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1.  NFAT restricts osteochondroma formation from entheseal progenitors 
JCI insight  2016;1(4):e86254-.
Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.
doi:10.1172/jci.insight.86254
PMCID: PMC4855520  PMID: 27158674
2.  NFAT restricts osteochondroma formation from entheseal progenitors 
JCI Insight  null;1(4):e86254.
Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.
NFAT transcription factors repress spontaneous osteochondroma formation from mesenchymal progenitor cells located within ligaments and tendons and, in particular, where these structures insert into bone.
doi:10.1172/jci.insight.86254
PMCID: PMC4855520  PMID: 27158674
3.  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.
doi:10.1002/dvg.20573
PMCID: PMC2806493  PMID: 20014345
cardiomyocyte; Cre recombinase; doxycycline; rtTA; Tnnt2
4.  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.
doi:10.1172/JCI38723
PMCID: PMC2689123  PMID: 19411759
5.  Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α 
PLoS ONE  2016;11(11):e0164207.
Objective
To examine the impact of 5-Aza-2ʹ-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.
Materials and Methods
FLS were isolated from seven RA-derived synovial tissues and cultured in vitro. The expression of miR-124a was measured by real time quantitative polymerase chain reaction (PCR) in FLS with or without 5-AzadC treatment. MiR-124a gene methylation was detected by methylation-specific PCR. FLS were divided into three groups as control, IL-1β and IL-1β/5-AzadC, respectively. The cells in the IL-1β group were treated with 5 μg/L of IL-1β for 24 hours, whereas the cells in the IL-1β/5-AzadC group were first treated with IL-1β exactly as those in the IL-1β group for 24 h but further treated with 1μM 5-AzadC for additional 3 days. The cell growth was estimated based on absorbance at UV450nm. Secreted TNF-α from the cells was evaluated by enzyme-linked immunosorbent assay. After that, RA-FLS treated with IL-1β plus 5-AzadC were further transfected with miR-124a inhibitor or scrambled control. After culturing for 3 days, cell growth and TNF-α concentrations were measured.
Results
After 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).
Conclusion
Methylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.
doi:10.1371/journal.pone.0164207
PMCID: PMC5100945  PMID: 27824863
6.  Pathologically decreased expression of miR-193a contributes to metastasis by targeting WT1-E-cadherin axis in non-small cell lung cancers 
Background
The metastatic cascade is a complex and multistep process with many potential barriers. Recently, miR-193a has been reported to be a suppressive miRNA in multiple types of cancers, but its underlying anti-oncogenic activity in non-small cell lung cancers (NSCLC) is not fully elucidated.
Methods
The expressions of miR-193a (miR-193a-5p) in human lung cancer tissues and cell lines were detected by real-time PCR. Dual-luciferase reporter assay was used to identify the direct target of miR-193a. Cell proliferation, apoptosis, and metastasis were assessed by CCK-8, flow cytometry, and Transwell assay, respectively.
Results
The expression of miR-193a in lung cancer tissues was decreased comparing to adjacent non-tumor tissues due to DNA hypermethylation in lung cancer tissues. Ectopic expression of miR-193a inhibited cell proliferation, colony formation, migration, and invasion in A549 and H1299 cells. Moreover, overexpression of miR-193a partially reversed tumor growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in NSCLC cells. Mechanistically, miR-193a reduced the expression of WT1, which negatively regulated the protein level of E-cadherin, suggesting that miR-193a might prevent EMT via modulating WT1-E-cadherin axis. Importantly, knockdown of WT1 resembled the anti-cancer activity by miR-193a and overexpression of WT1 partially reversed miR-193a-induced anti-cancer activity, indicating that WT1 plays an important role in miR-193a-induced anti-cancer activity. Finally, overexpression of miR-193a decreased the growth of tumor xenografts in mice.
Conclusion
Collectively, our results have revealed an important role of miR-193a-WT1-E-cadherin axis in metastasis, demonstrated an important molecular cue for EMT, and suggested a therapeutic strategy of restoring miR-193a expression in NSCLC.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-016-0450-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13046-016-0450-8
PMCID: PMC5100283  PMID: 27821145
MiR-193a; Wilm’s tumor-1; E-cadherin; Epithelial-to-mesenchymal transition
7.  Probing chromatin landscape reveals roles of endocardial TBX20 in septation 
The Journal of Clinical Investigation  null;126(8):3023-3035.
Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20–binding site–dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation.
doi:10.1172/JCI85350
PMCID: PMC4966318  PMID: 27348591
8.  Odor-induced mood state modulates language comprehension by affecting processing strategies 
Scientific Reports  2016;6:36229.
It is controversial whether mood affects cognition by triggering specific processing strategies or by limiting processing resources. The current event-related potential (ERP) study pursued this issue by examining how mood modulates the processing of task relevant/irrelevant information. In question-answer pairs, a question context marked a critical word in the answer sentence as focus (and thus relevant) or non-focus (thereby irrelevant). At the same time, participants were exposed to either a pleasant or unpleasant odor to elicit different mood states. Overall, we observed larger N400s when the critical words in the answer sentences were semantically incongruent (rather than congruent) with the question context. However, such N400 effect was only found for focused words accompanied by a pleasant odor and for both focused and non-focused words accompanied by an unpleasant odor, but not for non-focused words accompanied by a pleasant odor. These results indicate top-down attentional shift to the focused information in a positive mood state and non-selective attention allocated to the focused and non-focused information in a less positive mood state, lending support to the “processing strategy” hypothesis. By using a novel approach to induce mood states, our study provides fresh insights into the mechanisms underlying mood modulation of language comprehension.
doi:10.1038/srep36229
PMCID: PMC5087082  PMID: 27796356
9.  Comparative and parallel genome-wide association studies for metabolic and agronomic traits in cereals 
Nature Communications  2016;7:12767.
The plant metabolome is characterized by extensive diversity and is often regarded as a bridge between genome and phenome. Here we report metabolic and phenotypic genome-wide studies (mGWAS and pGWAS) in rice grain that, in addition to previous metabolic GWAS in rice leaf and maize kernel, show both distinct and overlapping aspects of genetic control of metabolism within and between species. We identify new candidate genes potentially influencing important metabolic and/or morphological traits. We show that the differential genetic architecture of rice metabolism between different tissues is in part determined by tissue specific expression. Using parallel mGWAS and pGWAS we identify new candidate genes potentially responsible for variation in traits such as grain colour and size, and provide evidence of metabotype-phenotype linkage. Our study demonstrates a powerful strategy for interactive functional genomics and metabolomics in plants, especially the cloning of minor QTLs for complex phenotypic traits.
The plant metabolome is often considered as a bridge between genomic and phenotypic variation. Here, the authors perform metabolic and phenotypic genome-wide association studies in rice grain, and compare to prior studies on rice leaf and maize kernel, to demonstrate a new strategy to investigate complex traits.
doi:10.1038/ncomms12767
PMCID: PMC5059443  PMID: 27698483
10.  Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation 
Scientific Reports  2016;6:34299.
Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.
doi:10.1038/srep34299
PMCID: PMC5035925  PMID: 27667023
11.  Effect of matrine combined with cisplatin on the expression of XIAP in human rhabdomyosarcoma RD cells 
Oncology Letters  2016;12(5):3793-3798.
The combined effects of matrine (Mat) and cisplatin on the survival and apoptosis of rhabdomyosarcoma (RMS) RD cells, as well as the possible mechanism of the synergistic effect of Mat and cisplatin were investigated in the present study. RMS RD cells were divided and treated as follows: control group, 5 mg/l cisplatin group, Mat groups (0.5, 1.0 and 1.5 g/l), and Mat (0.5, 1.0 and 1.5 g/l) combined with 5 mg/l cisplatin groups. An MTT assay and flow cytometry were applied to detect the survival and apoptotic rates, respectively, while RT-PCR was applied to detect the expression levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA in the RD cells of each group. The survival rates of RD cells in each experimental group were lower than in the control group, and the apoptotic rates were higher than those in the control group (P<0.05). An increase in drug concentrations led to the cell proliferation inhibitory and apoptotic rates of the single Mat groups increasing as a function of dose (pairwise comparison among the groups, P<0.05), while the proliferation inhibitory and apoptotic rates of Mat combined with the cisplatin groups under different concentration were significantly higher than those of the single Mat and single cisplatin groups under the same concentration (P<0.01). The expression levels of XIAP mRNA in the RD cells of each experimental group were lower than those in the control group (P<0.05). Additionally, the expression levels of XIAP mRNA in the group treated with Mat and cisplatin were significantly lower than those of the single cisplatin and single Mat groups (P<0.01). In conclusion, Mat and cisplatin are capable of inhibiting the proliferation of RD cells and inducing apoptosis by suppressing the XIAP mRNA expression levels.
doi:10.3892/ol.2016.5150
PMCID: PMC5104167  PMID: 27895732
human rhabdomyosarcoma RD cells; matrine; cisplatin; X-linked apoptosis inhibition gene; chemotherapy; children
12.  Similar frequency of the McGurk effect in large samples of native Mandarin Chinese and American English speakers 
Experimental brain research  2015;233(9):2581-2586.
Humans combine the visual information from mouth movements with auditory information from the voice to recognize speech. A common method for assessing multisensory speech perception is the McGurk effect: when presented with particular pairings of incongruent auditory and visual speech syllables (e.g., the auditory speech sounds for “ba” dubbed onto the visual mouth movements for “ga”) individuals perceive a third syllable, distinct from the auditory and visual components. Chinese and American cultures differ in the prevalence of direct facial gaze and in the auditory structure of their languages, raising the possibility of cultural and language-related group differences in the McGurk effect. There is no consensus in the literature about the existence of these group differences, with some studies reporting less McGurk effect in native Mandarin Chinese speakers than in English speakers and others reporting no difference. However, these studies sampled small numbers of participants tested with a small number of stimuli. Therefore, we collected data on the McGurk effect from large samples of Mandarin-speaking individuals from China and English-speaking individuals from the USA (total n = 307) viewing 9 different stimuli. Averaged across participants and stimuli, we found similar frequencies of the McGurk effect between Chinese and American participants (48% vs. 44%). In both groups, we observed a large range of frequencies both across participants (range from 0% to 100%) and stimuli (15% to 83%) with the main effect of culture and language accounting for only 0.3% of the variance in the data. The high variability in the McGurk effect necessitates the use of large sample sizes to accurately estimate group differences.
doi:10.1007/s00221-015-4324-7
PMCID: PMC4536079  PMID: 26041554
McGurk effect; cultural differences; audiovisual speech; multisensory integration
13.  Hepatitis C virus Genotype 1a core gene nucleotide patterns associated with hepatocellular carcinoma risk 
The Journal of General Virology  2015;96(Pt 9):2928-2937.
Specific sequence changes in codons 70 and 91 of the hepatitis C virus genotype 1b (HCV GT1b) core gene have been associated with increased risk of hepatocellular carcinoma (HCC). Essentially all previous studies were conducted in Asian populations with a wide range of liver disease, and none were conducted specifically in GT1a-infected individuals. We conducted a pilot study in a multiethnic population in the USA with HCV-related cirrhosis to determine if this association extended to GT1a-infected individuals and to determine if other sequence changes in the HCV core gene were associated with HCC risk. HCV core gene sequences from sera of 90 GT1 HCV carriers with cirrhosis (42 with HCC) were analysed using standard RT-PCR-based procedures. Nucleotide sequence data were compared with reference sequences available from GenBank. The frequency of sequence changes in codon 91 was not statistically different between HCC (7/19) and non-HCC (11/22) GT1b carriers. In GT1a carriers, sequence changes in codon 91 were observed less often than in GT1b carriers but were not observed in non-HCC subjects (4/23 vs 0/26, P = 0.03, Fisher's exact test). Sequence changes in codon 70 were not distributed differently between HCC and non-HCC GT1a and 1b carriers. Most importantly, for GT1a carriers, a panel of specific nucleotide changes in other codons was collectively present in all subjects with HCC, but not in any of the non-HCC patients. The utility of this test panel for early detection of HCC in GT1a-infected individuals needs to be assessed in larger populations, including longitudinal studies.
doi:10.1099/jgv.0.000219
PMCID: PMC4857454  PMID: 26296571
14.  Distinct tissue mineral density in plate and rod-like trabeculae of human trabecular bone 
Trabecular bone quality includes both microstructural and intrinsic tissue mineralization properties. However, the tissue mineralization in individual trabeculae of different trabecular types and orientations has not yet been investigated. The aim of this study was to develop an individual trabecula mineralization (ITM) analysis technique to determine tissue mineral density (TMD) distributions in plate- and rod-like trabeculae, respectively, and to compare the TMD of trabeculae along various orientations in μCT images of trabecular bone samples from the femoral neck, greater trochanter, and proximal tibia. ITM analyses indicated that trabecular plates, on average, had significantly higher TMD than trabecular rods. In addition, the distribution of TMD in trabecular plates depended on trabecular orientation with the lowest TMD in longitudinal plates and the highest TMD in transverse plates. Conversely, there was a relatively uniform distribution of TMD among trabecular rods, with respect to trabecular orientation. Further analyses of TMD distribution revealed that trabecular plates had higher mean and peak TMD, whereas trabecular rods had a wider TMD distribution and a larger portion of low mineralized trabeculae. Comparison of apparent Young's moduli derived from micro finite element models with and without heterogeneous TMD demonstrated that heterogeneous TMD in trabecular plates had a significant influence on the elastic mechanical property of trabecular bone. In conclusion, this study revealed differences in TMD between plate and rod-like trabeculae and among various trabecular orientations. The observation of less mineralized longitudinal trabecular plates suggests interesting implications of these load-bearing plates in bone remodeling. The newly developed ITM analysis can be a valuable technique to assess the influence of metabolic bone diseases and their pharmaceutical treatments on not only microstructure of trabecular bone, but also the microarchitectural heterogeneity of tissue mineralization.
doi:10.1002/jbmr.2498
PMCID: PMC4540699  PMID: 25736715
Tissue Mineral Density; Individual Trabecula Mineralization; Individual Trabecula Segmentation; Micro Computed Tomography
16.  Rubberband Effect in Temporal Control of Mismatch Negativity 
Frontiers in Psychology  2016;7:1299.
Mismatch negativity (MMN) is a difference event-related potential (ERP) wave reflecting the brain’s automatic reaction to deviant sensory stimuli, and it has been proven to be a useful tool in research on cognitive functions or clinical disorders. In most MMN studies, amplitude, peak latency, or the integral of the responses, in rare cases also the slopes of the responses, have been employed as parameters of the ERP responses for quantitative analyses. However, little is known about correlations between these parameters. To better understand the relations between different ERP parameters, we extracted and correlated several different parameters characterizing the MMN waves. We found an unexpected correlation which gives new insight into the temporal control of MMN: response amplitudes are positively correlated with downside slopes, whereas barely correlated with upside slopes. This result suggests an efficient feedback mechanism for the MMN to return to the baseline within a predefined time window, contradicting an exponential decay function as one might expect. As a metaphor we suggest a rubberband effect for the MMN responses, i.e., the larger the distance of the response from neural equilibrium, the stronger the return force to equilibrium.
doi:10.3389/fpsyg.2016.01299
PMCID: PMC5015478  PMID: 27642285
event related potential; mismatch negativity; oddball paradigm; time window; correlation
17.  Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy 
Background
Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens.
Methods
IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice.
Results
Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg).
Conclusions
IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabris venom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.
doi:10.1186/s40409-016-0078-3
PMCID: PMC4997716  PMID: 27563307
IgY; Egg yolk; Snake venom; Trimeresurus albolabris; LD50
18.  Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells 
Nature Communications  2016;7:12422.
Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial–mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions.
Pericytes and vascular smooth muscle cells are crucial for functional blood vessels, but the developmental sources of these cells are incompletely understood. Here, the authors show that endocardial endothelial cells give rise to cardiac mural cells, which are controlled by Wnt signalling.
doi:10.1038/ncomms12422
PMCID: PMC4990645  PMID: 27516371
19.  A Novel Single-Excitation Capacitive Angular Position Sensor Design 
Sensors (Basel, Switzerland)  2016;16(8):1196.
This paper presents a high-precision capacitive angular position sensor (CAPS). The CAPS is designed to be excited by a single voltage to eliminate the matching errors of multi-excitations, and it is mainly composed of excitation electrodes, coupling electrodes, petal-form sensitive electrodes and a set of collection electrodes. A sinusoidal voltage is applied on the excitation electrodes, then the voltage couples to the coupling electrodes and sensitive electrodes without contact. The sensitive electrodes together with the set of collection electrodes encode the angular position to amplitude-modulated signals, and in order to increase the scale factor, the sensitive electrodes are patterned in the shape of petal-form sinusoidal circles. By utilizing a resolver demodulation method, the amplitude-modulated signals are digitally decoded to get the angular position. A prototype of the CAPS is fabricated and tested. The measurement results show that the accuracy of the sensor is 0.0036°, the resolution is 0.0009° and the nonlinearity over the full range is 0.008° (after compensation), indicating that the CAPS has great potential to be applied in high-precision applications with a low cost.
doi:10.3390/s16081196
PMCID: PMC5017362  PMID: 27483278
angular position sensor; capacitance sensitive; single-excitation; sinusoidal circular electrodes; amplitude modulation
20.  Developmental origin of age-related coronary artery disease 
Cardiovascular Research  2015;107(2):287-294.
Aim
Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well.
Methods and results
Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1+, Gata5-Cre+ cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1+, Gata5-Cre− population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre+ population resulted in coronary arteries consisting solely of Gata5-Cre− caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5–8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality.
Conclusion
Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.
doi:10.1093/cvr/cvv167
PMCID: PMC4565989  PMID: 26054850
Epicardium; Smooth muscle; Coronary artery disease; Pathology
21.  Quantifying influenza virus diversity and transmission in humans 
Nature genetics  2016;48(2):195-200.
Influenza A virus is characterized by high genetic diversity.1–3 However, most of what we know about influenza evolution has come from consensus sequences sampled at the epidemiological scale4 that only represent the dominant virus lineage within each infected host. Less is known about the extent of intra-host virus diversity and what proportion is transmitted between individuals.5 To characterize those virus variants that achieve sustainable transmission in new hosts, we examined intra-host virus genetic diversity within household donor/recipient pairs from the first wave of the 2009 H1N1 pandemic when seasonal H3N2 was co-circulating. While the same variants were found in multiple members of the community, the relative frequencies of variants fluctuated, with patterns of genetic variation more similar within than between households. We estimated the effective population size of influenza A virus across donor/recipient pairs to be approximately 100–200 contributing members, which enabled the transmission of multiple lineages including antigenic variants.
doi:10.1038/ng.3479
PMCID: PMC4731279  PMID: 26727660
Influenza A virus; evolution; diversity; virus transmission; next generation sequencing
22.  Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability 
Nature Communications  2016;7:12040.
The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.
A major goal of HIV-1 vaccine development is to produce antigens that can induce broadly neutralizing antibodies. Here the authors examine the underlying causes of HIV-1 envelope metastability and design uncleaved, prefusion-optimized gp140 trimers with potential for use as HIV-1 vaccine antigens.
doi:10.1038/ncomms12040
PMCID: PMC4931249  PMID: 27349805
23.  Presenting native-like trimeric HIV-1 antigens with self-assembling nanoparticles 
Nature Communications  2016;7:12041.
Structures of BG505 SOSIP.664 trimer in complex with broadly neutralizing antibodies (bNAbs) have revealed the critical role of trimeric context for immune recognition of HIV-1. Presentation of trimeric HIV-1 antigens on nanoparticles may thus provide promising vaccine candidates. Here we report the rational design, structural analysis and antigenic evaluation of HIV-1 trimer-presenting nanoparticles. We first demonstrate that both V1V2 and gp120 can be presented in native-like trimeric conformations on nanoparticles. We then design nanoparticles presenting various forms of stabilized gp140 trimer based on ferritin and a large, 60-meric E2p that displays 20 spikes mimicking virus-like particles (VLPs). Particle assembly is confirmed by electron microscopy (EM), while antigenic profiles are generated using representative bNAbs and non-NAbs. Lastly, we demonstrate high-yield gp140 nanoparticle production and robust stimulation of B cells carrying cognate VRC01 receptors by gp120 and gp140 nanoparticles. Together, our study provides an arsenal of multivalent immunogens for HIV-1 vaccine development.
The development of native-like envelope trimers has been a major focus in the efforts to produce HIV vaccines. Here the authors demonstrate the production and characterization of virus-like nanoparticles displaying trimeric HIV-1 antigens with the potential to elicit broadly neutralizing antibodies.
doi:10.1038/ncomms12041
PMCID: PMC4931238  PMID: 27349934
24.  Potential Role of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Regulation of Matrix Metalloproteinases (MMPs) Expression in Periodontal Diseases 
Periodontal diseases are characterized by pathological destruction of extracellular matrix (ECM) of periodontal tissues. Matrix metalloproteinases (MMPs) are a significant part of the degradation of ECM. However, the regulation of MMPs expression level in periodontal diseases is as yet undetermined.
RECK (reversion-inducing cysteine-rich protein with Kazal motifs), a novel membrane-anchored inhibitor of MMPs, could regulate the expressions of MMP-2, 9 and MT1-MMP as a cell surface-signaling molecule. Thus, we propose that RECK may play an important role in regulating MMPs in the ECM degradation of periodontal diseases. The RECK/MMPs signaling pathway could provide a new approach for prevention and treatment of RECK in periodontal diseases by blocking MMPs.
doi:10.12659/MSM.896546
PMCID: PMC4913808  PMID: 27272560
Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Periodontal Diseases
25.  Differential Susceptibilities of Human Lung Primary Cells to H1N1 Influenza Viruses 
Journal of Virology  2015;89(23):11935-11944.
ABSTRACT
Human alveolar epithelial cells (AECs) and alveolar macrophages (AMs) are the first lines of lung defense. Here, we report that AECs are the direct targets for H1N1 viruses that have circulated since the 2009 pandemic (H1N1pdm09). AMs are less susceptible to H1N1pdm09 virus, but they produce significantly more inflammatory cytokines than AECs from the same donor. AECs form an intact epithelial barrier that is destroyed by H1N1pdm09 infection. However, there is significant variation in the cellular permissiveness to H1N1pdm09 infection among different donors. AECs from obese donors appear to be more susceptible to H1N1pdm09 infection, whereas gender, smoking history, and age do not appear to affect AEC susceptibility. There is also a difference in response to different strains of H1N1pdm09 viruses. Compared to A/California04/09 (CA04), A/New York/1682/09 (NY1682) is more infectious and causes more epithelial barrier injury, although it stimulates less cytokine production. We further determined that a single amino acid residue substitution in NY1682 hemagglutinin is responsible for the difference in infectivity. In conclusion, this is the first study of host susceptibility of human lung primary cells and the integrity of the alveolar epithelial barrier to influenza. Further elucidation of the mechanism of increased susceptibility of AECs from obese subjects may facilitate the development of novel protection strategies against influenza virus infection.
IMPORTANCE Disease susceptibility of influenza is determined by host and viral factors. Human alveolar epithelial cells (AECs) form the key line of lung defenses against pathogens. Using primary AECs from different donors, we provided cellular level evidence that obesity might be a risk factor for increased susceptibility to influenza. We also compared the infections of two closely related 2009 pandemic H1N1 strains in AECs from the same donor and identified a key viral factor that affected host susceptibility, the dominance of which may be correlated with disease epidemiology. In addition, primary human AECs can serve as a convenient and powerful model to investigate the mechanism of influenza-induced lung injury and determine the effect of genetic and epigenetic factors on host susceptibility to pandemic influenza virus infection.
doi:10.1128/JVI.01792-15
PMCID: PMC4645340  PMID: 26378172

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