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1.  Antigenic evolution of H9N2 chicken influenza viruses isolated in China during 2009–2013 and selection of a candidate vaccine strain with broad cross-reactivity 
Veterinary microbiology  2015;182:1-7.
We previously demonstrated that H9N2 subtype avian influenza viruses (AIVs) isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E) and then underwent antigenic drift from commercial vaccines, causing a country-wide outbreak during 2010–2013. In this study, H9N2 AIVs isolated from chickens during 2009–2013 were antigenically analyzed by performing hemagglutination inhibition and neutralization assays using a panel of polyclonal antibodies. Our findings confirmed the antigenic drift of recent H9N2 viruses from the commercial vaccine and showed that most of these antigenic variants form a novel HI antigenic group, F, with a few belonging to groups D and E. Slight antigenic variation was observed in group F viruses. Genetic analysis of amino acid sequences deduced from hemagglutinin (HA) gene sequences indicated that 9 of 15 mutations predominant in the 2009–2013 viruses can be mapped to known antigenic sites, which might be responsible for the novel antigenicity of group F. These antigenic changes make it necessary to modify the influenza vaccine to ensure efficient protection. A vaccine candidate, Ck/HeB/YT/10, was selected and provided significant protection against viruses from different antigenic groups in terms of reduction in virus shedding, suggesting broad cross-reactivity. Taken together, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak and now seems to be undergoing new antigenic divergence. Systematic surveillance and timely updating of vaccine strains are important for viral prevention and control in the future.
doi:10.1016/j.vetmic.2015.10.031
PMCID: PMC5029119  PMID: 26711021
H9N2; chicken influenza virus; antigenic evolution; vaccine
2.  Small molecules efficiently reprogram human astroglial cells into functional neurons 
Cell stem cell  2015;17(6):735-747.
Summary
We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by single neural transcription factor NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of 9 small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemical reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.
Graphical Abstract
doi:10.1016/j.stem.2015.09.012
PMCID: PMC4675726  PMID: 26481520
3.  Physiological Adjustments and Circulating MicroRNA Reprogramming Are Involved in Early Acclimatization to High Altitude in Chinese Han Males 
Background: Altitude acclimatization is a physiological process that restores oxygen delivery to the tissues and promotes oxygen use under high altitude hypoxia. High altitude sickness occurs in individuals without acclimatization. Unraveling the molecular underpinnings of altitude acclimatization could help understand the beneficial body responses to high altitude hypoxia as well as the altered biological events in un-acclimatized individuals. This study assessed physiological adjustments and circulating microRNA (cmiRNA) profiles in individuals exposed to high altitude, aiming to explore altitude acclimatization in humans.
Methods: Ninety volunteers were enrolled in this study. Among them, 22 individuals provided samples for microRNA arrays; 68 additional individuals constituted the validation set. Un-acclimatized individuals were identified by the Lake Louise Scoring System. Thirty-three phenotypes were recorded pre- and post-exposure to high altitude, including stress hormones, lipid profiles, hematological indices, myocardial enzyme spectrum, and liver and kidney function related enzymes. CmiRNA expression profiles were assessed using miRCURYTM LNA Array (v.18.0) screening, with data validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then, associations of plasma microRNA expression with physiological adjustments were evaluated. The biological relevance of the main differentially expressed cmiRNAs was explored by bioinformatics prediction.
Results: Nineteen of the 33 phenotypes were significantly altered during early altitude acclimatization, including hematological indices, lipid profiles, and stress hormones; meanwhile, 86 cmiRNAs (79 up-regulated and 7 down-regulated) showed differential expression with statistical significance. Among them, 32 and 25 microRNAs were strongly correlated with low-density lipoprotein-cholesterol and total cholesterol elevations, respectively. In addition, 22 microRNAs were closely correlated with cortisol increase. In un-acclimatized individuals, 55 cmiRNAs were up-regulated and 36 down-regulated, compared with acclimatized individuals. The HIF signaling pathway was suppressed in un-acclimatized individuals.
Conclusion: Physiological adjustments, including the hematological system, stress hormones, and lipid molecules contributed to early altitude acclimatization, and showed strong correlations with cmiRNA reprogramming. Moreover, acclimatized and un-acclimatized individuals showed different cmiRNA profile. Suppression of the HIF-1 signaling pathway by microRNA regulation may play a key role in the pathogenesis of un-acclimatization with high altitude hypoxia.
doi:10.3389/fphys.2016.00601
PMCID: PMC5133430  PMID: 27994555
acclimatization; high altitude; hypoxia; circulating microRNAs; Chinese Han males
4.  Germline Genetic Variation in ETV6 and Risk of Childhood Acute Lymphoblastic Leukemia: a Systematic Genetic Study 
The Lancet. Oncology  2015;16(16):1659-1666.
Background
Hereditary predisposition is rarely suspected for childhood acute lymphoblastic leukemia (ALL). Recent studies identified germline ETV6 variations associated with marked familial clustering of hematologic malignancies, pointing to this gene as a potentially important genetic determinant for ALL susceptibility. The aims of the current study are to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which they contribute to the overall risk of childhood ALL.
Methods
Whole-exome sequencing of an index family with multiple cases of ALL was performed to identify causal variants for ALL predisposition. Targeted sequencing of ETV6 was done in 4,405 children from the Children's Oncology Group (COG) and St. Jude Children's Research Hospital frontline ALL trials. Patients were included in this study on the basis of their enrollment in these clinical trials and the availability of germline DNA. ETV6 variant genotypes were compared with non-ALL controls to define ALL-related germline risk variants. ETV6 variant function was characterized bioinformatically and correlated with clinical and demographic features in 2,021 children with ALL.
Findings
We identified a novel nonsense ETV6 variant (p.R359X) with a high penetrance of familial ALL. Subsequent targeted sequencing of ETV6 in 4,405 childhood ALL cases discovered 31 exonic variants (4 nonsense, 21 missense, 1 splice site, and 5 frame shift variants) that are potentially related to ALL risk in 35 cases (0.79%). Fifteen (48%) of the 31 ALL-related ETV6 variants clustered in the ETS domain and predicted to be highly deleterious. Children with ALL-related ETV6 variants were significantly older at leukemia diagnosis than others (10.2 years [IQR 5.3-13.8] vs 4.7 years [IQR 3.0-8.7], P=0.017). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (9 of 14 cases [64.3%] vs 538 of 2,007 cases [26.8%]; P=0.0050).
Interpretation
Our findings indicated germline ETV6 variations as the basis of a novel genetic syndrome associated with predisposition to childhood ALL.
Funding
This study was supported by the National Institutes of Health and by the American Lebanese Syrian Associated Charities.
doi:10.1016/S1470-2045(15)00369-1
PMCID: PMC4684709  PMID: 26522332
5.  Screening the Expression Changes in MicroRNAs and Their Target Genes in Mature Cementoblasts Stimulated with Cyclic Tensile Stress 
Cementum is a thin layer of cementoblast-produced mineralized tissue covering the root surfaces of teeth. Mechanical forces, which are produced during masticatory activity, play a paramount role in stimulating cementoblastogenesis, which thereby facilitates the maintenance, remodeling and integrity of cementum. However, hitherto, the extent to which a post-transcriptional modulation mechanism is involved in this process has rarely been reported. In this study, a mature murine cementoblast cell line OCCM-30 cells (immortalized osteocalcin positive cementoblasts) was cultured and subjected to cyclic tensile stress (0.5 Hz, 2000 µstrain). We showed that the cyclic tensile stress could not only rearrange the cell alignment, but also influence the proliferation in an S-shaped manner. Furthermore, cyclic tensile stress could significantly promote cementoblastogenesis-related genes, proteins and mineralized nodules. From the miRNA array analyses, we found that 60 and 103 miRNAs were significantly altered 6 and 18 h after the stimulation using cyclic tensile stress, respectively. Based on a literature review and bioinformatics analyses, we found that miR-146b-5p and its target gene Smad4 play an important role in this procedure. The upregulation of miR-146b-5p and downregulation of Smad4 induced by the tensile stress were further confirmed by qRT-PCR. The direct binding of miR-146b-5p to the three prime untranslated region (3′ UTR) of Smad4 was established using a dual-luciferase reporter assay. Taken together, these results suggest an important involvement of miR-146b-5p and its target gene Smad4 in the cementoblastogenesis of mature cementoblasts.
doi:10.3390/ijms17122024
PMCID: PMC5187824  PMID: 27941605
cementoblast; tensile stress; miRNA; microarray; miR-146b-5p; Smad4
6.  miR-199a-3p inhibits cell proliferation and induces apoptosis by targeting YAP1, suppressing Jagged1-Notch signaling in human hepatocellular carcinoma 
Background
miR-199a-3p was significantly downregulated in the majority of human hepatocellular carcinoma (HCC) tissues and HCC cell lines. Yes associated protein 1 (YAP1) was overexpressed in human HCC, which promoted HCC development and progression by upregulating Jagged1 and activating the Notch pathway. We searched potential targets of miR-199a-3p with DIANA, TargetScan and PicTar tools, and found that YAP1 is one of the potential targets. Based on these findings, we speculated that miR-199a-3p might suppress HCC growth by targeting YAP1, downregulating Jagged1 and suppressing the Notch pathway.
Results
We determined the expression of miR-199a-3p and YAP1 by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found downregulation of miR-199a-3p and upregulation of YAP1 in HCC cell lines. Cell proliferation and apoptosis assays showed that miR-199a-3p suppresses HCC cell proliferation and promotes apoptosis, and knockdown of YAP1 has similar role. Furthermore, we verified that miR-199a-3p can directly target YAP1. We further investigated and confirmed that miR-199a-3p and YAP1 regulate HCC cell proliferation and apoptosis through Jagged1-Notch signaling.
Conclusion
miR-199a-3p targets YAP1, downregulates Jagged1 and suppresses the Notch signaling to inhibit HCC cell proliferation and promote apoptosis. These findings provide new insights into the mechanism by which miR-199a-3p suppresses HCC cell proliferation and induces apoptosis.
doi:10.1186/s12929-016-0295-7
PMCID: PMC5103406  PMID: 27832779
Hepatocellular carcinoma; miR-199a-3p; Yes associated protein 1; Jagged1; Notch signaling
7.  High‐Performance Direct Methanol Fuel Cells with Precious‐Metal‐Free Cathode 
Advanced Science  2016;3(11):1600140.
Direct methanol fuel cells (DMFCs) hold great promise for applications ranging from portable power for electronics to transportation. However, apart from the high costs, current Pt‐based cathodes in DMFCs suffer significantly from performance loss due to severe methanol crossover from anode to cathode. The migrated methanol in cathodes tends to contaminate Pt active sites through yielding a mixed potential region resulting from oxygen reduction reaction and methanol oxidation reaction. Therefore, highly methanol‐tolerant cathodes must be developed before DMFC technologies become viable. The newly developed reduced graphene oxide (rGO)‐based Fe‐N‐C cathode exhibits high methanol tolerance and exceeds the performance of current Pt cathodes, as evidenced by both rotating disk electrode and DMFC tests. While the morphology of 2D rGO is largely preserved, the resulting Fe‐N‐rGO catalyst provides a more unique porous structure. DMFC tests with various methanol concentrations are systematically studied using the best performing Fe‐N‐rGO catalyst. At feed concentrations greater than 2.0 m, the obtained DMFC performance from the Fe‐N‐rGO cathode is found to start exceeding that of a Pt/C cathode. This work will open a new avenue to use nonprecious metal cathode for advanced DMFC technologies with increased performance and at significantly reduced cost.
doi:10.1002/advs.201600140
PMCID: PMC5102660  PMID: 27980990
direct methanol fuel cells; electrocatalysis; graphene; nonprecious metal catalysts; oxygen reduction
8.  Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway 
Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway.
doi:10.3390/ijms17111847
PMCID: PMC5133847  PMID: 27827993
integrin; FAK; ERK; PI3K; mandibular condyle; chondrocyte; apoptosis
9.  The diagnostic value of non-contrast enhanced quiescent interval single shot (QISS) magnetic resonance angiography at 3T for lower extremity peripheral arterial disease, in comparison to CT angiography 
Background
The high incidence of renal insufficiency in patients with Peripheral Arterial Disease raises the concern for nephrogenic systemic fibrosis (NSF) with respect to contrast enhanced MRA. The risk of NSF is eliminated with non-contrast enhanced magnetic resonance angiography. The purpose of the current study is to compare image quality and diagnostic performance of non-contrast enhanced Quiescent Interval Single Shot (QISS) magnetic resonance angiography at 3 T versus CT angiography for evaluation of lower extremity Peripheral Arterial Disease (PAD).
Methods
32 consecutive patients (23 male, 9 female, age range 40–81 years, average age 61.97 years) with clinically suspected lower extremity PAD underwent QISS MRA and CTA. 19 of 32 patients underwent Digital Subtraction Angiography (DSA). Image quality of MRA was compared with CTA by two radiologists with 10 and 8 years’ experience according to a 4-point scale. The Kappa test was used to determine the intermodality agreement between MRA and CTA in stenosis assessment, and interobserver agreement with each method. Sensitivity and specificity of CTA and MRA in detecting hemodynamically significant stenosis (≥50 %) were compared, with DSA serving as reference standard when available.
Results
Image quality of QISS MRA was rated 3.70 ± 0.49 by reader 1, and 3.72 ± 0.47 by reader 2, significantly lower than that of CTA (3.80 ± 0.44 and 3.82 ± 0.42, P < 0.001 for both readers). Intermodality agreement between MRA and CTA was excellent for assessment of stenosis (Kappa = 0.923 ± 0.013 for reader 1, 0.930 ± 0.012 for reader 2). Interobserver agreement was 0.936 ± 0.012 for CTA and 0.935 ± 0.011 for MRA. For readers 1 and 2 respectively, the sensitivity of QISS was 94.25 and 93.26 % (versus 90.11 and 89.13 % for CTA, P > 0.05), and specificity of QISS was 96.70 and 97.75 % (versus 96.55 and 96.51 % for CTA, P > 0.05). For heavily calcified segments, sensitivity of QISS (95.83 and 95.83 %) was significantly higher than that of CTA (74.19 and 76.67 %, P < 0.05).
Conclusion
QISS is a reliable alternative to CTA for evaluation of lower extremity PAD, and may be suitable as a first-line screening examination in patients with contraindications to intravenous contrast administration.
doi:10.1186/s12968-016-0294-6
PMCID: PMC5072342  PMID: 27760564
Non-contrast enhanced; Magnetic resonance angiography; Lower extremity; Peripheral arterial disease
10.  KLHL21, a novel gene that contributes to the progression of hepatocellular carcinoma 
BMC Cancer  2016;16:815.
Background
Hepatocellular carcinoma (HCC) has very high prevalence and associated-mortality. However, targeted therapies that are currently used in clinical practice for HCC have certain limitations, in part because of the lack of reliable and clinically applicable biomarkers that can be used for diagnosis and prognosis assessments and for the surveillance of treatment effectiveness.
Methods
Meta-analysis was used to analyze the integrated microarray data for global identification of a set of robust biomarkers for HCC. Quantitative RT-PCR (qRT-PCR) was performed to validate the expression levels of selected genes. Gene expression was inhibited by siRNA. CellTiter 96® AQueous One Solution Cell Proliferation assays were used to determine cell proliferation, and Transwell assays were used to determine cell migration and invasion potential.
Results
Meta-analysis of the expression data provided a gene expression signature from a total of 1525 patients with HCC, showing 1529 up-regulated genes and 478 down-regulated genes in cancer samples. The expression levels of genes having strong clinical significance were validated by qRT-PCR using primary HCC tissues and the paired adjacent noncancerous liver tissues. Up-regulation of VPS45, WIPI1, TTC1, IGBP1 and KLHL21 genes and down-regulation of FCGRT gene were confirmed in clinical HCC samples. KLHL21 was the most promising gene for potential use as a bioclinical marker in this analysis. Abrogating expression of it significantly inhibited cell proliferation, migration and invasion.
Conclusions
Our study suggests that KLHL21 is a potential target for therapeutic intervention. Our findings also provide novel candidate genes on a genome-wide scale, which may have significant impact on the design and execution of effective therapy of HCC patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2851-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-016-2851-7
PMCID: PMC5073891  PMID: 27769251
KLHL21; Bioinformatics; HCC; Biomarker
11.  MicroRNA-574-5p promotes metastasis of non-small cell lung cancer by targeting PTPRU 
Scientific Reports  2016;6:35714.
Dysregulation of microRNAs (miRNAs) has been associated with malignant behavior in a variety of cancers. Our previous study demonstrated that miRNA expression profiles are predictors for patients with advanced non-small cell lung cancer (NSCLC). We also showed that miRNAs are involved in small-cell lung cancer metastasis. Here, we used qRT-PCR to re-analyze our previous microarray results using serum samples from 75 patients with NSCLC. Surprisingly, we found that miR-574-5p and miR-874 were overexpressed in patients with metastatic advanced NSCLC but not in patients with non-metastatic advanced NSCLC. Additionally, miR-574-5p expression was correlated between matched serum and tissue samples from 68 patients. However, these 2 miRNAs are not prognostic factors for NSCLC. Transwell and wound-healing assays showed that miR-574-5p promotes the migration and invasion of NSCLC cells. Furthermore, miR-574-5p enhanced the tyrosine phosphorylation of β-catenin by repressing PTPRU expression in vitro. In conclusion, this study explored the expression of miR-574-5p in clinical samples and its molecular mechanisms in the metastasis of advanced NSCLC.
doi:10.1038/srep35714
PMCID: PMC5071770  PMID: 27761023
12.  Significant association of SNP rs2106261 in the ZFHX3 gene with atrial fibrillation in a Chinese Han GeneID population 
Human genetics  2010;129(3):239-246.
Atrial fibrillation (AF) is the most common cardiac rhythm disorder at the clinical setting and accounts for up to 15% of all strokes. Recent genome-wide association studies (GWAS) identified two single nucleotide polymorphisms (SNPs), rs2106261 and rs7193343 in ZFHX3 (zinc finger homeobox 3 gene) and rs13376333 in KCNN3 (encoding a potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3) that showed significant association with AF in multiple populations of European ancestry. Here, we studied a Chinese Han, GeneID cohort consisting of 650 AF patients and 1,447 non-AF controls to test whether the GWAS findings on ZFHX3/KCNN3 and AF can be expanded to a different ethnic population. No significant association was detected for rs7193343 in ZFHX3 and rs13376333 in KCNN3. However, significant association was identified between rs2106261 in ZFHX3 and AF in the GeneID population for both allelic frequencies (P = 0.001 after adjusting for covariates of age, gender, hypertension, coronary artery disease, and diabetes mellitus; OR = 1.32), and genotypic frequencies assuming either an additive or recessive model (OR = 1.29, P = 0.001 and OR = 1.77, P = 0.00018, respectively). When only lone AF cases were analyzed, the association remained significant (OR = 1.50, P = 0.001 for allelic association; OR = 1.45, P = 0.001 for an additive model; OR = 2.24, P = 0.000043 for a recessive model). Our results indicate that rs2106261 in ZFHX3 confers a significant risk of AF in a Chinese Han population. The study expands the association between ZFHX3 and AF to a non-European ancestry population and provides the first evidence of a cross-race susceptibility of the 16q22 AF locus.
doi:10.1007/s00439-010-0912-6
PMCID: PMC5069458  PMID: 21107608
13.  Ovarian metastasis from non-small cell lung cancer with ALK and EGFR mutations: A report of two cases 
Oncology Letters  2016;12(6):4361-4366.
Ovarian metastasis from non-small cell lung cancer (NSCLC) is a rare condition. The current study presents the cases of 2 female patients aged 38 and 47 years old, respectively, who were initially diagnosed with NSCLC adenocarcinoma on histology. Both patients initially presented with chest pain and a cough, and subsequently developed ovarian metastases following multiple treatments. The 38-year old patient exhibited an epidermal growth factor receptor mutation, confirmed by scorpion/amplified refractory mutation system analysis from a lung biopsy. The 47-year old exhibited an anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement, revealed by fluorescence in situ hybridization analysis of the breast tissue biopsy, confirming a diagnosis of ALK rearrangement-positive NSCLC. These patients developed ovarian metastasis in the course of the disease. The current study reports the diagnostic challenges and clinical management of the disease, and provides a review of the literature.
doi:10.3892/ol.2016.5292
PMCID: PMC5228381
non-small cell lung cancer; ovarian metastasis; epidermal growth factor receptor mutation; anaplastic lymphoma receptor tyrosine kinase rearrangement-positive
14.  Antiperovskite Li3OCl Superionic Conductor Films for Solid‐State Li‐Ion Batteries 
Advanced Science  2016;3(3):1500359.
Antiperovskite Li3OCl superionic conductor films are prepared via pulsed laser deposition using a composite target. A significantly enhanced ionic conductivity of 2.0 × 10−4 S cm−1 at room temperature is achieved, and this value is more than two orders of magnitude higher than that of its bulk counterpart. The applicability of Li3OCl as a solid electrolyte for Li‐ion batteries is demonstrated.
doi:10.1002/advs.201500359
PMCID: PMC5067573  PMID: 27812460
antiperovskite phase; solid‐state batteries; superionic conductor; sustainable chemistry; thin films
15.  Mathematical evaluation of the influence of multiple factors on implant stability quotient values in clinical practice: a retrospective study 
Objectives
The objective of this study is to mathematically evaluate the influence of multiple factors on implant stability quotient values in clinical practice.
Patients and methods
Resonance frequency analysis was performed at T1 (measured immediately at the time of implant placement) and at T2 (measured before dental restoration) in 177 patients (329 implants). Using a multivariate linear regression model, we analyzed the influence of the following eleven candidate factors: sex, age, maxillary/mandibular location, bone type, immediate/delayed implantation, bone grafting (presence or absence), insertion torque, I-/II-stage healing pattern, implant diameter, implant length, and T1–T2 time interval.
Results
The following factors were identified to significantly influence the implant stability quotient (ISQ) values at T1: insertion torque, bone grafting, I-/II-stage healing pattern, immediate/delayed implantation, maxillary/mandibular location, implant diameter, and sex. In contrast, the ISQ values at T2 were significantly influenced only by three factors: implant diameter, T1–T2 time interval, and insertion torque.
Conclusion
Among the eleven candidate factors, seven key factors were found to influence the T1-ISQ values, while only three key factors influenced the T2-ISQ values. Both T1 and T2-ISQ values were found to be influenced by implant diameter and insertion torque. T1 was influenced specifically by the sex of the patient, the location (maxillary or mandibular), the implantation mode (immediate/delayed implantation), the healing stage, and the absence or presence of bone graft materials.
doi:10.2147/TCRM.S113764
PMCID: PMC5066996  PMID: 27785040
resonance frequency analysis; implant stability quotient; dental implant; immediate implantation; delayed implantation; insertion torque
16.  Genome-Wide Identification and Characterization of MicroRNAs and Target Genes in Lonicera japonica 
PLoS ONE  2016;11(10):e0164140.
MiRNAs function in post-transcriptional regulation of gene expression and play very important roles in plant development. Lonicera japonica is one of the important medicinal plants in China. However, few studies on the discovery of conserved and novel miRNAs from L. japonica were reported. In this study, we employed deep sequencing technology to identify miRNAs in leaf and flower tissues of L. japonica. A total of 22.97 million clean reads from flower and leaf tissues were obtained, which generated 146 conserved miRNAs distributed in 20 families and 110 novel miRNAs. Accordingly, 72 differentially expressed miRNAs (P≤0.001) between leaves and flowers and their potential target genes were identified and validated. The qRT-PCR validation showed that majority of the differentially expressed miRNAs showed significant tissue-specific expression in L. japonica. Furthermore, the miRNA-mRNA and mRNA-mRNA regulatory networks were constructed using Cytoscape software. Taken together, this study identified a large number of miRNAs and target genes in L. japonica, which not only provides the first global miRNA expression profiles, but also sheds light on functional genomics research on L. japonica in the future.
doi:10.1371/journal.pone.0164140
PMCID: PMC5053492  PMID: 27711182
17.  Aberrant Meiotic Prophase I Leads to Genic Male Sterility in the Novel TE5A Mutant of Brassica napus 
Scientific Reports  2016;6:33955.
Genic male sterility (GMS) has already been extensively utilized for hybrid rapeseed production. TE5A is a novel thermo-sensitive dominant GMS line in Brassica napus, however, its mechanisms of GMS remain largely unclear. Histological and Transmission electron microscopy (TEM) analyses of anthers showed that the male gamete development of TE5A was arrested at meiosis prophase I. EdU uptake of S-phase meiocytes revealed that the TE5A mutant could accomplish DNA replication, however, chromosomal and fluorescence in situ hybridization (FISH) analyses of TE5A showed that homologous chromosomes could not pair, synapse, condense and form bivalents. We then analyzed the transcriptome differences between young floral buds of sterile plants and its near-isogenic fertile plants through RNA-Seq. A total of 3,841 differentially expressed genes (DEGs) were obtained, some of which were associated with homologous chromosome behavior and cell cycle control during meiosis. Dynamic expression changes of selected candidate DEGs were then analyzed at different anther developmental stages. The present study not only demonstrated that the TE5A mutant had defects in meiotic prophase I via detailed cytological analysis, but also provided a global insight into GMS-associated DEGs and elucidated the mechanisms of GMS in TE5A through RNA-Seq.
doi:10.1038/srep33955
PMCID: PMC5037387  PMID: 27670217
18.  Correlation between serum parathyroid hormone levels and coronary artery calcification in patients without renal failure 
Biomedical Reports  2016;5(5):601-606.
The aim of the present study was to investigate the correlation between serum parathyroid hormone (PTH) levels and coronary artery calcification (CAC) in patients without renal failure, as well as to determine independent risk factors of CAC score (CACS). A total of 157 patients who underwent coronary computed tomography angiographic examination at the 101th Hospital of the People's Liberation Army between December 2013 and February 2015 were retrospectively evaluated. The correlation between PTH levels and CACS was determined using a Pearson correlation analysis. A receiver operating characteristic (ROC) curve was drawn to determine the best cutoff PTH level for prediction of CAC. The independent association between serum PTH levels and CAC was analyzed by using a logistic regression analysis model with the response variable Be binary class. The results revealed that PTH levels in patients in the CAC group were significantly higher than those of patients in the non-calcification group. PTH levels were positively correlated with CACS (r=0.288, P<0.001). The ROC curve suggested that a PTH level of ≥31.05 pg/ml was the best cut-off point for the prediction of CAC, with a sensitivity of 80.88%, specificity of 60.67% and an area under the curve of 0.761. After including predictive factors for CAC (gender, age, smoking status, diabetes, hypertension, hyperlipidemia, body mass index, glomerular filtration rate and calcium, phosphorus, calcium-phosphorus product, magnesium, PTH, total cholesterol, low-density lipoprotein cholesterol, triglyceride, high-density lipoprotein cholesterol and C-reactive protein levels), the odds ratio of the serum PTH levels regarding the prediction of CAC was 1.050 (95% confidence interval, 1.027–1.074; P<0.001). In conclusion, the present study suggested that serum PTH levels are correlated with CAC in patients without renal failure and may thus be used as a reliable predictor of CAC.
doi:10.3892/br.2016.761
PMCID: PMC5103683  PMID: 27882224
parathyroid hormone; coronary artery disease; calcinosis; computed tomography angiography
19.  JUMPg: an Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells 
Journal of proteome research  2016;15(7):2309-2320.
Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by researching only unmatched high quality spectra, and re-using amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then the JUMPg program is applied to process a label-free mass spectrometry dataset of Alzheimer’s disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and “non-coding gene” translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled dataset of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multi-omics data integration.
Graphical abstract
doi:10.1021/acs.jproteome.6b00344
PMCID: PMC5033046  PMID: 27225868
Genomics; proteomics; mass spectrometry; proteogenomics; RNA-seq; database search; multistage analysis; spectrum quality control
21.  Slowly Delivered Icariin/Allogeneic Bone Marrow-Derived Mesenchymal Stem Cells to Promote the Healing of Calvarial Critical-Size Bone Defects 
Stem Cells International  2016;2016:1416047.
Bone tissue engineering technique is a promising strategy to repair large-volume bone defects. In this study, we developed a 3-dimensional construct by combining icariin (a small-molecule Chinese medicine), allogeneic bone marrow-derived mesenchymal stem cells (BMSCs), and a siliceous mesostructured cellular foams-poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SMC-PHBHHx) composite scaffold. We hypothesized that the slowly released icariin could significantly promote the efficacy of SMC-PHBHHx/allogeneic BMSCs for repairing critical-size bone defects in rats. In in vitro cellular experiments, icariin at optimal concentration (10−6 mol/L) could significantly upregulate the osteogenesis- and angiogenesis-related genes and proteins, such as Runx2, ALP, osteocalcin, vascular endothelial growth factors, and fibroblast growth factors, as well as the mineralization of BMSCs. Icariin that was adsorbed onto the SMC-PHBHHx scaffold showed a slow release profile within a 2-week monitoring span. Eight weeks after implantation in calvarial critical-size bone defects, the constructs with icariin were associated with significantly higher bone volume density, trabecular thickness, trabecular number, and significantly lower trabecular separation than the constructs without icariin. Histomorphometric analysis showed that icariin was also associated with a significantly higher density of newly formed blood vessels. These data suggested a promising application potential of the icariin/SMC-PHBHHx/allogeneic BMSCs constructs for repairing large-volume bone defects in clinic.
doi:10.1155/2016/1416047
PMCID: PMC5040948  PMID: 27721833
22.  Integrin α6/Akt/Erk signaling is essential for human breast cancer resistance to radiotherapy 
Scientific Reports  2016;6:33376.
Integrin α6 (ITGA6), a transmembrane glycoprotein adhesion receptor protein, is widely upregulated in many types of tumors and promotes migration and invasion in cancer cells. However, the role that the ITGA6-associated signaling network plays in radiosensitivity in breast cancer has not been described. The expression of ITGA6 was examined in human breast cancer and normal breast cell lines using western blot analysis. We also explored the role of ITGA6 in the regulation of radiation sensitivity in breast cancer using the colony formation assays, cell cycle analyses, apoptosis assays and immunofluorescence analyses. The results showed that the protein and mRNA expression levels of ITGA6 was higher in breast cancer cells than in normal cells. ITGA6 protectived responses to radiotherapy in breast cancer cells by altering cell apoptosis, DNA damage repair and cell-cycle regulation. Furthermore, ITGA6 enhanced radiation resistance via PI3K/Akt and MEK/Erk signaling. In addition, overexpressing ITGA6 promoted radiation resistance in cells, and this effect was neutralized by the PI3K inhibitor LY294002 and MEK inhibitor U0126. Taken together, these findings indicate that ITGA6 might be involved in a mechanism that underlies radiation resistance and that ITGA6 could be a potential target for therapies aimed at overcoming radiation resistance in breast cancer.
doi:10.1038/srep33376
PMCID: PMC5022055  PMID: 27624978
23.  Serum Amylase Levels in Relation to Islet β Cell Function in Patients with Early Type 2 Diabetes 
PLoS ONE  2016;11(9):e0162204.
Objective
The insulin-pancreatic acinar axis may play a major role in pancreatic function. Amylase is an exocrine enzyme that is produced by pancreatic acinar cells, and low serum amylase levels may be associated with endocrine diseases, such as metabolic syndrome and diabetes. We hypothesized that low serum amylase levels may be associated with impaired islet β cell function in type 2 diabetes. Therefore, we investigated the relationship between the serum amylase levels and islet β cell function in patients with early type 2 diabetes.
Methods
The cross-sectional study recruited 2327 patients with a mean of 1.71±1.62 years since their diagnosis of type 2 diabetes, and all participants were treated with lifestyle intervention alone. Serum amylase levels, the 75-g oral glucose tolerance test (OGTT) and metabolic risk factors were examined in all participants. The insulin sensitivity index (Matsuda index, ISIMatsuda) and insulin secretion index (ratio of total area-under-the-insulin-curve to glucose-curve, AUCins/glu) were derived from the OGTT. Integrated islet β cell function was assessed by the Insulin Secretion-Sensitivity Index-2 (ISSI-2) (ISIMatsuda multiplied by AUCins/glu).
Results
Serum amylase levels in the normal range were significantly correlated with ISIMatsuda, AUCins/glu and ISSI-2 (r = 0.203, 0.246 and 0.413, respectively, p<0.001). The association of the serum amylase levels with ISSI-2 (adjusted r = 0.363, p<0.001) was closer than the association with ISIMatsuda (adjusted r = 0.191, p<0.001) and AUCins/glu (adjusted r = 0.174, p<0.001) after adjusting for the anthropometric indices, time since the diagnosis of diabetes, lipid profiles, uric acid levels, estimated glomerular filtration rate, HbA1c levels, smoking and drinking using the partial correlation test. After adjusting for these metabolic risk factors in the multivariate regression analysis with the amylase levels as the dependent variable, ISSI-2 was the major independent contributor to the serum amylase levels (β = 0.416, t = 21.72, p<0.001). Meanwhile, in a comparison of the groups with the highest and lowest quartiles of serum amylase levels, the mean difference in logISSI-2 was 0.902 (95% CI 0.823 to 0.982), and after adjusting for metabolic risk factors, the mean difference in logISSI-2 was 0.610 (0.537 to 0.683).
Conclusions
Serum amylase levels in the normal range are positively associated with integrated islet β cell function in patients with early type 2 diabetes, as assessed by ISSI-2.
doi:10.1371/journal.pone.0162204
PMCID: PMC5015989  PMID: 27606813
24.  The diagnostic value of time-resolved MR angiography with Gadobutrol at 3 T for preoperative evaluation of lower extremity tumors: Comparison with computed tomography angiography 
Purpose
To evaluate the diagnostic value of time resolved magnetic resonance angiography with interleaved stochastic trajectory (TWIST) using Gadobutrol for preoperative evaluation of lower extremity tumors.
Materials and methods
This prospective study was approved by the local Institutional Review Board. 50 consecutive patients (31 men, 19 women, age range 18–80 years, average age 42.7 years) with lower extremity tumors underwent TWIST and computed tomography angiography (CTA). Image quality of TWIST and CTA were evaluated by two radiologists according to a 4-point scale. The degree of arterial stenosis caused by tumor was assessed using TWSIT and CTA separately, and the intra-modality agreement was determined using a kappa test. The number of feeding arteries identified by TWIST was compared with that by CTA using Wilcoxon signed rank test. The ability to identify arterio-venous fistulae (AVF) were compared using a chi-square test.
Results
Image quality of TWIST and CTA were rated as 3.88 ± 0.37 and 3.97 ± 0.16, without statistically significant difference (P = 0.135). Intra-modality agreement was excellent for the assessment of arterial stenosis (kappa = 0.806 ± 0.073 for Reader 1, kappa = 0.805 ± 0.073 for Reader 2). Readers identified AVF with TWIST in 27 of 50 cases, and identified AVF with CTA in 14 of 50 (P < 0.001). Mean feeding arteries identified with TWIST was significantly more than that with CTA (2.08 ± 1.72 vs 1.62 ± 1.52, P = 0.02).
Conclusion
TWIST is a reliable imaging modality for the assessment of lower extremity tumors. TWIST is comparable to CTA for the identification of AVF and feeding arteries.
doi:10.1016/j.ejro.2016.08.006
PMCID: PMC5009234  PMID: 27622202
TWIST, time resolved magnetic resonance angiography with interleaved stochastic trajectory; CTA, computed tomography angiography; AVF, arterio-venous fistulae; Time-resolved; Magnetic resonance angiography; Lower extremity; Tumors
25.  Vismodegib Exerts Targeted Efficacy Against Recurrent Sonic Hedgehog–Subgroup Medulloblastoma: Results From Phase II Pediatric Brain Tumor Consortium Studies PBTC-025B and PBTC-032 
Journal of Clinical Oncology  2015;33(24):2646-2654.
Purpose
Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB).
Patients and Methods
Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available.
Results
A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH–MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses.
Conclusion
Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH–MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.
doi:10.1200/JCO.2014.60.1591
PMCID: PMC4534527  PMID: 26169613

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