtTA activated TetO-Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte-specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2-rtTA;TetO-Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods.

Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte-restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8–14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.

Rationale

Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well established, the molecular events that inhibit or repress cardiac hypertrophy are less known.

Objective

To identify and investigate novel regulators that modulate cardiac hypertrophy.

Methods and Results

Here, we report the identification, characterization and functional examination of CIP, a novel cardiac Isl1-interacting protein. CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast-two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a co-factor of CIP. CIP directly interacted with Isl1 and we mapped the domains of these two proteins which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the MEF2C enhancer. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy.

Conclusions

Our studies therefore identify CIP a novel regulator of cardiac hypertrophy.

Chronic inflammation-promoted metastasis has been considered as a major challenge in cancer therapy. Pro-inflammatory cytokine TNFα can induce cancer invasion and metastasis associated with epithelial–mesenchymal transition (EMT). However, the underlying mechanisms are not entirely clear. In this study, we showed that TNFα induces EMT in human HCT116 cells and thereby promotes colorectal cancer (CRC) invasion and metastasis. TNFα-induced EMT was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin and fibronectin with a concomitant decrease of E-cadherin and Zona occludin-1(ZO-1). TNFα treatment also increased the expression of transcription factor Snail, but not Slug, ZEB1 and Twist. Overexpression of Snail induced a switch from E-cadherin to N-cadherin expression in HCT116 cells, which is a characteristic of EMT. Conversely, knockdown of Snail significantly attenuated TNFα-induced EMT in HCT116 cells, suggesting that Snail plays a crucial role in TNFα-induced EMT. Interestingly, exposure to TNFα rapidly increased Snail protein expression and Snail nuclear localization but not mRNA level upregulation. Finally, we demonstrated that TNFα elevated Snail stability by activating AKT pathway and subsequently repressing GSK-3β activity and decreasing the association of Snail with GSK-3β. Knockdown of GSK-3β further verified our finding. Taken together, these results revealed that AKT/GSK-3β-mediated stabilization of Snail is required for TNFα-induced EMT in CRC cells. Our study provides a better understanding of inflammation-induced CRC metastasis.

Ghrelin, an enteric peptide hormone linked to the pathophysiology of obesity has been a therapeutic target of great interest over the past decade. Many research efforts have focused on the antagonism of ghrelin’s endogenous receptor GHSR1a, which is found along ascending vagal afferent fibers, as well as in the arcuate nucleus of the hypothalamus. Additionally, peptidic inhibitors against ghrelin O-acyltransferase, the enzyme responsible for the paracrine activation of ghrelin, have recently been studied. Our research has taken an alternative immunological approach, studying both active and passive vaccination as a means to sequester ghrelin in the periphery, with the original discovery in rat of decreased feed efficiency and adiposity, as well as increased metabolic activity. Using our previous hapten designs as a stepping-stone, three monoclonal antibodies (JG2, JG3, and JG4) were procured against ghrelin and tested in vivo. While mAb JG4 had the highest affinity for ghrelin, it failed to attenuate the orexigenic effects of food deprivation on energy metabolism or food intake in mice. However, animals that were administered a combination of JG3:JG4, (termed a doublet), or JG2:JG3:JG4, (termed a triplet), demonstrated higher heat dispersion and rate of respiration (higher CO2 emission and O2 consumption) during a 24-hr fast refeed. Mice administered the triplet cocktail of JG2:JG3:JG4 also demonstrated decreased food intake upon refeeding as compared to control animals. Recently, Lu and colleagues reported that a passive approach using a single, high affinity N-terminally directed monoclonal antibody did not abrogate the effects of endogenous ghrelin. Our current report corroborates this finding, yet, refutes that a monoclonal antibody approach cannot be efficacious. Rather, we find that a multiple monoclonal antibody (oligoclonal) approach can reproduce the underlying logic to previously reported efficacies using active vaccinations.

Metabolomics aims at detection and quantitation of all metabolites in biological samples. The presence of metabolites with a wide variety of physicochemical properties and different levels of abundance challenges existing analytical platforms used for identification and quantitation of metabolites. Significant efforts have been made to improve analytical and computational methods for metabolomics studies.

This review focuses on the use of liquid chromatography with tandem mass spectrometry (LC-MS/MS) for quantitative and qualitative metabolomics studies. It illustrates recent developments in computational methods for metabolite identification, including ion annotation, spectral interpretation and spectral matching. We also review selected reaction monitoring and high-resolution MS for metabolite quantitation. We discuss current challenges in metabolite identification and quantitation as well as potential solutions.

Osteoporosis is typically diagnosed by dual energy x-ray absorptiometry (DXA) measurements of areal bone mineral density (aBMD). Emerging technologies, such as high-resolution peripheral quantitative computed tomography (HR-pQCT), may increase the diagnostic accuracy of DXA and enhance our mechanistic understanding of decreased bone strength in osteoporosis. Women with (n=68) and without (n=101) a history of postmenopausal fragility fracture had aBMD measured by DXA, trabecular plate and rod microarchitecture measured by HR-pQCT image-based individual trabeculae segmentation (ITS) analysis, and whole bone and trabecular bone stiffness by micro finite element analysis (μFEA) of HR-pQCT images at the radius and tibia. DXA T-scores were similar in women with and without fractures at the spine, hip and 1/3 radius, but lower in fracture subjects at the ultradistal radius. Trabecular microarchitecture of fracture subjects was characterized by preferential reductions in trabecular plate bone volume, number, and connectivity over rod trabecular parameters, loss of axially aligned trabeculae, and a more rod-like trabecular network. In addition, decreased thickness and size of trabecular plates were observed at the tibia. The differences between groups were greater at the radius than the tibia for plate number, rod bone volume fraction and number and plate-rod and rod-rod junction densities. Most differences between groups remained after adjustment for T-score by DXA. At a fixed bone volume fraction, trabecular plate volume, number and connectivity were directly associated with bone stiffness. In contrast, rod volume, number and connectivity were inversely associated with bone stiffness. In summary, HR-pQCT-based ITS and μFEA measurements discriminate fracture status in postmenopausal women independent of DXA measurements. Moreover, these results suggest that preferential loss of plate-like trabeculae contribute to lower trabecular bone and whole bone stiffness in women with fractures. We conclude that HR-pQCT-based ITS and μFEA measurements increase our understanding of the microstructural pathogenesis of fragility fracture in postmenopausal women.

Chinese-American women have lower rates of hip and forearm fracture than white women despite lower areal bone density (aBMD) by dual X-ray absorptiometry (DXA). We recently reported higher trabecular (Dtrab) and cortical (Dcomp) bone density as well as greater trabecular (Tb.Th) and cortical thickness (C.Th) but smaller bone area (CSA), as measured by high-resolution peripheral quantitative computed tomography (HR-pQCT), in premenopausal Chinese-American compared with white women. These findings may help to account for the lower fracture rate among Chinese-American women but were limited to measurements in premenopausal women. This study was designed to extend these investigations to postmenopausal Chinese-American (n = 29) and white (n = 68) women. Radius CSA was 10% smaller in the Chinese-American versus the white group (p = .008), whereas their C.Th and Dcomp values were 18% and 6% greater (p < .001 for both). Tibial HR-pQCT results for cortical bone were similar to the radius, but Tb.Th was 11% greater in Chinese-American versus white women (p = .007). Tibial trabecular number and spacing were 17% lower and 20% greater, respectively, in Chinese-American women (p < .0001 for both). There were no differences in trabecular or whole-bone stiffness estimated by microstructural finite-element analysis, but Chinese-American women had a greater percentage of load carried by the cortical bone compartment at the distal radius and tibia. There was no difference in load distribution at the proximal radius or tibia. Whole-bone finite-element analysis may indicate that the thicker, more dense cortical bone and thicker trabeculae in postmenopausal Chinese-American women compensate for fewer trabeculae and smaller bone size.

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.

Cre-LoxP-mediated genetic lineage trace has been used to illuminate the cell fate of progenitor cells in vivo. Application of this strategy to the epicardium, a sheet of cells covering the surface of heart, revealed that it dynamically participates in both heart development and postnatal heart repair and regeneration. After myocardial infarction, epicardial cells undergo epithelial-to-mesenchymal transition (EMT) and mainly adopt myofibroblast, fibroblast and smooth muscle cell fates. Here we present the wholemount images that map epicardial EMT following myocardial infarction, taking advantage of an inducible epicardial Cre line and a double fluorescence reporter. While remote epicardium retained its epithelial cell shape, reactivated epicardium in the infarcted region showed significant EMT. This image supports active involvement of the epicardium in repair and regeneration of infarcted myocardium.

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

miR-34b and -c inhibit osteoblast proliferation and differentiation by decreasing the levels of cell cycle proteins and of the nuclear matrix protein SATB2.

A screen of microRNAs preferentially expressed in osteoblasts identified members of the miR-34 family as regulators of osteoblast proliferation and/or differentiation. Osteoblast-specific gain- and loss-of-function experiments performed in vivo revealed that miR-34b and -c affected skeletogenesis during embryonic development, as well as bone mass accrual after birth, through two complementary cellular and molecular mechanisms. First, they inhibited osteoblast proliferation by suppressing Cyclin D1, CDK4, and CDK6 accumulation. Second, they inhibited terminal differentiation of osteoblasts, at least in part through the inhibition of SATB2, a nuclear matrix protein that is a critical determinant of osteoblast differentiation. Genetic evidence obtained in the mouse confirmed the importance of SATB2 regulation by miR-34b/c. These results are the first to identify a family of microRNAs involved in bone formation in vivo and to identify a specific genetic pathway by which these microRNAs regulate osteoblast differentiation.

Most malaria drug development focuses on parasite stages detected in red-blood cells even though to achieve eradication next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4,000 commercially available compounds with previously demonstrated blood stage activity (IC50 < 1 μM), and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. Our orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 mg/kg) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Background

Mild cognitive impairment (MCI), defined as a transitional zone between normal cognition and dementia, requires a battery of formal neuropsychological tests administered by a trained rater for its diagnosis. The objective of this study was to develop a screening tool for MCI.

Methods

One hundred ninety seven cognitively normal controls (NC), one hundred sixteen patients with amnestic MCI –single domain (aMCI-sd), one hundred ninety five patients with amnestic MCI-multiple domain (aMCI-md), and two hundred twenty eight patients with mild Alzheimer’s disease (AD) were evaluated by comprehensive neuropsychological tests and by the Memory and Executive Screening (MES).

Results

Correlation analysis showed that the three indicators of the MES were significantly negatively related with age (P<0.05), yet not related with education (P>0.05). There was no ceiling or floor effect. Test completion averaged seven minutes (421.14±168.31 seconds). The receiver operating characteristics (ROC) analyses performed on the aMCI-sd group yielded 0.89 for the area under the curve (AUC) (95% CI, 0.85–0.92) for the MES-total score, with sensitivity of 0.795 and specificity of 0.828. There was 81% correct classification rate when the cut-off was set at less than 75. Meanwhile, the aMCI-md group yielded 0.95 for the AUC (95% CI, 0.93–0.97) for the MES-total score, with sensitivity of 0.87 and specificity of 0.91, and 90% correct classification rate when the cut-off was set at less than 72.

Conclusion

The MES, minimally time-consuming, may be a valid and easily administered cognitive screening tool with high sensitivity and specificity for aMCI, with single or multiple domain impairment.

Interleukin-17 (IL-17) is a proinflammatory cytokine that is associated with inflammation, autoimmune disorders, and even tumors. Previous studies revealed that a large group of human malignant tumors have abnormally high IL-17 expression. In the present study, we analyzed two single-nucleotide polymorphisms (SNPs) in the IL17A (rs2275913) and IL17F (rs763780) in 311 cervical cancer patients and 463 controls using TaqMan assays. Our results indicated that the frequencies of AA genotype and A allele of rs2275913 were significantly different between the cervical cancer patients and controls (P = 0.008, OR = 1.32, 95% CI, 1.07–1.62). Stratified analyses revealed that the polymorphism of rs2275913 was also associated with positive peritumor intravascular cancer emboli and high clinical stage. The genotype and allele frequencies of rs763780 did not show any difference between patients and controls or relate to patient clinical characteristics. Collectively, these findings suggested that IL17 gene polymorphism rs2275913 was associated with the susceptibility as well as positive peritumor intravascular cancer emboli and high clinical stage of cervical cancer in Chinese women.

Epitope-based vaccination might play an important role in the protective immunity against Japanese encephalitis virus (JEV) infection. The purpose of the study is to evaluate the immune characteristics of recombinant MVA carrying multi-epitope gene of JEV (rMVA-mep). The synthetic gene containing critical epitopes (B-cell, CTL and Th) of JEV was cloned into the eukaryotic expression vector pGEM-K1L, and the rMVA-mep was prepared. BALB/c mice were immunized with different dosages of purified rMVA-mep and the immune responses were determined in the form of protective response against JEV, antibodies titers (IgG1 and IgG2a), spleen cell lymphocyte proliferation, and the levels of interferon-γ and interleukin-4 cytokines. The results showed that live rMVA-mep elicited strongly immune responses in dose-dependent manner, and the highest level of immune responses was observed from the groups immunized with 107 TCID50 rMVA-mep among the experimental three concentrations. There were almost no difference of cytokines and neutralizing antibody titers among 107 TCID50 rMVA-mep, recombinant ED3 and inactivated JEV vaccine. It was noteworthy that rMVA-mep vaccination potentiates the Th1 and Th2-type immune responses in dose-dependent manner, and was sufficient to protect the mice survival against lethal JEV challenge. These findings demonstrated that rMVA-mep can produce adequate humoral and cellular immune responses, and protection in mice, which suggested that rMVA-mep might be an attractive candidate vaccine for preventing JEV infection.

Serotonin is a critical regulator of bone mass, fulfilling different functions depending on its site of synthesis. Brain-derived serotonin promotes osteoblast proliferation, whereas duodenal-derived serotonin suppresses it. To understand the molecular mechanisms of duodenal-derived serotonin action on osteoblasts, we explored its transcriptional mediation in mice. We found that the transcription factor FOXO1 is a crucial determinant of the effects of duodenum-derived serotonin on bone formation We identified two key FOXO1 complexes in osteoblasts, one with the transcription factor cAMP-responsive element–binding protein 1 (CREB) and another with activating transcription factor 4 (ATF4). Under normal levels of circulating serotonin, the proliferative activity of FOXO1 was promoted by a balance between its interaction with CREB and ATF4. However, high circulating serotonin levels prevented the association of FOXO1 with CREB, resulting in suppressed osteoblast proliferation. These observations identify FOXO1 as the molecular node of an intricate transcriptional machinery that confers the signal of duodenal-derived serotonin to inhibit bone formation.

Background

Osteoprotegerin (OPG), a key regulatory factor in bone metabolism, was documented also a potential pro-angiogenic factor, which acts an important role in protecting vascular endothelial cells. Since preeclampsia has gradually been employed to be vascular diseases, we speculated that OPG might be associated with preeclampsia. The study was to evaluate the level of OPG protein and mRNA in placenta, and investigate the relationship between OPG and the pathogenesis of preeclampsia.

Methodology/Principal Findings

Placental specimens from 30 term normal pregnancy, 30 severe preeclampsia and 30 mild cases were studied. The expression and levels of OPGs’ protein and mRNA were detected by immunohistochemisty, western blot analysis and real-time quantitative PCR analysis respectively. The expression of OPG protein was found in cytoplasm of placenta cytotrophoblasts and syncytiotrophoblasts in three groups. There were no significant differences of OPG protein between the maternal and fetal side in each group. The OPG protein and mRNA levels in severe preeclampsia were significantly higher than those in mild cases and normal pregnancy. However, there were no markedly differences of the OPG protein and mRNA levels between term delivery and preterm delivery in severe cases. In preeclampsia, the OPG protein and mRNA level was positively correlated with systolic blood pressure and 24 h urinary protein respectively.

Conclusions/Significance

OPG protein and mRNA level in placentas of preeclampsia were found abnormal compared with normal pregnancy. In preeclampsia, the OPG protein and mRNA levels were closely related with its important clinical parameters. Taken together, OPG might be closely correlated with the pathogenesis of preeclampsia.

An epithelial sheet, the epicardium, lines the surface of the heart. In the developing embryo, the epicardium expresses the transcriptional regulator Wilm’s Tumor Gene 1 (Wt1). Through incompletely understood mechanisms, Wt1 inactivation derails normal heart development. We investigated mechanisms by which Wt1 regulates heart development and epicardial epithelial to mesenchymal transition (EMT). We used genetic lineage tracing approaches to track and isolate epicardium and epicardium derivatives in hearts lacking Wt1 (Wt1KO). Wt1KO hearts had diminished proliferation of compact myocardium and impaired coronary plexus formation. Wt1KO epicardium failed to undergo EMT. Wt1KO epicardium expressed reduced Lef1 and Ctnnb1 (β-catenin), key components of the canonical Wnt/β-catenin signaling pathway. Wt1KO epicardium expressed decreased levels of canonical Wnt downstream targets Axin2, Cyclin D1, and Cyclin D2 and exhibited decreased activity of the Batgal Wnt/b-catenin reporter transgene, suggestive of diminished canonical Wnt signaling. Hearts with epicardium-restricted Ctnnb1 loss of function resembled Wt1KO hearts and also failed to undergo epicardial EMT. However, Ctnnb1 inactivation did not alter WT1 expression, positioning Wt1 upstream of canonical Wnt/β-catenin signaling. Wnt5a, a prototypic non-canonical Wnt with enriched epicardial expression, and Raldh2, a key regulator of retinoic acid signaling confined to the epicardium, were also markedly downregulated in Wt1KO epicardium. Hearts lacking Wnt5a or Raldh2 shared phenotypic features with Wt1KO. Although Wt1 has been proposed to regulate EMT by repressing E-cadherin, we detected no change in E-cadherin in Wt1KO epicardium. Collectively, our study shows that Wt1 regulates epicardial EMT and heart development through canonical Wnt, non-canonical Wnt, and retinoic acid signaling pathways.

Yellow Sea green tides have occurred in coastal China almost every year from 2007 to 2011. Ulva prolifera (Müller) J. Agardh has been identified as the causative macroalgal species. U. intestinalis, however, has been observed in the bloom areas, co-occurring with U. prolifera, but it has not been found to be causative. The Yellow Sea green tide has shown consistent phases of development that match corresponding environmental changes. U. prolifera, not U. intestinalis, is dominant. Our experimental design was based on these observed phenomena, and the results of our field investigation indicated a close relationship between changes in principal environmental factors (irradiance, temperature, and salinity) and the development of each phase of the bloom. These main environmental factors were simulated to allow estimation and comparison of the physiological responses of U. prolifera and U. intestinalis. Ecophysiological differences were found between these two species. (1) More photosynthetic activity and plasticity were detected in U. prolifera. (2) U. prolifera was found to be more sensitive to dynamic environments, especially harsh and changing environmental conditions. U. intestinalis was found to be more stable, probably due to the higher stress tolerance given by its antioxidant system. (3) Markedly higher nutrient absorption activity was observed in U. prolifera. Comparisons of the ecophysiological traits of these two species in this present study may foster understanding of their natural ecological processes. Specifically, U. prolifera seemed to be more engaged with the ephemeral blooms, while U. intestinalis seemed to be directed toward persistence. This also suggests that the ecological success of U. prolifera may be inextricably linked to its higher capacity for photosynthesis, nutrient absorption, and nutrient assimilation.

Searching metabolites against databases according to their masses is often the first step in metabolite identification for a mass spectrometry-based untargeted metabolomics study. Major metabolite databases include Human Metabolome DataBase (HMDB), Madison Metabolomics Consortium Database (MMCD), Metlin, and LIPID MAPS. Since each one of these databases covers only a fraction of the metabolome, integration of the search results from these databases is expected to yield a more comprehensive coverage. However, the manual combination of multiple search results is generally difficult when identification of hundreds of metabolites is desired. We have implemented a web-based software tool that enables simultaneous mass-based search against the four major databases, and the integration of the results. In addition, more complete chemical identifier information for the metabolites is retrieved by cross-referencing multiple databases. The search results are merged based on IUPAC International Chemical Identifier (InChI) keys. Besides a simple list of m/z values, the software can accept the ion annotation information as input for enhanced metabolite identification. The performance of the software is demonstrated on mass spectrometry data acquired in both positive and negative ionization modes. Compared with search results from individual databases, MetaboSearch provides better coverage of the metabolome and more complete chemical identifier information. Availability: The software tool is available at http://omics.georgetown.edu/MetaboSearch.html.

Background

Analysis of multiple LC-MS based metabolomic studies is carried out to determine overlaps and differences among various experiments. For example, in large metabolic biomarker discovery studies involving hundreds of samples, it may be necessary to conduct multiple experiments, each involving a subset of the samples due to technical limitations. The ions selected from each experiment are analyzed to determine overlapping ions. One of the challenges in comparing the ion lists is the presence of a large number of derivative ions such as isotopes, adducts, and fragments. These derivative ions and the retention time drifts need to be taken into account during comparison.

Results

We implemented an ion annotation-assisted method to determine overlapping ions in the presence of derivative ions. Following this, each ion is represented by the monoisotopic mass of its cluster. This mass is then used to determine overlaps among the ions selected across multiple experiments.

Conclusion

The resulting ion list provides better coverage and more accurate identification of metabolites compared to the traditional method in which overlapping ions are selected on the basis of individual ion mass.

Two hepatitis B virus (HBV) C/D recombinants were isolated from western China. No direct evidence indicates that these new viruses arose as a result of recombination between genotype C and D or a result of convergence. In this study, we search for evidence of intra-individual recombination in the family cluster cases with co-circulation of genotype C, D and C/D recombinants. We studied 68 individuals from 15 families with HBV infections in 2006, identified individuals with mixed HBV genotype co-infections by restriction fragment length polymorphism and proceeded with cloning and DNA sequencing. Recombination signals were detected by RDP3 software and confirmed by split phylogenetic trees. Families with mixed HBV genotype co-infections were resampled in 2007. Three of 15 families had individuals with different HBV genotype co-infections in 2006. One individual (Y2) had a triple infection of HBV genotype C, D and C/D recombinant in 2006, but only genotype D in 2007. Further clonal analysis of this patient indicated that the C/D recombinant was not identical to previously isolated CD1 or CD2, but many novel recombinants with C2, D1 and CD1 were simultaneously found. All parental strains could recombine with each other to form new recombinant in this patient. This indicates that the detectable mixed infection and recombination have a limited time window. Also, as the recombinant nature of HBV precludes the possibility of a simple phylogenetic taxonomy, a new standard may be required for classifying HBV sequences.

Retrograded starch (RS3) was produced from indica rice starch with three kinds of gums (konjac glucomannan, KGM; carrageenan, CA, USA; and gellan, GA, USA) by autoclaving, respectively, and the effect of the gums on the retrogradation behavior of starch was estimated. The influences of polysaccharide concentration, sodium chloride concentration, autoclaving time, refrigerated time, and pH value on RS3 formation were discussed. Except for sodium chloride’s persistent restraint on RS3, the others all forced RS3 yields higher at first, but lowered it after the peak value. The influencing sequence of these impact factors was: sodium chloride concentration > polysaccharide concentration > autoclaving time > refrigerated time > pH value. The results also proved that in the three gums, KGM plays the most significant role in RS3 changing. It was concluded that the incorporation of each of these three gums into starch, especially KGM, results in an increase or decrease of RS3 under different conditions. This phenomenon could be taken into consideration when developing starchy food with appropriate amount of RS3.

Background

Chimonanthus nitens (family Calycanthaceae), Shanlamei in Chinese, is an unique species in China. The extract of dried leaves of Chimonanthus nitens has anti-inflammatory, antipyretic and antitussive effects. Terpenes, coumarins, and flavonoids are usually regarded as the main active components. Therefore, simultaneous determination of these compounds is very important to control the quality of Chimonanthus nitens.

Results

A double-development TLC method was developed for simultaneous analysis of five compounds in Chimonanthus nitens. The chromatography was performed on silica gel 60 plate with chloroform-methanol (9∶1, v/v) and petroleum ether-ethyl acetate (10∶1, v/v) as mobile phase for twice development. Their characteristic TLC profiles were observed under UV light at 365 nm and the bands were then revealed by reaction with 1% vanillin-H2SO4 solution. Quantification of three monoterpenes was achieved by densitometry at 545 nm (β-caryophyllene) or 606 nm (cineole and linalool). Two coumarins (scopoletin and scoparone) were determined by densitometry at 340 nm with filter wavelength of 370 nm. The investigated compounds had good linearity (R2 >0.99) within test ranges.

Conclusions

The developed double-development TLC method is helpful to control the quality of Chimonanthus nitens, which is simple and accurate.