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1.  Inducible Cardiomyocyte-Specific Gene Disruption Directed by the Rat Tnnt2 Promoter in the Mouse 
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.
PMCID: PMC2806493  PMID: 20014345
cardiomyocyte; Cre recombinase; doxycycline; rtTA; Tnnt2
2.  Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart  
The Journal of Clinical Investigation  2009;119(6):1462-1476.
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.
PMCID: PMC2689123  PMID: 19411759
3.  Dependence of Mechanical Properties of Trabecular Bone on Plate-Rod Microstructure Determined by Individual Trabecula Segmentation (ITS) 
Journal of biomechanics  2013;47(3):702-708.
Individual trabecula segmentation (ITS) technique can decompose the trabecular bone network into individual trabecular plates and rods and is capable of quantifying the plate/rod-related microstructural characteristics of trabecular bone. This novel technique has been shown to be able to provide in-depth insights into micromechanics and failure mechanisms of human trabecular bone, as well as to distinguish the fracture status independent of area bone mineral density in clinical applications. However, the plate/rod microstructural parameters from ITS have never been correlated to experimentally determined mechanical properties of human trabecular bone. In this study, on-axis cylindrical trabecular bone samples from human proximal tibia (n=30), vertebral body (n=10), and proximal femur (n=30) were harvested, prepared, scanned using micro computed-tomography (μCT), analyzed with ITS and mechanically tested. Regression analyses showed that the plate bone volume fraction (pBV/TV) and axial bone volume fraction (aBV/TV) calculated by ITS analysis correlated the best with elastic modulus (R2=0.96-0.97) and yield strength (R2=0.95-0.96). Trabecular plate-related microstructural parameters correlated highly with elastic modulus and yield strength, while most rod-related parameters were found inversely and only moderately correlated with the mechanical properties. In addition, ITS analysis also identified that trabecular bone at human femoral neck has the highest trabecular plate-related parameters while the other sites are similar with each other in terms of plate-rod microstructure.
PMCID: PMC3925244  PMID: 24360196
4.  Genetic targeting of sprouting angiogenesis using Apln-CreER 
Nature Communications  2015;6:6020.
Under pathophysiological conditions in adults, endothelial cells (ECs) sprout from pre-existing blood vessels to form new ones by a process termed angiogenesis. During embryonic development, Apelin (APLN) is robustly expressed in vascular ECs. In adult mice, however, APLN expression in the vasculature is significantly reduced. Here we show that APLN expression is reactivated in adult ECs after ischaemia insults. In models of both injury ischaemia and tumor angiogenesis, we find that Apln-CreER genetically labels sprouting but not quiescent vasculature. By leveraging this specific activity, we demonstrate that abolishment of the VEGF–VEGFR2 signalling pathway as well as ablation of sprouting ECs diminished tumour vascularization and growth without compromising vascular homeostasis in other organs. Collectively, we show that Apln-CreER distinguishes sprouting vessels from stabilized vessels in multiple pathological settings. The Apln-CreER line described here will greatly aid future mechanistic studies in both vascular developmental biology and adult vascular diseases.
Apelin expression is robust in embryonic but not in adult endothelial cells (ECs), where it can be reactivated by hypoxia. Liu et al. show that apelin-driven expression of Cre recombinase in mice can be used for labelling of, or gene ablation in, sprouting but not quiescent ECs in pathologies characterized by hypoxia.
PMCID: PMC4309445  PMID: 25597280
5.  Analysis of Recombinant H7N9 Wild-Type and Mutant Viruses in Pigs Shows that the Q226L Mutation in HA Is Important for Transmission 
Journal of Virology  2014;88(14):8153-8165.
The fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/1/2013 (H7N9) virus and variants in vitro and in vivo using a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission.
IMPORTANCE The genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the α-2,6-linked receptor and enables transmission in pigs; (ii) wild-type A/Anhui/1/2013 (H7N9) shows modest replication, virulence, and transmissibility in pigs, suggesting that it is not well adapted to the mammalian host; and (iii) both wild-type and variant H7N9 viruses rapidly develop additional mammalian-signature mutations in pigs, indicating that they represent an important potential intermediate host. This is the first study analyzing the phenotypic effects of specific mutations within the HA and PB2 genes of the novel H7N9 viruses created by reverse genetics in an important mammalian host model. Finally, this study illustrates that loss-of-function mutations can be used to effectively identify residues critical to zoonosis/transmission.
PMCID: PMC4097782  PMID: 24807722
6.  Epicardial calcineurin–NFAT signals through Smad2 to direct coronary smooth muscle cell and arterial wall development 
Cardiovascular Research  2013;101(1):120-129.
Congenital coronary artery anomalies produce serious events that include syncope, arrhythmias, myocardial infarction, or sudden death. Studying the mechanism of coronary development will contribute to the understanding of the disease and help design new diagnostic or therapeutic strategies. Here, we characterized a new calcineurin–NFAT signalling which specifically functions in the epicardium to regulate the development of smooth muscle wall of the coronary arteries.
Methods and results
Using tissue-specific gene deletion, we found that calcineurin–NFAT signals in the embryonic epicardium to direct coronary smooth muscle cell development. The smooth muscle wall of coronary arteries fails to mature in mice with epicardial deletion of calcineurin B1 (Cnb1), and accordingly these mutant mice develop cardiac dysfunction with reduced exercise capacity. Inhibition of calcineurin at various developmental windows shows that calcineurin–NFAT signals within a narrow time window at embryonic Day 12.5–13.5 to regulate coronary smooth muscle cell development. Within the epicardium, NFAT transcriptionally activates the expression of Smad2, whose gene product is critical for transducing transforming growth factor β (TGFβ)–Alk5 signalling to control coronary development.
Our findings demonstrate new spatiotemporal and molecular actions of calcineurin–NFAT that dictate coronary arterial wall development and a new mechanism by which calcineurin–NFAT integrates with TGFβ signalling during embryonic development.
PMCID: PMC3868347  PMID: 23946498
Epicardial; Calcineurin–NFAT; Coronary artery; Smooth muscle cell Differentiation; Smad2
7.  Evaluation of Metabolite Biomarkers for Hepatocellular Carcinoma through Stratified Analysis by Gender, Race and Alcoholic Cirrhosis 
The effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating metabolites have been subjected to continuing investigation. This study compares the levels of selected metabolites in sera of HCC cases versus patients with liver cirrhosis and evaluates the influence of gender, race, and alcoholic cirrhosis on the performance of the metabolites as candidate biomarkers for HCC.
Targeted quantitation of 15 metabolites is performed by selected research monitoring (SRM) in sera from 89 Egyptian subjects (40 HCC cases and 49 cirrhotic controls) and 110 US subjects (56 HCC cases and 54 cirrhotic controls). Logistic regression models are used to evaluate the ability of these metabolites in distinguishing HCC cases from cirrhotic controls. The influences of gender, race, and alcoholic cirrhosis on the performance of the metabolites are analyzed by stratified logistic regression.
Two metabolites are selected based on their significance to both cohorts. While both metabolites discriminate HCC cases from cirrhotic controls in males and Caucasians, they are insignificant in females and African Americans. One metabolite is significant in patients with alcoholic cirrhosis and the other in non-alcoholic cirrhosis.
The study demonstrates the potential of two metabolites as candidate biomarkers for HCC by combining them with α-fetoprotein and gender. Stratified statistical analyses reveal that gender, race, and alcoholic cirrhosis affect the relative levels of small molecules in serum.
The findings of this study contribute to a better understanding of the influence of gender, race, and alcoholic cirrhosis in investigating small molecules as biomarkers for HCC.
PMCID: PMC3947117  PMID: 24186894
Mass spectrometry; metabolomics; cancer biomarker; liver cirrhosis; health disparity
8.  Bidirectional selection between two classes in complex social networks 
Scientific Reports  2014;4:7577.
The bidirectional selection between two classes widely emerges in various social lives, such as commercial trading and mate choosing. Until now, the discussions on bidirectional selection in structured human society are quite limited. We demonstrated theoretically that the rate of successfully matching is affected greatly by individuals' neighborhoods in social networks, regardless of the type of networks. Furthermore, it is found that the high average degree of networks contributes to increasing rates of successful matches. The matching performance in different types of networks has been quantitatively investigated, revealing that the small-world networks reinforces the matching rate more than scale-free networks at given average degree. In addition, our analysis is consistent with the modeling result, which provides the theoretical understanding of underlying mechanisms of matching in complex networks.
PMCID: PMC4271259  PMID: 25524835
9.  Effect of self-management training on adherence to medications among community residents with chronic schizophrenia: a singleblind randomized controlled trial in Shanghai, China 
Shanghai Archives of Psychiatry  2014;26(6):332-338.
Many community dwelling individuals with schizophrenia do not take medications regularly and, thus, are prone to frequent relapses.
Evaluate the effectiveness of self-management training on adherence to medications and relapse among individuals with chronic schizophrenia living in the community.
A total of 201 individuals with chronic schizophrenia living in the urban and rural communities of Shanghai Municipality were randomized into a treatment as usual control group (n=98) or a selfmanagement intervention group (n=103) that received weekly self-management skills training for 6 months followed by 24 months of monthly group booster sessions in which a community health worker reviewed patients’ self-management checklists. Two psychiatrists blind to the treatment status of patients, assessed adherence to medications using the Morisky Medication Adherence Scale and patients’ insight into their illness using the Scale to Assess Unawareness of Mental Disorders (SAUMD) at baseline and 30 months after baseline. A total of 194 individuals (95.6%) completed the study.
There were no differences between groups at baseline, but after 30 months the intervention group had significantly better medication compliance, significantly greater insight into their illness, and (by self-report) were using significantly higher dosages of antipsychotic medication. Only 2 (1.9%) of the 103 intervention group participants relapsed (i.e., experienced one or more re-hospitalizations) over the 30 months of follow-up, but 14 (14.3%) of the 98 control group subjects relapsed (X2=8.83, p=0.003).
Given the large sample size, relatively long follow-up, randomized design, and single-blind evaluation of outcomes the dramatic reduction in relapse and improvements in medication adherence and insight identified in this study are robust findings. These results extended our previous findings, which demonstrated the benefit of self-management training on improving the symptoms and social functioning of individuals with chronic schizophrenia living in the community. Cost-benefit studies are now needed to assess the feasibility of up-scaling this self-management intervention to a wide range of communities.
PMCID: PMC4311106  PMID: 25642107
schizophrenia; community mental health services; medication adherence; insight; relapse; re-hospitalization; randomized controlled trial; China
10.  Intracranial multiple germ cell tumors: a case report and review of literature 
Intracranial multiple germ cell tumors (GCTs) are rare. In this article, we reported a case of intracranial multiple GCTs in an 18-year-old boy with symptoms of psychosis for 8 months also. Tumors in the pineal, sellar region, corpus callosum, bilateral lateral ventricles and fourth ventricle were confirmed by enhanced magnetic resonance imaging (MRI) and stereotactic biopsy. Immunohistochemical analysis results demonstrated that the tumor cells were positive for CD117 and placental alkaline phosphatase (PLAP). The patient was treated by radiotherapy and the prescribed radiation doses were 18 Gy. After near 24 months of follow-up, no local recurrence and distant metastasis has been found.
PMCID: PMC4314009
Intracranial multiple germ cell tumors; symptoms of psychosis; pineal; sellar region; corpus callosum; lateral ventricles
11.  Hypoxic Conditioned Medium from Rat Cerebral Cortical Cells Enhances the Proliferation and Differentiation of Neural Stem Cells Mainly through PI3-K/Akt Pathways 
PLoS ONE  2014;9(11):e111938.
To investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.
Cerebral cortical cells from neonatal Sprague–Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named ‘hypoxic conditioned medium’ (HCM) and ‘normoxic conditioned medium’ (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.
The protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by LY294002 and SP600125, with LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by LY294002 and SP600125.
4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.
PMCID: PMC4227679  PMID: 25386685
12.  Study on the Traffic Air Pollution inside and outside a Road Tunnel in Shanghai, China 
PLoS ONE  2014;9(11):e112195.
To investigate the vehicle induced air pollution situations both inside and outside the tunnel, the field measurement of the pollutants concentrations and its diurnal variations was performed inside and outside the Xiangyin tunnel in Shanghai from 13:00 on April 24th to 13:00 on April 25th, 2013. The highest hourly average concentrations of pollutants were quantified that CO, NO, NO2 and NOX inside the tunnel were 13.223 mg/m3, 1.829 mg/m3, 0.291 mg/m3 and 3.029 mg/m3, respectively, while the lowest ones were 3.086 mg/m3, 0.344 mg/m3, 0.080 mg/m3 and 0.619 mg/m3. Moreover, the concentrations of pollutants were higher during the daytime, and lower at night, which is relevant to the traffic conditions inside the tunnel. Pollutants concentrations inside the tunnel were much higher than those outside the tunnel. Then in a case of slow wind, the effect of wind is much smaller than the impact of pollution sources. Additionally, the PM2.5 concentrations climbed to the peak sharply (468.45 µg/m3) during the morning rush hours. The concentrations of organic carbon (OC) and elemental carbon (EC) in PM2.5 inside the tunnel were 37.09–99.06 µg/m3 and 22.69–137.99 µg/m3, respectively. Besides, the OC/EC ratio ranged from 0.72 to 2.19 with an average value of 1.34. Compared with the results of other tunnel experiments in Guangzhou and Shenzhen, China, it could be inferred that the proportion of HDVs through the Xiangyin tunnel is relatively lower.
PMCID: PMC4227705  PMID: 25386920
13.  Ginsenoside Rb1 Prevents H2O2-Induced HUVEC Senescence by Stimulating Sirtuin-1 Pathway 
PLoS ONE  2014;9(11):e112699.
We have previously reported that Ginsenoside Rb1 may effectively prevent HUVECs from senescence, however, the detailed mechanism has not demonstrated up to now. Recent studies have shown that sirtuin-1 (Sirt1) plays an important role in the development of endothelial senescence. The purpose of this study was to explore whether Sirt1 is involved in the action of Ginsenoside Rb1 regarding protection against H2O2-induced HUVEC Senescence.
Methods and Results
Senescence induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs) was examined by analyzing plasminogen activator inhibitor-1 (PAI-1) expression, cell morphology, and senescence-associated beta-galactosidase (SA-β-gal) activity. The results revealed that 42% of control-treated HUVECs were SA-β-gal positive after treatment by 60 µmol/L H2O2, however, this particular effect of H2O2 was decreased more than 2-fold (19%) in the HUVECs when pretreated with Rb1 (20 µmol/L) for 30 min. Additionally, Rb1 decreased eNOS acetylation, as well as promoted more NO production that was accompanied by an increase in Sirt1 expression. Furthermore, upon knocking down Sirt1, the effect of Rb1 on HUVEC senescence was blunted.
The present study indicated that Ginsenoside Rb1 acts through stimulating Sirt1 in order to protect against endothelial senescence and dysfunction. As such, Sirt1 appears to be of particular importance in maintaining endothelial functions and delaying vascular aging.
PMCID: PMC4227851  PMID: 25386949
14.  Nfatc1 Directs the Endocardial Progenitor Cells to Make Heart Valve Primordium 
Trends in cardiovascular medicine  2013;23(8):294-300.
Heart valves arise from the cardiac endocardial cushions located at the atrioventricular canal (AVC) and cardiac outflow tract (OFT) during development. A subpopulation of cushion endocardial cells undergoes endocardial to mesenchymal transformation (EMT) and generates the cushion mesenchyme, which is then remodeled into the interstitial tissue of the mature valves. The cushion endocardial cells that do not undertake EMT proliferate to elongate valve leaflets. During EMT and the post-EMT valve remodeling, endocardial cells at the cushions highly express nuclear factor in activated T-cell, cytoplasmic 1 (Nfatc1), a transcription factor required for valve formation in mice. In this review, we present the current knowledge of Nfatc1 roles in the ontogeny of heart valves with a focus on the fate decision of the endocardial cells in the processes of EMT and valve remodeling.
PMCID: PMC3766465  PMID: 23669445
15.  Fabp4-CreER lineage tracing revealstwo distinctive coronary vascular populations 
Over the last two decades, genetic lineage tracing has allowed for the elucidation of the cellular origins and fates during both embryogenesis and in pathological settings in adults. Recent lineage tracing studies using Apln-CreER tool indicated that a large number of post-natal coronary vessels do not form from pre-existing vessels. Instead, they form de novo after birth, which represents a coronary vascular population (CVP) distinct from the pre-existing one. Herein, we present new coronary vasculature lineage tracing results using a novel tool, Fabp4-CreER. Our results confirm the distinct existence of two unique CVPs. The 1st CVP, which is labelled by Fabp4-CreER, arises through angiogenic sprouting of pre-existing vessels established during early embryogenesis. The 2nd CVP is not labelled by Fabp4, suggesting that these vessels form de novo, rather than through expansion of the 1st CVP. These results support the de novo formation of vessels in the post-natal heart, which has implications for studies in cardiovascular disease and heart regeneration.
PMCID: PMC4224549  PMID: 25265869
coronary vessel; angiogenesis; coronary artery diseases; heart regeneration
16.  BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development 
PLoS ONE  2014;9(10):e109493.
ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.
PMCID: PMC4192121  PMID: 25299188
17.  Olfaction spontaneously highlights visual saliency map 
Attention is intrinsic to our perceptual representations of sensory inputs. Best characterized in the visual domain, it is typically depicted as a spotlight moving over a saliency map that topographically encodes strengths of visual features and feedback modulations over the visual scene. By introducing smells to two well-established attentional paradigms, the dot-probe and the visual-search paradigms, we find that a smell reflexively directs attention to the congruent visual image and facilitates visual search of that image without the mediation of visual imagery. Furthermore, such effect is independent of, and can override, top-down bias. We thus propose that smell quality acts as an object feature whose presence enhances the perceptual saliency of that object, thereby guiding the spotlight of visual attention. Our discoveries provide robust empirical evidence for a multimodal saliency map that weighs not only visual but also olfactory inputs.
PMCID: PMC3757990  PMID: 23945694
olfaction; saliency map; attention; multi-sensory integration
18.  Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus 
PLoS Pathogens  2014;10(10):e1004420.
Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.
Author Summary
The identification of influenza virus-like sequences in two different bat species has generated great interest in understanding their biology, ability to mix with other influenza viruses, and their public health threat. Unfortunately, bat-influenza viruses couldn't be cultured from the samples containing the influenza-like nucleic acids. We used synthetic genomics strategies to create wild type bat-influenza, or bat-influenza modified by substituting the surface glycoproteins with those of model influenza A viruses. Although influenza virus-like particles were produced from both synthetic genomes, only the modified bat-influenza viruses could be cultured. The modified bat-influenza viruses replicated efficiently in vitro and an H1N1 modified version caused severe disease in mice. Collectively our data show: (1) the two bat-flu genomes identified in other studies are replication competent, suggesting that host cell specificity is the major limitation for propagation of bat-influenza, (2) bat-influenza NS1 antagonizes host interferon response more efficiently than that of a model influenza A virus, (3) bat-influenza has both genetic and protein incompatibility with influenza A or B viruses, and (4) that these bat-influenza lineages pose little pandemic threat.
PMCID: PMC4183581  PMID: 25275541
19.  Osteogenic fate of hypertrophic chondrocytes 
Cell Research  2014;24(10):1266-1269.
PMCID: PMC4185343  PMID: 25145361
20.  Prognostic value of serum nicotinamide phosphoribosyltransferase in patients with bladder cancer 
Croatian Medical Journal  2014;55(5):507-513.
To analyze the serum nicotinamide phosphoribosyltransferase (Nampt) level and its prognostic value in bladder cancer (BC).
The study included 131 patients with transitional cell BC and 109 healthy controls from the West China Hospital of Sichuan University in the period between 2007 and 2013. Nampt concentration in serum was measured by commercial ELISA kits for human Nampt.
The serum Nampt protein level in patients with BC (mean ± standard deviation, 16.02 ± 7.95 ng/mL) was significantly higher than in the control group (6.46 ± 2.08 ng/mL) (P < 0.001). Serum Nampt level was an independent prognostic marker of non-muscle-invasive BC, with a higher serum Nampt level (>14.74 ng/mL) indicating shorter recurrence-free survival rate (hazard ratio = 2.85, 95% confidence interval, 1.01-8.06; P = 0.048).
Our results suggest that serum Nampt level may serve as a biomarker of BC and an independent prognostic marker of non-muscle-invasive BC.
PMCID: PMC4228298  PMID: 25358883
21.  Multifrequency Excitation Method for Rapid and Accurate Dynamic Test of Micromachined Gyroscope Chips 
Sensors (Basel, Switzerland)  2014;14(10):19507-19516.
A novel multifrequency excitation (MFE) method is proposed to realize rapid and accurate dynamic testing of micromachined gyroscope chips. Compared with the traditional sweep-frequency excitation (SFE) method, the computational time for testing one chip under four modes at a 1-Hz frequency resolution and 600-Hz bandwidth was dramatically reduced from 10 min to 6 s. A multifrequency signal with an equal amplitude and initial linear-phase-difference distribution was generated to ensure test repeatability and accuracy. The current test system based on LabVIEW using the SFE method was modified to use the MFE method without any hardware changes. The experimental results verified that the MFE method can be an ideal solution for large-scale dynamic testing of gyroscope chips and gyroscopes.
PMCID: PMC4239857  PMID: 25330052
micromachined gyroscope chip; dynamic test; multifrequency excitation
22.  Generation and Efficacy Evaluation of Recombinant Classical Swine Fever Virus E2 Glycoprotein Expressed in Stable Transgenic Mammalian Cell Line 
PLoS ONE  2014;9(9):e106891.
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1∶40 to 1∶320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1∶10,240 to 1∶81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1∶80 to 1∶10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1×105 TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3–4 days after challenge and were euthanized from 7–9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.
PMCID: PMC4157854  PMID: 25198669
23.  Dynamic behaviors of approximately ellipsoidal microbubbles photothermally generated by a graphene oxide-microheater 
Scientific Reports  2014;4:6086.
Thermal microbubbles generally grow directly from the heater and are spherical to minimize surface tension. We demonstrate a novel type of microbubble indirectly generated from a graphene oxide-microheater. Graphene oxide's photothermal properties allowed for efficient generation of a thermal gradient field on the microscale. A series of approximately ellipsoidal microbubbles were generated on the smooth microwire based on heterogeneous nucleation. Other dynamic behaviors induced by the microheater such as constant growth, directional transport and coalescence were also investigated experimentally and theoretically. The results are not only helpful for understanding the bubble dynamics but also useful for developing novel photothermal bubble-based devices.
PMCID: PMC4133711  PMID: 25124694
24.  In the jungle of time: the concept of identity as a way out 
What could be a unifying principle for the manifold of temporal experiences: the simultaneity or temporal order of events, the subjective present, the duration of experiences, or the impression of a continuity of time? Furthermore, we time travel to the past visiting in imagination previous experiences in episodic memory, and we also time travel to the future anticipating actions or plans. For such time traveling we divide time into three domains: past, present, and future. What could be an escape out of this “jungle of time” characterized by many different perceptual and conceptual phenomena? The key concept we want to propose is “identity” which is derived from homeostasis as a fundamental biological principle. Within this conceptual frame two modes of identity are distinguished: individual or self-identity required because of homeostatic demands, and object-related identity necessary for the reliability and efficiency of neuro-cognitive processing. With this concept of self- and object-identity, the different temporal experiences can be conceptualized within a common frame. Thus, we propose a fundamental biological principle to conceptually unify temporal phenomena on the psychological level.
PMCID: PMC4114202  PMID: 25120528
time; identity; homeostasis; circadian rhythm; subjective present; simultaneity; episodic memory
25.  Quantitative Analysis of Adulterations in Oat Flour by FT-NIR Spectroscopy, Incomplete Unbalanced Randomized Block Design, and Partial Least Squares 
This paper developed a rapid and nondestructive method for quantitative analysis of a cheaper adulterant (wheat flour) in oat flour by NIR spectroscopy and chemometrics. Reflectance FT-NIR spectra in the range of 4000 to 12000 cm−1 of 300 oat flour objects adulterated with wheat flour were measured. The doping levels of wheat flour ranged from 5% to 50% (w/w). To ensure the generalization performance of the method, both the oat and the wheat flour samples were collected from different producing areas and an incomplete unbalanced randomized block (IURB) design was performed to include the significant variations that may be encountered in future samples. Partial least squares regression (PLSR) was used to develop calibration models for predicting the levels of wheat flour. Different preprocessing methods including smoothing, taking second-order derivative (D2), and standard normal variate (SNV) transformation were investigated to improve the model accuracy of PLS. The root mean squared error of Monte Carlo cross-validation (RMSEMCCV) and root mean squared error of prediction (RMSEP) were 1.921 and 1.975 (%, w/w) by D2-PLS, respectively. The results indicate that NIR and chemometrics can provide a rapid method for quantitative analysis of wheat flour in oat flour.
PMCID: PMC4131071  PMID: 25143857

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