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1.  Prediction of Links and Weights in Networks by Reliable Routes 
Scientific Reports  2015;5:12261.
Link prediction aims to uncover missing links or predict the emergence of future relationships from the current network structure. Plenty of algorithms have been developed for link prediction in unweighted networks, but only a few have been extended to weighted networks. In this paper, we present what we call a “reliable-route method” to extend unweighted local similarity indices to weighted ones. Using these indices, we can predict both the existence of links and their weights. Experiments on various real-world networks suggest that our reliable-route weighted resource-allocation index performs noticeably better than others with respect to weight prediction. For existence prediction it is either the highest or very close to the highest. Further analysis shows a strong positive correlation between the clustering coefficient and prediction accuracy. Finally, we apply our method to the prediction of missing protein-protein interactions and their confidence scores from known PPI networks. Once again, our reliable-route method shows the highest accuracy.
doi:10.1038/srep12261
PMCID: PMC4510530  PMID: 26198206
2.  Correction: Comparison of Current Diagnostic Criteria for Acute-On-Chronic Liver Failure 
PLoS ONE  2015;10(7):e0133817.
doi:10.1371/journal.pone.0133817
PMCID: PMC4508032  PMID: 26193100
3.  Generation of Wheat Transcription Factor FOX Rice Lines and Systematic Screening for Salt and Osmotic Stress Tolerance 
PLoS ONE  2015;10(7):e0132314.
Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.
doi:10.1371/journal.pone.0132314
PMCID: PMC4503417  PMID: 26176782
4.  TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR 
PLoS ONE  2015;10(7):e0132666.
The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.
doi:10.1371/journal.pone.0132666
PMCID: PMC4501803  PMID: 26172450
5.  The molecular cloning and clarification of a photorespiratory mutant, oscdm1, using enhancer trapping 
Frontiers in Genetics  2015;6:226.
Enhancer trap systems have been demonstrated to increase the effectiveness of gene identification in rice. In this study, a chlorophyll-deficient mutant, named oscdm1, was screened and characterized in detail from a T-DNA enhancer-tagged population. The oscdm1 plants were different from other chlorophyll-deficient mutants; they produced chlorotic leaves at the third leaf stage, which gradually died with further growth of the plants. However, the oscdm1 plants were able to survive exposure to elevated CO2 levels, similar to photorespiratory mutants. An analysis of the T-DNA flanking sequence in the oscdm1 plants showed that the T-DNA was inserted into the promoter region of a serine hydroxymethyltransferase (SHMT) gene. OsSHMT1 is a key enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for the interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Full-length OsSHMT1 complemented the oscdm1 phenotype, and the downregulation of OsSHMT1 in wild-type plants by RNA interference (RNAi) produced plants that mimicked the oscdm1 phenotype. GUS assays and quantitative PCR revealed the preferential expression of OsSHMT1 in young leaves. TEM revealed serious damage to the thylakoid membrane in oscdm1 chloroplasts. The oscdm1 plants showed more extensive damage than wild type using an IMAGING-PAM fluorometer, especially under high light intensities. OsSHMT1-GFP localized exclusively to mitochondria. Further analysis revealed that the H2O2 content in the oscdm1 plants was twice that in wild type at the fourth leaf stage. This suggests that the thylakoid membrane damage observed in the oscdm1 plants was caused by excessive H2O2. Interestingly, OsSHMT1-overexpressing plants exhibited increased photosynthetic efficiency and improved plant productivity. These results lay the foundation for further study of the OsSHMT1 gene and will help illuminate the functional role of OsSHMT1 in photorespiration in rice.
doi:10.3389/fgene.2015.00226
PMCID: PMC4490251  PMID: 26191072
rice; serine hydroxymethyltransferase; photorespiratory; mitochondria; hydrogen peroxide
6.  OM85-BV Induced the Productions of IL-1β, IL-6, and TNF-α via TLR4- and TLR2-Mediated ERK1/2/NF-κB Pathway in RAW264.7 Cells 
Broncho-Vaxom (OM85-BV) is an extract mixture from 8 strains of Gram+ and Gram− bacteria and plays an important role in anti-infection immune response by regulating macrophage activity and cytokine productions. However, the mechanism by which OM85-BV enhances the cytokine expression is still obscure. In this study, we evaluated the effects of OM85-BV on the productions of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in RAW264.7 murine macrophages. Exposure of RAW264.7 cells to 100 μg/mL OM85-BV upregulated the expression of IL-1β, IL-6, and TNF-α at the mRNA and protein levels in a time- and dose-dependent manner. In addition, OM85-BV induced extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor-kappa B (NF-κB) phosphorylation. Pretreatment with U0126 or Bay11-7082, respectively, could decrease IL-1β, IL-6, and TNF-α productions induced by OM85-BV. Application of Toll-like receptor (TLR) 4 or TLR2 small-interfering RNA (siRNA) into RAW264.7 cells could inhibit the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Consistent with this, downregulating either myeloid differentiation factor 88 (MyD88) or TRIF-related adaptor molecule (TRAM) gene with MyD88-siRNA or TRAM-siRNA separately could reduce the productions of cytokines and ERK1/2 and NF-κB phosphorylation induced by OM85-BV. Our study demonstrated that the productions of IL-1β, IL-6, and TNF-α induced by OM85-BV in RAW264.7 cells were through TLR4 and TLR2 signaling pathway-mediated activation of ERK1/2 and NF-κB.
doi:10.1089/jir.2013.0077
PMCID: PMC4080859  PMID: 24605772
7.  IKKα is involved in kidney recovery and regeneration of acute ischemia/reperfusion injury in mice through IL10-producing regulatory T cells 
Disease Models & Mechanisms  2015;8(7):733-742.
ABSTRACT
The recovery phase after kidney ischemia/reperfusion (IR) injury is often associated with the suppression of inflammation and the proliferation of tubular epithelial cells (TECs). The duration of this phase is often determined by the suppression of inflammation and the proliferation of TECs. Several lines of evidence suggest that IκB kinase α (IKKα) not only promotes the production of anti-inflammatory factors and/or prevents the production of inflammatory factors, but also induces the accompanying cell differentiation and regeneration, and suppresses inflammation. We therefore hypothesized that IKKα could participate in the kidney repair after IR injury and have used a mouse model of acute kidney injury (AKI) to test this. We found that IKKα mediated the repair of the kidney via infiltrated regulatory T (Treg) cells, which can produce anti-inflammatory cytokine IL10, and that IKKα also increased the proliferation of the surviving TECs and suppressed of inflammation. In addition, the expression of indoleamine 2,3-dioxygenase (IDO) in TECs is consistent with the infiltration of IL10-producing Treg cells. We conclude that IKKα is involved in kidney recovery and regeneration through the Treg cells that can produce IL10, which might be a potential therapeutic target that can be used to promote kidney repair after IR injury.
Summary: IKKα in the kidney recovery and regeneration via regulatory T cells secreting IL10.
doi:10.1242/dmm.018200
PMCID: PMC4486855  PMID: 26035380
IκB kinase α; Kidney; Ischemia-reperfusion injury; Repair; IL10; T regular cell; Indoleamine 2; 3-dioxygenase
8.  The Role of Long-term Acute Care Hospitals in Treating the Critically Ill and Medically Complex 
Medical Care  2015;53(7):582-590.
Supplemental Digital Content is available in the text.
Background:
Little evidence exists on the effects of receiving care in a long-term acute care hospital (LTCH).
Objective:
To examine LTCH effects on mortality and Medicare payments overall and among high-acuity patients.
Research Design:
A retrospective cohort study of Medicare beneficiaries using probit and generalized linear models. An instrumental variable technique was used to adjust for selection bias.
Subjects:
Medicare beneficiaries within 5 major diagnostic categories and not on prolonged mechanical ventilation.
Measures:
Mortality (365 d) and Medicare payments (180 d) during an episode of care.
Results:
LTCH care is associated with increases in Medicare payments ranging from $3146 to $17,589 (P<0.01) with no mortality benefit for 3 categories and payment reductions of $5419 and $5962 (P<0.01) at lower or similar mortality for 2 categories. LTCH patients with multiple organ failure experience lower mortality at similar or lower payments (3 categories) or similar mortality at lower payments (1 category) compared with patients in other settings, with mortality benefits between 5.4 and 9.7 percentage points (P<0.05) and payment reductions between $13,806 and $20,809 (P<0.01). For 1 category, we found no difference in mortality or payments between LTCH and non-LTCH patients with multiple organ failure. For patients with ≥3 days in intensive care, LTCH care is associated with improved mortality and lower payments in 4 and 3 categories, respectively.
Conclusions:
Receiving care in an LTCH may improve outcomes for some patients. Further research is needed to better define patients for whom care in these hospitals is beneficial.
doi:10.1097/MLR.0000000000000382
PMCID: PMC4470738  PMID: 26067882
Medicare; cost; payments; mortality; hospital; post acute care; intensive care; organ failure; effectiveness
9.  Subgingival microbiome in patients with healthy and ailing dental implants 
Scientific Reports  2015;5:10948.
Dental implants are commonly used to replace missing teeth. However, the dysbiotic polymicrobial communities of peri-implant sites are responsible for peri-implant diseases, such as peri-implant mucositis and peri-implantitis. In this study, we analyzed the microbial characteristics of oral plaque from peri-implant pockets or sulci of healthy implants (n = 10), peri-implant mucositis (n = 8) and peri-implantitis (n = 6) sites using pyrosequencing of the 16S rRNA gene. An increase in microbial diversity was observed in subgingival sites of ailing implants, compared with healthy implants. Microbial co-occurrence analysis revealed that periodontal pathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, were clustered into modules in the peri-implant mucositis network. Putative pathogens associated with peri-implantitis were present at a moderate relative abundance in peri-implant mucositis, suggesting that peri-implant mucositis an important early transitional phase during the development of peri-implantitis. Furthermore, the relative abundance of Eubacterium was increased at peri-implantitis locations, and co-occurrence analysis revealed that Eubacterium minutum was correlated with Prevotella intermedia in peri-implantitis sites, which suggests the association of Eubacterium with peri-implantitis. This study indicates that periodontal pathogens may play important roles in the shifting of healthy implant status to peri-implant disease.
doi:10.1038/srep10948
PMCID: PMC4468443  PMID: 26077225
10.  Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla 
Stem Cells and Development  2014;23(12):1405-1416.
Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.
doi:10.1089/scd.2013.0580
PMCID: PMC4046201  PMID: 24517722
11.  DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity 
The Journal of Experimental Medicine  2014;211(13):2549-2566.
Zhang et al. show that DOCK8-deficient T and NK cells develop cell and nuclear shape abnormalities that do not impair chemotaxis but contribute to a form of cell death they term cytothripsis. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells resulting in impaired immune response to skin infection.
DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.
doi:10.1084/jem.20141307
PMCID: PMC4267229  PMID: 25422492
12.  Measuring multiple evolution mechanisms of complex networks 
Scientific Reports  2015;5:10350.
Numerous concise models such as preferential attachment have been put forward to reveal the evolution mechanisms of real-world networks, which show that real-world networks are usually jointly driven by a hybrid mechanism of multiplex features instead of a single pure mechanism. To get an accurate simulation for real networks, some researchers proposed a few hybrid models by mixing multiple evolution mechanisms. Nevertheless, how a hybrid mechanism of multiplex features jointly influence the network evolution is not very clear. In this study, we introduce two methods (link prediction and likelihood analysis) to measure multiple evolution mechanisms of complex networks. Through tremendous experiments on artificial networks, which can be controlled to follow multiple mechanisms with different weights, we find the method based on likelihood analysis performs much better and gives very accurate estimations. At last, we apply this method to some real-world networks which are from different domains (including technology networks and social networks) and different countries (e.g., USA and China), to see how popularity and clustering co-evolve. We find most of them are affected by both popularity and clustering, but with quite different weights.
doi:10.1038/srep10350
PMCID: PMC4464182  PMID: 26065382
13.  3.0T 1H magnetic resonance spectroscopy for assessment of steatosis in patients with chronic hepatitis C 
AIM: To investigate the utility of 1H magnetic resonance spectroscopy (1H MRS) as a noninvasive test for steatosis in patients infected with hepatitis C virus.
METHODS: Ninety patients with chronic hepatitis C and pathology data underwent 3.0T 1H MRS, and the results of MRS and pathological analysis were compared.
RESULTS: This group of patients included 26 people with mild fatty liver (28.89%), 16 people with moderate fatty liver (17.78%), 18 people with severe fatty liver (20.0%), and 30 people without fatty liver (33.33%). The water peak was near 4.7 parts per million (ppm), and the lipid peak was near 1.3 ppm. Analysis of variance revealed that differences in the lipid peak, the area under the lipid peak, ratio of the lipid peak to the water peak, and ratio of the area under the lipid peak to the area under the water peak were statistically significant among the groups. Specifically, as the severity of fatty liver increased, the value of each index increased correspondingly. In the pairwise comparisons, the mean lipid peak, area under the lipid peak, ratio of the lipid peak to the water peak, and ratio of the area under the lipid peak to the area under the water peak were significantly different between the no fatty liver and moderate fatty liver groups, whereas no differences were noted between the severe fatty liver group and the mild or moderate fatty liver group. Area under the ROC curve (AUC) of area ratio in lipid and water and ratio in lipid and water in the no fatty liver group to mild fatty liver group, mild fatty liver group to moderate fatty liver group, and moderate fatty liver disease group to severe fatty liver group, were 0.705, 0.900, and 0.975, respectively.
CONCLUSION: 1H MRS is a noninvasive technique that can be used to provide information on the effect of liver steatosis on hepatic metabolic processes. This study indicates that the 1H MRS can be used as an indicator of steatosis in patients with chronic hepatitis C.
doi:10.3748/wjg.v21.i21.6736
PMCID: PMC4458784  PMID: 26074712
1H; Magnetic resonance spectroscopy; Hepatitis C; Antiviral therapy
14.  The effects of phosphodiesterase-5 inhibitor sildenafil against post-resuscitation myocardial and intestinal microcirculatory dysfunction by attenuating apoptosis and regulating microRNAs expression: essential role of nitric oxide syntheses signaling 
Background
Recent experimental and clinical studies have indicated the cardioprotective role of sildenafil during ischemia/reperfusion (I/R) injury. Sildenafil has been shown to attenuate postresuscitation myocardial dysfunction in piget models of ventricular fibrillation. This study was designed to investigate if administration of sildenafil will attenuate post-resuscitation myocardial dysfunction by attenuating apoptosis and regulating miRNA expressions, furthermore, ameliorating the severity of post-microcirculatory dysfunction.
Methods
Twenty-four male pigs (weighing 30 ± 2 kg) were randomly divided into groups, sildenafil pretreatment (n = 8), saline (n = 8) and sham operation (sham, n = 8). Sildenafil pretreatment consisted of 0.5 mg/kg sildenafil, administered once intraperitoneally 30 min prior to ventricular fibrillation (VF). Eight minutes of untreated VF was followed by defibrillation in anesthetized, closed-chest pigs. Hemodynamic status and blood samples were obtained at 0 min, 0.5, 1, 2, 4 and 6 h after return of spontaneous circulation (ROSC). Surviving pigs were euthanatized at 24 h after ROSC, and hearts were removed for analysis by electron microscopy, western blotting, quantitative real-time polymerase chain reaction (PCR), and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Intestinal microcirculatory blood flow was visualized by a sidestream dark-field imaging device at baseline and 0.5, 1, 2, 4, and 6 h after ROSC.
Results
Compared with the saline group, the sildenafil group had a higher 24-hour survival (7/8 versus 3/8 survivors, p < 0.05) and a better outcome in hemodynamic parameters. The protective effect of sildenafil also correlated with reduced cardiomyocyte apoptosis, as evidenced by reduced TUNEL-positive cells, increased anti-apoptotic Bcl-2/Bax ratio and inhibited caspase-3 activity in myocardium. Additionally, sildenafil treatment inhibited the increases in the microRNA-1 levels and alleviated the decreases in the microRNA-133a levels which negatively regulates pro-apoptotic genes. At 6 h after ROSC, post-resuscitation perfused vessel density and microcirculatory flow index were significantly lower in the saline group than in the sildenafil group.
Conclusions
The major findings of this study are as follows: (1) sildenafil improved post-resuscitation perfusion of the heart, and thus reduced cardiac myocyte apoptosis and improved cardiac function; (2) sildenafil treatment inhibited the increases in the microRNA-1 levels, but alleviated the decreases in the microRNA-133a levels.
doi:10.1186/s12967-015-0550-9
PMCID: PMC4467614  PMID: 26040988
Sildenafil; iNOS; eNOS; Post-resuscitation myocardial dysfunction; MicroRNAs; Apoptosis
15.  Anticancer Effect and Apoptosis Induction of Gambogic Acid in Human Leukemia Cell Line K562 In Vitro 
Background
The aim of this study was to investigate the anticancer effect and related mechanisms of gambogic acid (GA), a traditional Chinese medicine, on human leukemia cell line K562, together with the effect on bone marrow mononuclear cells (MNCs).
Material/Methods
K562 cells and MNCs were treated with various concentrations and treatment times of GA. Inhibitory rate was detected by use of the Cell Counting Kit-8 (CCK-8) assay. Apoptosis was analyzed by morphological detection, Annexin-V/PI doubling staining, and TUNEL assays. The expression changes of pivotal proteins were evaluated by Western blotting.
Results
GA not only suppressed cell proliferation, but also induced apoptosis of K562 cells in a dose-dependent manner. While it did not significantly inhibit cell proliferation of MNCs, it did induce apoptosis in a dose-dependent manner. CCK-8 assay revealed that the proliferation of K562 cells was significantly inhibited when the concentration of GA was more than 0.5 μM. Morphological detection showed the nuclei became denser and more intense orange in K562 cells after GA treatment compared with the untreated group. The expression levels of BCL-2, nuclear factor-κB (NF-κB), c-myc, phosphatidylinositol3-kinase (PI3K), and phosphorylation of serine-threonine kinase (p-AKT) were down-regulated by GA.
Conclusions
GA significantly suppressed the proliferation of K562 cells, but has less effect on MNCs. The inhibition of K562 cells proliferation and apoptosis induced by GA might be related to the down-regulation of BCL-2, NF-κB, c-myc, PI3K, and p-AKT.
doi:10.12659/MSM.893004
PMCID: PMC4463775  PMID: 26049398
Apoptosis; Bone Marrow Mononuclear Cells; Gambogic Acid; Gene Expression; Human Leukemia Cell Line K562
16.  Somatic reversion in DOCK8 immunodeficiency modulates disease phenotype 
Background
Autosomal recessive, loss-of-function mutations in DOCK8 cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking.
Objective
We investigated whether reversions contributed to the variable disease expression.
Methods
Patients followed at the NIH Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets.
Results
We identified 17 out of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion due to somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or in NK cells, but less so in naïve T cells or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation.
Conclusions
In DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival, but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8 immunodeficient patients have mutable mosaic genomes that may modulate disease phenotype over time.
doi:10.1016/j.jaci.2014.03.025
PMCID: PMC4132167  PMID: 24797421
DOCK8; reversion; somatic repair; recombination; gene conversion; intragenic single crossover; T cell; NK cell; allergy; immunodeficiency
17.  GX1-conjugated poly(lactic acid) nanoparticles encapsulating Endostar for improved in vivo anticolorectal cancer treatment 
Tumor angiogenesis plays a key role in tumor growth and metastasis; thus, targeting tumor-associated angiogenesis is an important goal in cancer therapy. However, the efficient delivery of drugs to tumors remains a key issue in antiangiogenesis therapy. GX1, a peptide identified by phage-display technology, is a novel tumor vasculature endothelium-specific ligand and possesses great potential as a targeted vector and antiangiogenic agent in the diagnosis and treatment of human cancers. Endostar, a novel recombinant human endostatin, has been shown to inhibit tumor angiogenesis. In this study, we developed a theranostic agent composed of GX1-conjugated poly(lactic acid) nanoparticles encapsulating Endostar (GPENs) and labeled with the near-infrared dye IRDye 800CW to improve colorectal tumor targeting and treatment efficacy in vivo. The in vivo fluorescence molecular imaging data showed that GPENs (IRDye 800CW) more specifically targeted tumors than free IRDye 800CW in colorectal tumor-bearing mice. Moreover, the antitumor efficacy was evaluated by bioluminescence imaging and immunohistology, revealing that GPENs possessed improved antitumor efficacy on subcutaneous colorectal xenografts compared to other treatment groups. Thus, our study showed that GPENs, a novel GX1 peptide guided form of nanoscale Endostar, can be used as a theranostic agent to facilitate more efficient targeted therapy and enable real-time monitoring of therapeutic efficacy in vivo.
doi:10.2147/IJN.S82029
PMCID: PMC4454195  PMID: 26060399
GX1 peptide; Endostar; colorectal cancer; angiogenesis; IRDye 800CW; molecular imaging
18.  Microbiota prevents cholesterol loss from the body by regulating host gene expression in mice 
Scientific Reports  2015;5:10512.
We have previously observed that knockout of Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol transporter essential for intestinal cholesterol absorption, reduces the output of dry stool in mice. As the food intake remains unaltered in NPC1L1-knockout (L1-KO) mice, we hypothesized that NPC1L1 deficiency may alter the gut microbiome to reduce stool output. Consistently, here we demonstrate that the phyla of fecal microbiota differ substantially between L1-KO mice and their wild-type controls. Germ-free (GF) mice have reduced stool output. Inhibition of NPC1L1 by its inhibitor ezetimibe reduces stool output in specific pathogen-free (SPF), but not GF mice. In addition, we show that GF versus SPF mice have reduced intestinal absorption and increased fecal excretion of cholesterol, particularly after treatment with ezetimibe. This negative balance of cholesterol in GF mice is associated with reduced plasma and hepatic cholesterol, and likely caused by reduced expression of NPC1L1 and increased expression of ABCG5 and ABCG8 in small intestine. Expression levels of other genes in intestine and liver largely reflect a state of cholesterol depletion and a decrease in intestinal sensing of bile acids. Altogether, our findings reveal a broad role of microbiota in regulating whole-body cholesterol homeostasis and its response to a cholesterol-lowering drug, ezetimibe.
doi:10.1038/srep10512
PMCID: PMC4444975  PMID: 26015368
19.  Spin Polarization Inversion at Benzene-Absorbed Fe4N Surface 
Scientific Reports  2015;5:10602.
We report a first-principle study on electronic structure and simulation of the spin-polarized scanning tunneling microscopy graphic of a benzene/Fe4N interface. Fe4N is a compound ferromagnet suitable for many spintronic applications. We found that, depending on the particular termination schemes and interface configurations, the spin polarization on the benzene surface shows a rich variety of properties ranging from cosine-type oscillation to polarization inversion. Spin-polarization inversion above benzene is resulting from the hybridizations between C pz and the out-of-plane d orbitals of Fe atom.
doi:10.1038/srep10602
PMCID: PMC4445049  PMID: 26012892
20.  MALDI-TOF Mass Array Analysis of Nell-1 Promoter Methylation Patterns in Human Gastric Cancer 
BioMed Research International  2015;2015:136941.
Mass spectrometry (MS) enables rapid and sensitive qualitative and quantitative analyses of biomolecules (proteins, peptides, oligosaccharides, lipids, DNA, and RNA), drugs, and metabolites. MS has become an essential tool in modern biomedical research, including the analysis of DNA methylation. DNA methylation has been reported in many cancers, suggesting that it can be utilized as an early biomarker to improve the early diagnosis rate. Using matrix-assisted laser desorption/ionization time-of-flight MS and MassCLEAVE reagent, we compared Nell-1 hypermethylation levels among tumor tissues, paracarcinoma tissues, and normal tissues from gastric cancer patients. Almost 80% of the CpG sites in the amplicons produced were covered by the analysis. Our results indicate a significant difference in methylation status between gastric cancer tissue (a higher level) and normal tissue. The same trend was identified in gastric cancer tissue versus paracarcinoma tissue. We also detected lower relative expression of Nell-1 by real-time PCR. Furthermore, immunohistochemical analyses revealed that Nell-1 staining was less intense in cancer tissue relative to normal tissue and that the tumor cells had spread to the muscle layer. These findings may serve as a guide for the early diagnosis of gastric cancer.
doi:10.1155/2015/136941
PMCID: PMC4452250  PMID: 26090379
21.  Natural Products for Antithrombosis 
Thrombosis is considered to be closely related to several diseases such as atherosclerosis, ischemic heart disease and stroke, as well as rheumatoid arthritis, hyperuricemia, and various inflammatory conditions. More and more studies have been focused on understanding the mechanism of molecular and cellular basis of thrombus formation as well as preventing thrombosis for the treatment of thrombotic diseases. In reality, there is considerable interest in the role of natural products and their bioactive components in the prevention and treatment of thrombosis related disorders. This paper briefly describes the mechanisms of thrombus formation on three aspects, including coagulation system, platelet activation, and aggregation, and change of blood flow conditions. Furthermore, the natural products for antithrombosis by anticoagulation, antiplatelet aggregation, and fibrinolysis were summarized, respectively.
doi:10.1155/2015/876426
PMCID: PMC4449941  PMID: 26075003
22.  Identification and Characterization of MicroRNAs in Ginkgo biloba var. epiphylla Mak 
PLoS ONE  2015;10(5):e0127184.
Ginkgo biloba, a dioecious plant known as a living fossil, is an ancient gymnosperm that stands distinct from other gymnosperms and angiosperms. Ginkgo biloba var. epiphylla (G. biloba var. epiphylla), with ovules borne on the leaf blade, is an unusual germplasm derived from G. biloba. MicroRNAs (miRNAs) are post-transcriptional gene regulators that play critical roles in diverse biological and metabolic processes. Currently, little is known about the miRNAs involved in the key stage of partly epiphyllous ovule germination in G. biloba var. epiphylla. Two small RNA libraries constructed from epiphyllous ovule leaves and normal leaves of G. biloba var. epiphylla were sequenced on an Illumina/Solexa platform. A total of 82 miRNA sequences belonging to 23 families and 53 putative novel miRNAs were identified in the two libraries. Differential expression analysis showed that 25 conserved and 21 novel miRNAs were differentially expressed between epiphyllous ovule leaves and normal leaves. The expression patterns of partially differentially expressed miRNAs and the transcript levels of their predicted target genes were validated by quantitative real time RT-PCR. All the expression profiles of the 21 selected miRNAs were similar to those detected by Solexa deep sequencing. Additionally, the transcript levels of almost all the putative target genes of 9 selected miRNAs were opposite to those of the corresponding miRNAs. The putative target genes of the differentially expressed miRNAs were annotated with Gene Ontology terms related to reproductive process, metabolic process and responding to stimulus. This work presents a broad range of small RNA transcriptome data obtained from epiphyllous ovule and normal leaves of G. biloba var. epiphylla, which may provide insights into the miRNA-mediated regulation in the epiphyllous ovule germination process.
doi:10.1371/journal.pone.0127184
PMCID: PMC4433266  PMID: 25978425
23.  Radical cascade reaction of alkynes with N-fluoroarylsulfonimides and alcohols 
Nature Communications  2015;6:7011.
Cascade reactions initiated by radical addition to alkynes are synthetically very attractive because they enable access to highly complex molecular skeletons in only few synthetic steps under usually mild conditions. Here we report a general radical cascade reaction of alkynes, N-fluoroarylsulfonimides and alcohols, enabling the efficient synthesis of important α-amino-α-aryl ketones from readily available starting materials via a single operation. During this process, the highly regioselective nitrogen-centred radical addition to internal and terminal alkynes generating vinyl radicals and the next explicit migration of aryl group from the nitrogen source lead the following efficient desulfonylation, oxygenation, and semi-pinacol rearrangement. In addition, the semi-pinacol rearrangement precursors, α-alkyloxyl-α,α-diaryl imines, could also be efficiently obtained under milder conditions. This methodology might open a new entry for designing intermolecular radical cascade reaction of alkynes.
Cascade and multi-component reactions allow highly functionalized molecules to be built from simpler precursors. Here, the authors report a three-component coupling giving α-amino-α-aryl ketones, initiated by the regioselective addition of nitrogen radicals to alkynes.
doi:10.1038/ncomms8011
PMCID: PMC4421815  PMID: 25901840
24.  Malaria Imported from Ghana by Returning Gold Miners, China, 2013 
Emerging Infectious Diseases  2015;21(5):864-867.
During May-August 2013, a malaria outbreak comprising 874 persons in Shanglin County, China, was detected among 4,052 persons returning from overseas. Ghana was the predominant destination country, and 92.3% of malarial infections occurred in gold miners. Preventive measures should be enhanced for persons in high-risk occupations traveling to malaria-endemic countries.
doi:10.3201/eid2105.141712
PMCID: PMC4412230  PMID: 25897805
imported malaria; disease outbreak; China; epidemiology; Ghana; parasites; vector-borne infections; Anopheles; Plasmodium; mosquitoes; malaria
25.  Maternal protein restriction induces early-onset glucose intolerance and alters hepatic genes expression in the peroxisome proliferator-activated receptor pathway in offspring 
Aims/Introduction
Maternal undernutrition during pregnancy and/or lactation can alter the offspring's response to environmental challenges, and thus increases the risk of the development of metabolic diseases at a later age. However, whether maternal protein restriction can modulate glucose metabolism in the early life of offspring is less understood. Furthermore, we explored the potential underlying mechanisms that illustrate this phenotype.
Materials and Methods
To test this hypothesis, we examined the offspring of C57BL/6J mice at weaning to determine the effects of feeding their mothers a low-protein diet or normal chow diet throughout pregnancy and lactation. Gene array experiments and quantitative real-time polymerase chain reaction were utilized to explore the altered hepatic genes expression.
Results
The offspring of dams fed a low-protein diet had a lower birthweight and bodyweight, impaired glucose tolerance, decreased insulin sensitivity, and decreased serum cholesterol at weaning. Using gene array experiments, 253 differentially expressed genes were identified in the liver tissues of the offspring between the two groups. Bioinformatic analyses showed that all differentially expressed genes were mapped to 11 pathways. We focused on the ‘peroxisome proliferator-activated receptor signaling pathway,’ because peroxisome proliferator-activated receptors have emerged as central regulators of glucose and lipid homeostasis. Quantitative real-time polymerase chain reaction was utilized for the validation of genes in the pathway.
Conclusions
A maternal low-protein diet during pregnancy and lactation promotes early-onset glucose intolerance in the offspring mice, and the altered hepatic genes expression in peroxisome proliferator-activated receptor signaling pathway could play role in regulating this phenomenon.
doi:10.1111/jdi.12303
PMCID: PMC4420558  PMID: 25969711
Early onset; Glucose intolerance; Maternal low-protein diet

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