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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.  Single cell lineage tracing reveals that oriented cell division contributes to trabecular morphogenesis and regional specification 
Cell reports  2016;15(1):158-170.
The cardiac trabeculae are sheet-like structures extending from the myocardium that function to increase surface area. A lack of trabeculation causes embryonic lethality due to compromised cardiac function. To understand the cellular and molecular mechanisms of trabecular formation, we genetically labeled individual cardiomyocytes prior to trabeculation via the brainbow multicolor system, and traced and analyzed the labeled cells during trabeculation by whole-embryo clearing and imaging. The clones derived from labeled single cells displayed four different geometric patterns that are derived from different patterns of oriented cell division (OCD) and migration. Of the four types of clones, the inner, transmural, and mixed clones contributed to trabecular cardiomyocytes. Further studies showed that perpendicular OCD is an extrinsic asymmetric cell division that putatively contributes to trabecular regional specification. Furthermore, N-Cadherin deletion in labeled clones disrupted the clonal patterns. In summary, our data demonstrate that OCD contributes to trabecular morphogenesis and specification.
PMCID: PMC4826812  PMID: 27052172
4.  The association of cigarette smoking and alcohol drinking with body mass index: a cross-sectional, population-based study among Chinese adult male twins 
BMC Public Health  2016;16:311.
Obesity is a multifactorial abnormality which has an underlying genetic control but requires environmental influences to trigger. Numerous epidemiological studies have examined the roles of physical inactivity and dietary factors in obesity development. Interactions between obesity-related genes and these lifestyles have also been confirmed. However, less attention has been paid to these complex relationship between cigarette smoking, alcohol drinking and obesity. The purpose of this study was to assess whether cigarette smoking and alcohol drinking were associated with body mass index (BMI), and whether these lifestyle factors modified the genetic variance of BMI.
Subjects were twins recruited through the Chinese National Twin Registry, aged 18 to 79 years, and the sample comprised 6121 complete male twin pairs. Information on height, weight, cigarette smoking and alcohol drinking status were assessed with self-report questionnaires. The associations of cigarette smoking and alcohol drinking with BMI were evaluated by linear regression models. Further, structure equation models were conducted to estimate whether cigarette smoking and alcohol drinking status modified the degree of genetic variance of BMI.
After adjustment for a variety of socio-demographic and lifestyle factors, former smokers had higher BMI (β = 0.475; 95 % CI, 0.196 to 0.754) whereas moderate to heavy smokers had lower BMI (β = −0.115; 95 % CI, −0.223 to −0.007) when compared with nonsmokers. BMI decreased with increased cigarette pack-years (β = −0.008; 95 % CI, −0.013 to −0.003). These effects still existed substantially in within-MZ twin pair analyses. By contrast, current alcohol drinking had no significant influence on BMI when additionally controlled for shared factors in within-pair analyses. Genetic modification by alcohol drinking was statistically significant for BMI (β = −0.137; 95 % CI, −0.215 to −0.058), with the intake of alcohol decreasing the additive genetic component of BMI.
Cigarette smoking was negatively associated with BMI independent of genetic influences. The influence of genes on BMI was moderated by alcohol drinking, such that for individuals who were regular drinkers, genetic factors became less influential. Our findings highlight gene-alcohol interaction in finding candidate genes of BMI and elucidating the etiological factors of obesity.
PMCID: PMC4827244  PMID: 27068329
Twin studies; Body mass index; Gene-environment interaction; Smoking; Alcohol drinking
5.  Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads 
BioData Mining  2016;9:13.
Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.
We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.
PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.
Electronic supplementary material
The online version of this article (doi:10.1186/s13040-016-0090-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4820869  PMID: 27051465
PacBio; Bioinformatics analysis; Gene isoforms; Repetitive sequences; Major surface glycoprotein; Pneumocystis; NGS; Uclust
6.  Identification of Multiple Stress Responsive Genes by Sequencing a Normalized cDNA Library from Sea-Land Cotton (Gossypium barbadense L.) 
PLoS ONE  2016;11(3):e0152927.
Plants often face multiple stresses including drought, extreme temperature, salinity, nutrition deficiency and biotic stresses during growth and development. All the stresses result in a series of physiological and metabolic reactions and then generate reversible inhibition of metabolism and growth and can cause seriously irreversible damage, even death. At each stage of cotton growth, environmental stress conditions pose devastating threats to plant growth and development, especially yield and quality. Due to the complex stress conditions and unclear molecular mechanisms of stress response, there is an urgent need to explore the mechanisms of cotton response against abiotic stresses.
Methodology and Principal Findings
A normalized cDNA library was constructed using Gossypium barbadense Hai-7124 treated with different stress conditions (heat, cold, salt, drought, potassium and phosphorus deficit and Verticillium dahliae infection). Random sequencing of this library generated 6,047 high-quality expressed sequence tags (ESTs). The ESTs were clustered and assembled into 3,135 uniESTs, composed of 2,497 contigs and 638 singletons. The blastx results demonstrated 2,746 unigenes showing significant similarity to known genes, 74 uniESTs displaying significant similarity to genes of predicted proteins, and 315 uniESTs remain uncharacterized. Functional classification unveiled the abundance of uniESTs in binding, catalytic activity, and structural molecule activity. Annotations of the uniESTs by the plant transcription factor database (PlantTFDB) and Plant Stress Protein Database (PSPDB) disclosed that transcription factors and stress-related genes were enriched in the current library. The expression of some transcription factors and specific stress-related genes were verified by RT-PCR under various stress conditions.
Annotation results showed that a huge number of genes respond to stress in our study, such as MYB-related, C2H2, FAR1, bHLH, bZIP, MADS, and mTERF. These results will improve our knowledge of stress tolerance in cotton. In addition, they are also helpful in discovering candidate genes related to stress tolerance. The publicly available ESTs from G. barbadense are a valuable genomic resource that will facilitate further molecular study and breeding of stress-tolerant cotton.
PMCID: PMC4816313  PMID: 27031331
7.  Effects of icotinib on early-stage non-small-cell lung cancer as neoadjuvant treatment with different epidermal growth factor receptor phenotypes 
OncoTargets and therapy  2016;9:1735-1741.
Epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs) have demonstrated efficacy in treating advanced non-small-cell lung cancer (NSCLC). Preliminary findings suggested that EGFR–TKIs might also be beneficial in neoadjuvant therapy in treating NSCLC. Therefore, this study aimed to evaluate the efficacy and safety of neoadjuvant therapy with icotinib in patients with early-stage NSCLC.
Patients and methods
We retrospectively reviewed the medical history of patients who were initially diagnosed with stage IA–IIIA NSCLC and were under icotinib administration before surgery between December 2011 and December 2014. Tumor assessment was conducted between the second and fourth week from initial icotinib treatment. The association between personal characteristics, smoking status, disease stage, EGFR mutation status, and clinical outcomes were investigated using multivariate logistic regression analysis.
A total of 67 patients with NSCLC were reviewed, and approximately half (38/67) of them were identified as having EGFR-mutant tumors. The overall response rate of all patients was 26.7% at 2–4 weeks’ assessment. Multivariate analysis showed that female sex (38.5% versus 10.7% in males, P=0.028) and EGFR mutation status (42.1% versus 6.9% in EGFR wild type, P=0.011) were independent predictive factors. The analysis also showed that the most common adverse effects were rash (43.3%) and dry skin (34.4%), which were tolerable.
Icotinib induced clinical response with minimal toxicity as neoadjuvant treatment in early NSCLC, especially in patients with common EGFR mutations. Further studies are warranted to confirm our findings.
PMCID: PMC4809345  PMID: 27042123
non-small-cell lung cancer; epidermal growth factor receptor; tyrosine kinase inhibitor; neoadjuvant
8.  Trabecular Plates and Rods Determine Elastic Modulus and Yield Strength of Human Trabecular Bone 
Bone  2014;0:71-80.
The microstructure of trabecular bone is usually perceived as a collection of plate-like and rod-like trabeculae, which can be determined from the emerging high-resolution skeletal imaging modalities such as micro computed tomography (μCT) or clinical high-resolution peripheral quantitative CT (HR-pQCT) using the individual trabecula segmentation (ITS) technique. It has been shown that the ITS-based plate and rod parameters are highly correlated with elastic modulus and yield strength of human trabecular bone. In the current study, plate-rod (PR) finite element (FE) models were constructed completely based on ITS-identified individual trabecular plates and rods. We hypothesized that PR FE can accurately and efficiently predict elastic modulus and yield strength of human trabecular bone. Human trabecular bone cores from proximal tibia (PT), femoral neck (FN) and greater trochanter (GT) were scanned by micro computed tomography (μCT). Specimen-specific ITS-based PR FE models were generated for each μCT image and corresponding voxel-based FE models were also generated in comparison. Both types of specimen-specific models were subjected to nonlinear FE analysis to predict the apparent elastic modulus and yield strength using the same trabecular bone tissue properties. Then, mechanical tests were performed to experimentally measure the apparent modulus and yield strength. Strong linear correlations for both elastic modulus (r2=0.97) and yield strength (r2=0.96) were found between the PR FE model predictions and experimental measures, suggesting that trabecular plates and rods morphology adequately captures three-dimensional (3D) microarchitecture of human trabecular bone. In addition, the PR FE model predictions in both elastic modulus and yield strength were highly correlated with the voxel-based FE models (r2=0.99, r2=0.98, respectively), resulted from the original 3D images without the PR segmentation. In conclusion, the ITS-based PR models predicted accurately both elastic modulus and yield strength determined experimentally across three distinct anatomic sites. Trabecular plates and rods accurately determine elastic modulus and yield strength of human trabecular bone.
PMCID: PMC4282941  PMID: 25460571
Individual trabecula segmentation; microarchitecture; plate and rod; finite element; elastic modulus; yield strength
9.  Tie1 is required for lymphatic valve and collecting vessel development 
Developmental biology  2015;399(1):117-128.
Tie1 is a receptor tyrosine kinase with broad expression in embryonic endothelium. Reduction of Tie1 levels in mouse embryos with a hypomorphic Tie1 allele resulted in abnormal lymphatic patterning and architecture, decreased lymphatic draining efficiency, and ultimately, embryonic demise. Here we report that Tie1 is present uniformly throughout the lymphatics and from late embryonic/early postnatal stages, becomes more restricted to lymphatic valve regions. To investigate later events of lymphatic development, we employed Cre-loxP recombination utilizing a floxed Tie1 allele and an Nfatc1Cre line, to provide loxP excision predominantly in lymphatic endothelium and developing valves. Interestingly, unlike the early prenatal defects previously described by ubiquitous endothelial deletion, excision of Tie1 with Nfatc1Cre resulted in abnormal lymphatic defects in postnatal mice and was characterized by agenesis of lymphatic valves and a deficiency of collecting lymphatic vessels. Attenuation of Tie1 signaling in lymphatic endothelium prevented initiation of lymphatic valve specification by Prox1 high expression lymphatic endothelial cells that is associated with the onset of turbulent flow in the lymphatic circulation. Our findings reveal a fundamental role for Tie signaling during lymphatic vessel remodeling and valve morphogenesis and implicate it as a candidate gene involved in primary lymphedema.
PMCID: PMC4374493  PMID: 25576926
Tie1; lymphatic valve; lymphovenous valve; lymphatic vessel remodeling; conditional knockout; Prox1
10.  Association of Plasma IL-32 Levels and Gene Polymorphisms with Systemic Lupus Erythematosus in Chinese Han Population 
Disease Markers  2016;2016:2460206.
Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease. IL-32, a secreted protein, has been reported to be associated with several autoimmune diseases. Our preliminary experiment showed different plasma IL-32 levels than that mentioned in a published report on the same population. In order to elucidate the correlation between IL-32 and SLE, we determined the plasma level and two single nucleotide polymorphisms (SNPs) of IL-32 in 152 patients with SLE and 310 healthy controls and analyzed the relationship based on the clinical parameters. The results showed that plasma IL-32 levels in patients with SLE were markedly lower than that in the healthy controls. In the SLE group, patients with detectable IL-32 presented low serum C3 concentrations. Further studies indicated that the rs28372698 SNP was associated with the susceptibility to SLE. Taken together, our results suggested that IL-32 could possibly be a candidate marker to monitor SLE disease stability and screening in future.
PMCID: PMC4789414  PMID: 27069296
11.  Epicardial FSTL1 reconstitution regenerates the adult mammalian heart 
Nature  2015;525(7570):479-485.
The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
PMCID: PMC4762253  PMID: 26375005
12.  Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis 
The Journal of Clinical Investigation  null;125(11):4122-4134.
Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.
PMCID: PMC4639982  PMID: 26436652
13.  Formosins A–F: Diterpenoids with Anti-microbial Activities from Excoecaria formosana 
Three new halimane-type diterpenoids formosins A–C (1–3), and three clerodane-type diterpenoids formosins D–F (4–6), were isolated from the twigs of Excoecaria formosana. Their structures were assigned on the basis of spectroscopic data analysis. Compounds 1 and 4 showed moderate anti-microbial activities against Bacillus subtilis (MIC = 50 and 50 μg/mL, respectively). Compound 6 exhibited moderate anti-microbial activities against two strains of Helicobacter pylori (Hp-SS1 and ATCC 43504) with MIC values of 50 and 50 μg/mL, respectively.
Graphical abstract
Electronic supplementary material
The online version of this article (doi:10.1007/s13659-016-0086-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4749522  PMID: 26815858
Excoecaria formosana; Halimane-type; Clerodane-type; Diterpenoid; Anti-microbial
14.  The cerebral cavernous malformation pathway controls embryonic endocardial gene expression through regulation of MEKK3 signaling and KLF expression 
Developmental cell  2015;32(2):168-180.
The cerebral cavernous malformation (CCM) pathway is required in endothelial cells for normal cardiovascular development and to prevent postnatal vascular malformations, but its molecular effectors are not well defined. Here we show that loss of CCM signaling in endocardial cells results in mid-gestation heart failure associated with premature degradation of cardiac jelly. CCM deficiency dramatically alters endocardial and endothelial gene expression, including increased expression of the Klf2 and Klf4 transcription factors and the Adamts4 and Adamts5 proteases that degrade cardiac jelly. These changes in gene expression result from increased activity of MEKK3, a mitogen-activated protein kinase that binds CCM2 in endothelial cells. MEKK3 is both necessary and sufficient for expression of these genes, and partial loss of MEKK3 rescues cardiac defects in CCM-deficient embryos. These findings reveal a molecular mechanism by which CCM signaling controls endothelial gene expression during cardiovascular development that may also underlie CCM formation.
Graphical Abstract
PMCID: PMC4589864  PMID: 25625206
15.  High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission 
Cell Host & Microbe  2016;19(1):114-126.
Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs.
Graphical Abstract
•Developed SaLSSA, a serum-free one-step assay for malaria transmission-blocking activity•13,983 known and new compounds analyzed by SaLSSA•>90% known antimalarial drugs do not show activity against late-stage gametocytes•Compounds with consistent low nanomolar transmission-blocking activity identified
Preventing human-mosquito transmission is important for malaria control. Plouffe et al. developed SaLSSA, a one-step high-throughput assay to screen for malaria transmission-blocking activity. A large panel of known and new small molecules was analyzed by SaLSSA. This provides starting points for the discovery and development of transmission-blocking drugs.
PMCID: PMC4723716  PMID: 26749441
transmission; malaria; chemotherapy; gametocytes; Plasmodium
16.  Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart 
Cardiovascular Research  2016;109(3):419-430.
Capillary and arterial endothelial cells share many common molecular markers in both the neonatal and adult hearts. Herein, we aim to establish a genetic tool that distinguishes these two types of vessels in order to determine the cellular mechanism underlying collateral artery formation.
Methods and results
Using Apln-GFP and Apln-LacZ reporter mice, we demonstrate that APLN expression is enriched in coronary vascular endothelial cells. However, APLN expression is reduced in coronary arterial endothelial cells. Genetic lineage tracing, using an Apln-CreER mouse line, robustly labelled capillary endothelial cells, but not arterial endothelial cells. We leveraged this differential activity of Apln-CreER to study collateral artery formation following myocardial infarction (MI). In a neonatal heart MI model, we found that Apln-CreER-labelled capillary endothelial cells do not contribute to the large collateral arteries. Instead, these large collateral arteries mainly arise from pre-existing, infrequently labelled coronary arteries, indicative of arteriogenesis. Furthermore, in an adult heart MI model, Apln-CreER activity also distinguishes large and small diameter arteries from capillaries. Lineage tracing in this setting demonstrated that most large and small coronary arteries in the infarcted myocardium and border region are derived not from capillaries, but from pre-existing arteries.
Apln-CreER-mediated lineage tracing distinguishes capillaries from large arteries, in both the neonatal and adult hearts. Through genetic fate mapping, we demonstrate that pre-existing arteries, but not capillaries, extensively contribute to collateral artery formation following myocardial injury. These results suggest that arteriogenesis is the major mechanism underlying collateral vessel formation.
PMCID: PMC4752045  PMID: 26768261
Apln-CreER; Lineage tracing; Collateral artery; Arteriogenesis; Arterialization
17.  Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence 
Journal of Virology  2015;89(13):6860-6873.
The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV.
IMPORTANCE Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.
PMCID: PMC4468500  PMID: 25903329
18.  Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes 
Cell Research  2015;26(1):119-130.
Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs.
PMCID: PMC4816131  PMID: 26634606
cardiac stem cell; c-kit; lineage tracing; cardiomyocyte
19.  Hand2 is an Essential Regulator for Two Notch-Dependent Functions within the Embryonic Endocardium 
Cell reports  2014;9(6):2071-2083.
The bHLH transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway, and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardiumto-endocardium based cell-signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.
PMCID: PMC4277501  PMID: 25497097
Hand2; Nrg1; EfnB2; Tricuspid atresia; endocardium; Notch signaling
20.  Plantamajoside, a potential anti-tumor herbal medicine inhibits breast cancer growth and pulmonary metastasis by decreasing the activity of matrix metalloproteinase-9 and -2 
BMC Cancer  2015;15:965.
Metastasis is the major cause of death in breast cancers. MMPs play a key role in tumor microenvironment that facilitates metastasis. The existing researches suggest that the high expression of gelatinase A and B (MMP2 and MMP9) promote the metastasis of breast cancer. Therefore, gelatinase inhibitor can effectively suppress tumor metastasis. However, at present, there is no dramatically effective gelatinase inhibitor against breast cancer.
We screened gelatinase inhibitor among Chinese herbal medicine by molecular docking technology; investigated the proliferation, migration and invasion of MDA-MB-231 human breast cancer cell line and 4T1 mouse breast cancer cell line in response to the treatment with the screened inhibitor by wound assay, invasion assay and gelatin zymography; then further examined the effects of inhibitor on allograft mammary tumors of mice by immunohistochemistry.
We successfully screened an Chinese herbal medicine-Plantamajoside(PMS)-which can reduce the gelatinase activity of MMP9 and MMP2. In vitro, PMS can inhibit the proliferation, migration and invasion of MDA-MB-231 human breast cancer cell line and 4T1 mouse breast cancer cell line by decreasing MMP9 and MMP2 activity. In vivo, oral administration of PMS to the mice bearing 4T1 cells induced tumors resulted in significant reduction in allograft tumor volume and weights, significant decrease in microvascular density and significant lower lung metastasis rate.
Our results indicate that as a promising anti-cancer agent, PMS may inhibit growth and metastasis of breast cancer by inhibiting the activity of MMP9 and MMP2.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1960-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4682252  PMID: 26674531
PMS; Herbal medicine; Breast cancer; Metastasis; MMP9 and MMP2; Angiogenesis
21.  Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle 
eLife  null;4:e10567.
Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation.
eLife digest
Many tissues and organs in an adult’s body – including bone marrow, skin and intestines – contain a small number of cells called adult stem cells. These cells usually stay dormant within these tissues (at a site called a ‘niche’) until they are required to repair damaged or lost cells. At this point, adult stem cells can specialize, or ‘differentiate’, into the many different cell types that make up the tissue or organ where they reside.
The cells that produce hairs are an example of adult stem cells. In mammals, hairs grow from structures called hair follicles that are found in the skin, and over the life of an animal, old hairs are shed and replaced. Previous research had suggested that certain embryonic cells are set to become hair follicle stem cells before the hair follicles emerge in the adult tissue. However it remained unclear how this decision is made, and which genes and molecules are involved in this process.
Xu et al. have now found that, in mice, the fate of hair follicle stem cells is decided at an early stage in development, when the hair follicle is a simpler structure called a ‘hair peg’. Cells near the upper part of the hair peg tend to become dormant and adopt an adult stem cell fate, while the ones in the lower part are more likely to differentiate straight away. This shows that the position, hence the niche environment, plays a key role in determining these different cells’ fates.
Xu et al. went on to discover that the decision for a cell to become a hair follicle stem cell relies on reduced signaling through the so-called Wnt signal pathway. Understanding how adult stem cells become established during development may help future efforts to grow tissues and organs in the laboratory for research purposes or organ transplantation.
PMCID: PMC4758985  PMID: 26653852
Wnt; hair follicle; regeneration; niche; stem cell; self-renewnal; Mouse
22.  Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL 
Nature medicine  2015;21(6):563-571.
Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in the backbone of ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapse B-ALL cases. All individuals who harbored PRPS1 mutations relapsed early on-treatment, and mutated ALL clones expanded exponentially prior to clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol can effectively abrogate PRPS1 mutant-driven drug resistance. Overall these results highlight the importance of constitutive activation of de novo purine pathway in thiopurine resistance, and offer therapeutic strategies for the treatment of relapsed and resistant ALL.
PMCID: PMC4670083  PMID: 25962120
23.  The role of speckle tracking echocardiography in assessment of lipopolysaccharide-induced myocardial dysfunction in mice 
Journal of Thoracic Disease  2015;7(12):2253-2261.
Sepsis-induced myocardial dysfunction is a common and severe complication of septic shock. Conventional echocardiography often fails to reveal myocardial depression in severe sepsis due to hemodynamic changes; in contrast, decline of strain measurements by speckle tracking echocardiography (STE) may indicate impaired cardiac function. This study investigates the role of STE in detecting lipopolysaccharide (LPS)-induced cardiac dysfunction with mouse models.
We evaluated cardiac function in 20 mice at baseline, 6 h (n=10) and 20 h (n=10) after LPS injection to monitor the development of heart failure induced by severe sepsis using 2-D and M-mode echocardiography. Ejection fraction (EF) and fractional shortening (FS) were measured with standard M-mode tracings, whereas circumferential and radial strain was derived from STE. Serum biochemical and cardiac histopathological examinations were performed to determine sepsis-induced myocardial injury.
Left ventricular (LV) myocardial function was significantly reduced at 6 h after LPS treatment assessed by circumferential strain (−14.65%±3.00% to −8.48%±1.72%, P=0.006), whereas there were no significant differences between 6 and 20 h group. Conversely, EF and FS were significantly increased at 20 h when comparing to 6 h (P<0.05) accompanied with marked decreases in EF and FS 6 h following LPS administration. Consistent with strain echocardiographic results, we showed that LPS injection leaded to elevated serum level of cardiac Troponin-T (cTnT), CK-MB and rising leucocytes infiltration into myocardium within 20 h.
Altogether, these results demonstrate that, circumferential strain by STE is a specific and reliable value for evaluating LPS-induced cardiac dysfunction in mice.
PMCID: PMC4703658  PMID: 26793347
Sepsis; septic cardiomyopathy; endotoxin-induced cardiac dysfunction; strain imaging; speckle tracking echocardiography (STE)
24.  Composition and Interactions of Hepatitis B Virus Quasispecies Defined the Virological Response During Telbivudine Therapy 
Scientific Reports  2015;5:17123.
Reverse transcriptase (RT) mutations contribute to hepatitis B virus resistance during antiviral therapy with nucleos(t)ide analogs. However, the composition of the RT quasispecies and their interactions during antiviral treatment have not yet been thoroughly defined. In this report, 10 patients from each of 3 different virological response groups, i.e., complete virological response, partial virological response and virological breakthrough, were selected from a multicenter trial of Telbivudine treatment. Variations in the drug resistance-related critical RT regions in 107 serial serum samples from the 30 patients were examined by ultra-deep sequencing. A total of 496,577 sequence reads were obtained, with an average sequencing coverage of 4,641X per sample. The phylogenies of the quasispecies revealed the independent origins of two critical quasispecies, i.e., the rtA181T and rtM204I mutants. Data analyses and theoretical modeling showed a cooperative-competitive interplay among the quasispecies. In particular, rtM204I mutants compete against other quasispecies, which eventually leads to virological breakthrough. However, in the absence of rtM204I mutants, synergistic growth of the drug-resistant rtA181T mutants with the wild-type quasispecies could drive the composition of the viral population into a state of partial virological response. Furthermore, we demonstrated that the frequency of drug-resistant mutations in the early phase of treatment is important for predicting the virological response to antiviral therapy.
PMCID: PMC4657086  PMID: 26599443
25.  Stimulation of autophagy promotes functional recovery in diabetic rats with spinal cord injury 
Scientific Reports  2015;5:17130.
In this study we examined the relationship between autophagy and apoptosis in diabetic rats after spinal cord injury (SCI), also we determined the role of autophagy in diabetes-aggravated neurological injury in vivo and in vitro. Our results showed that diabetes decreased the survival of neurons, promoted astrocytes proliferation, increased inflammatory cells infiltration and inhibited functional recovery after SCI. Diabetes was shown to confer increased activation of apoptotic pathways, along with an increase in autophagy; similar effects were also observed in vitro in neuronal PC12 cells. Treatment with rapamycin, an autophagy activator, partially abolished the adverse effect of diabetes, suggesting that diabetes may enhance neurological damage and suppress locomotor recovery after SCI, in addition to its effects on apoptosis and autophagy. In contrast, further stimulation of autophagy improved neurological function via inhibition of apoptosis. These results explained how diabetes exacerbates SCI in cellular level and suggested autophagy stimulation to be a new therapeutic strategy for diabetic SCI.
PMCID: PMC4657088  PMID: 26597839

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