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1.  Repeated functional convergent effects of NaV1.7 on acid insensitivity in hibernating mammals 
Hibernating mammals need to be insensitive to acid in order to cope with conditions of high CO2; however, the molecular basis of acid tolerance remains largely unknown. The African naked mole-rat (Heterocephalus glaber) and hibernating mammals share similar environments and physiological features. In the naked mole-rat, acid insensitivity has been shown to be conferred by the functional motif of the sodium ion channel NaV1.7. There is now an opportunity to evaluate acid insensitivity in other taxa. In this study, we tested for functional convergence of NaV1.7 in 71 species of mammals, including 22 species that hibernate. Our analyses revealed a functional convergence of amino acid sequences, which occurred at least six times independently in mammals that hibernate. Evolutionary analyses determined that the convergence results from both parallel and divergent evolution of residues in the functional motif. Our findings not only identify the functional molecules responsible for acid insensitivity in hibernating mammals, but also open new avenues to elucidate the molecular underpinnings of acid insensitivity in mammals.
doi:10.1098/rspb.2013.2950
PMCID: PMC3871328  PMID: 24352952
SCN9A; adaptation; convergent evolution; acid insensitivity
2.  Molecular characterization and copy number of SMN1, SMN2 and NAIP in Chinese patients with spinal muscular atrophy and unrelated healthy controls 
Background
Spinal muscular atrophy (SMA) is caused by SMN1 dysfunction, and the copy number of SMN2 and NAIP can modify the phenotype of SMA. The aim of this study was to analyze the copy numbers and gene structures of SMA-related genes in Chinese SMA patients and unrelated healthy controls.
Methods
Forty-two Chinese SMA patients and two hundred and twelve unrelated healthy Chinese individuals were enrolled in our study. The copy numbers and gene structures of SMA-related genes were measured by MLPA assay.
Results
We identified a homozygous deletion of SMN1 in exons 7 and 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of SMN1 exon 8. The proportions of the 212 unrelated healthy controls with different copy numbers for the normal SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). Three hybrid SMN genes and five genes that lack partial sequences were found in SMA patients and healthy controls. Distributions of copy numbers for normal SMN2 and NAIP were significantly different (P < 0.001) in people with and without SMA.
Conclusion
The copy numbers and gene structures of SMA-related genes were different in Chinese SMA patients and healthy controls.
doi:10.1186/s12891-015-0457-x
PMCID: PMC4328246
Chinese; MLPA; SMA; Gene copy number
3.  Assessing winter oilseed rape freeze injury based on Chinese HJ remote sensing data*  
The winter oilseed rape (Brassica napus L.) accounts for about 90% of the total acreage of oilseed rape in China. However, it suffers the risk of freeze injury during the winter. In this study, we used Chinese HJ-1A/1B CCD sensors, which have a revisit frequency of 2 d as well as 30 m spatial resolution, to monitor the freeze injury of oilseed rape. Mahalanobis distance-derived growing regions in a normal year were taken as the benchmark, and a mask method was applied to obtain the growing regions in the 2010–2011 growing season. The normalized difference vegetation index (NDVI) was chosen as the indicator of the degree of damage. The amount of crop damage was determined from the difference in the NDVI before and after the freeze. There was spatial variability in the amount of crop damage, so we examined three factors that may affect the degree of freeze injury: terrain, soil moisture, and crop growth before the freeze. The results showed that all these factors were significantly correlated with freeze injury degree (P<0.01, two-tailed). The damage was generally more serious in low-lying and drought-prone areas; in addition, oilseed rape planted on south- and west-oriented facing slopes and those with luxuriant growth status tended to be more susceptible to freeze injury. Furthermore, land surface temperature (LST) of the coldest day, soil moisture, pre-freeze growth and altitude were in descending order of importance in determining the degree of damage. The findings proposed in this paper would be helpful in understanding the occurrence and severity distribution of oilseed rape freeze injury under certain natural or vegetation conditions, and thus help in mitigation of this kind of meteorological disaster in southern China.
doi:10.1631/jzus.B1400150
PMCID: PMC4322424  PMID: 25644468
Brassica napus; Freeze injury; Remote sensing; Crop monitoring; HJ-CCD
4.  Discovery of Mosquito Saliva MicroRNAs during CHIKV Infection 
PLoS Neglected Tropical Diseases  2015;9(1):e0003386.
Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host.
Author Summary
Mosquito saliva contains a complex repertoire of bioactive factors that are secreted into blood feeding site, the skin. Infected mosquitoes transmit pathogens to the host during feeding via saliva. The bioactive factors in mosquito saliva are responsible for modulating host hemostasis, immune defenses and pain/itch responses, and have been implicated to enhance pathogen infection and establishment in the host. In our efforts to identify and characterize salivary immunomodulators that enhance Chikungunya virus (CHIKV) transmission, we have discovered, for the first time, exogenous microRNA in mosquito saliva. MicroRNAs (miRNAs) are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression. Short non-coding RNAs were extracted from the saliva of Chikungunya virus (CHIKV) infected and uninfected Aedes aegypti and Aedes albopictus saliva, and subjected to Illumina next generation sequencing. Bioinformatic analysis revealed the presence of miRNAs in the mosquito saliva. We have also identified several novel miRNAs that are expressed only during CHIKV infection. Though the functional roles of these miRNAs are yet to be established, our in-vitro data from testing 5 miRNAs demonstrate their role in the regulation of CHIKV infection. These miRNAs may play an important role in regulating the establishment of CHIKV infection in the mammalian host during blood feeding.
doi:10.1371/journal.pntd.0003386
PMCID: PMC4303268  PMID: 25612225
5.  Facile non-hydrothermal synthesis of oligosaccharides coated sub-5 nm magnetic iron oxide nanoparticles with dual MRI contrast enhancement effect 
Ultrafine sub-5 nm magnetic iron oxide nanoparticles coated with oligosaccharides (SIO) with dual T1-T2 weighted contrast enhancing effect and fast clearance has been developed as magnetic resonance imaging (MRI) contrast agent. Excellent water solubility, biocompatibility and high stability of such sub-5 nm SIO nanoparticles were achieved by using the “in-situ polymerization” coating method, which enables glucose forming oligosaccharides directly on the surface of hydrophobic iron oxide nanocrystals. Reported ultrafine SIO nanoparticles exhibit a longitudinal relaxivity (r1) of 4.1 mM−1s−1 and a r1/r2 ratio of 0.25 at 3 T (clinical field strength), rendering improved T1 or “brighter” contrast enhancement in T1-weighted MRI in addition to typical T2 or “darkening” contrast of conventional iron oxide nanoparticles. Such dual contrast effect can be demonstrated in liver imaging with T2 “darkening” contrast in the liver parenchyma but T1 “bright” contrast in the hepatic vasculature. More importantly, this new class of ultrafine sub-5 nm iron oxide nanoparticles showed much faster body clearance than those with larger sizes, promising better safety for clinical applications.
doi:10.1039/C4TB00811A
PMCID: PMC4147377  PMID: 25181490
6.  HASF is a stem cell paracrine factor that activates PKC epsilon mediated cytoprotection 
Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; tunel positive nuclei were fewer in number, caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ε (PKCε). Inhibition of PKCε blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKCε. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKCε.
doi:10.1016/j.yjmcc.2013.11.010
PMCID: PMC3897274  PMID: 24269490
cytoprotection; ischemia; paracrine; PKC epsilon; mesenchymal stem cell
7.  Influential Factors of Insufficient Physical Activity among Adolescents with Asthma in Taiwan 
PLoS ONE  2014;9(12):e116417.
Purpose
Little research has been reported concerning insufficient physical activity in Taiwanese adolescents with asthma. The aims of this paper are to compare the amount of physical activity between asthmatic and non-asthmatic adolescents in Taiwan, as well as to investigate the influential factors associated with insufficient physical activity in asthmatic adolescents.
Methods
Self-reporting structured questionnaires (socio-economic status, scale of family support for physical activity, amount of physical activity) and peak expiratory flow were assessed from 286 adolescents with asthma and 588 non-asthmatic adolescents in a cross-sectional design. Insufficient amount of physical activity was based on less than 300 minutes per week of moderate and vigorous physical activity.
Results
Adolescents with asthma have a greater amount of physical activity and a higher level of family support than those who are non-asthmatic. In Taiwan, adolescents with asthma, girls relative to boys, obesity relative to average weight, and low family support relative to high family support were found to be associated with insufficient physical activity.
Conclusion
Physical activity in adolescents with asthma is insufficient especially in girls, in asthmatics with obesity, and in those with low family support. We suggest that physical activity programs should be applied to Taiwan adolescents with asthma in order to match the criteria of 300 minutes per week of moderate and vigorous physical activity, especially for girls, the obese and those with a low level of family support.
doi:10.1371/journal.pone.0116417
PMCID: PMC4281071  PMID: 25551759
8.  Folate intake, serum folate levels, and prostate cancer risk: a meta-analysis of prospective studies 
BMC Public Health  2014;14:1326.
Background
Studies have reported inconsistent results concerning the existence of associations of folate intake and serum folate levels with prostate cancer risk. This study sought to summarise the evidence regarding these relationships using a dose–response meta-analysis approach.
Methods
In January 2014, we performed electronic searches of PubMed, Embase, and the Cochrane Library to identify studies examining the effect of folate on the incidence of prostate cancer. Only prospective studies that reported effect estimates with 95% confidence intervals (CIs) of the incidence of prostate cancer for more than 2 categories of folate were included.
Results
Overall, we included 10 prospective studies reporting data on 202,517 individuals. High dietary folate intake had little or no effect on prostate cancer risk (risk ratio [RR] = 1.02; 95% CI = 0.95–1.09; P = 0.598). The dose–response meta-analysis suggested that a 100 μg per day increase in dietary folate intake has no significant effect on the risk of prostate cancer (RR = 1.01; 95% CI = 0.99–1.02; P = 0.433). However, high serum folate levels were associated with an increased risk of prostate cancer (RR = 1.21; 95% CI = 1.05–1.39; P = 0.008). The dose–response meta-analysis indicated that a 5 nmol/L increment of serum folate levels was also associated with an increased risk of prostate cancer (RR = 1.04; 95% CI = 1.00–1.07; P = 0.042).
Conclusions
Our study indicated that dietary folate intake had little or no effect on prostate cancer risk. However, increased serum folate levels have potentially harmful effects on the risk of prostate cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1326) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2458-14-1326
PMCID: PMC4320532  PMID: 25543518
Folate; Prostate cancer; Dose–response; Meta-analysis
9.  Shenfu Injection for Intradialytic Hypotension: A Systematic Review and Meta-Analysis 
Objective. To evaluate the effectiveness and safety of Shenfu injection (SFI) for intradialytic hypotension (IDH). Methods. A systematic review of data sources published as of April 2014 was conducted. These included the Cochrane Central Register of Controlled Trials (2014 issue 4), Pubmed, Embase, CBM, CNKI, VIP, and Wangfang Data. Randomized controlled trials (RCTs) involving SFI for treatment and prevention of IDH were identified. Two researchers independently selected articles, extracted data, assessed quality, and cross checked the results. Revman 5.2 was used to analyze the results. Results. Eight RCTs were included. The meta-analysis indicated that compared with conventional therapies alone, SFI could elevate systolic blood pressure (SBP), increase the clinical effective rate, decrease the incidence of hypotension, increase serum albumin (ALB) levels, and reduce C-reactive protein (CRP) levels without serious adverse effects. GRADE Quality of Evidence. the quality of SBP, the effective rate, ALB, and CRP were low, and hypotension incidence and DBP were very low. Conclusions. SFI is more effective than conventional therapies for prevention and treatment of IDH. However, a clinical recommendation is not warranted due to the small number of studies included and low methodology quality. Multi-center and high-quality RCTs with large sample sizes are needed to provide stronger evidence.
doi:10.1155/2014/279853
PMCID: PMC4284991  PMID: 25587340
10.  Urinary LTE4 Levels as a Diagnostic Marker for IgE-Mediated Asthma in Preschool Children: A Birth Cohort Study 
PLoS ONE  2014;9(12):e115216.
Objectives
Leukotrienes play a central pathophysiological role in allergic asthma. The aim of this study was to investigate the utility of measuring urinary leukotriene E4 (LTE4) levels in the diagnosis of atopic diseases in early childhood.
Methods
Children aged 0 through 4 years from a birth cohort in the Prediction of Allergies in Taiwanese Children (PATCH) study were enrolled. Urinary LTE4 levels were measured and its association between total serum IgE levels, allergen-specific IgE sensitization and atopic diseases were assessed.
Results
A total of 182 children were regular followed up at clinics for a four-year follow-up period. Urinary LTE4 levels appeared to be elevated in children with total serum IgE levels exceeding 100 kU/L, allergen-specific IgE sensitization after 2 years of age. Elevation of urinary LTE4 levels (≥500 pg/mg of creatinine) significantly discriminated high serum total IgE levels (≥100 kU/L) at age 2 (P = 0.027). A higher level of total serum IgE or urinary LTE4 was significantly associated with the risk of developing allergic rhinitis and asthma at age 3. A significantly higher urinary LTE4 level was found in children with a combination of IgE sensitization and asthma at age 4.
Conclusions
Urinary LTE4 levels appear to be highly associated with IgE sensitization and its related allergic airway diseases after age 2. The measurement of urinary LTE4 (≥500 pg/mg of creatinine) could not only be a non-invasive method for atopic predisposition but also potentially provide a strategy for the diagnosis and management of asthma in preschool children.
doi:10.1371/journal.pone.0115216
PMCID: PMC4270744  PMID: 25521113
11.  Curcumin suppresses invasiveness and vasculogenic mimicry of squamous cell carcinoma of the larynx through the inhibition of JAK-2/STAT-3 signaling pathway 
To determine the role of JAK-2/STAT-3 signaling pathway in invasion and vasculogenic mimicry of laryngeal squamous cell carcinoma. HEp-2 cells were treated with 1 or 10 μmol/L curcumin and AG490 (the inhibitor of JAK-2) for 48 h, the invasion and vasculogenic mimicry of tumor cells were tested with Transwell chamber test and tube formation experiment. RT-PCR was used to measure the expression of MMP-2 and VEGF. Western blot assay was employed to determine the expression of JAK-2, STAT3, p-STAT3, MMP-2 and VEGF. Compared to control group,there were less tumor cells permeating membrane and less formed tubes after curcumin or AG490 treatment, RT-PCR showed that the expression of MMP-2 and VEGF at mRNA level were decreased (P < 0.01). Western blotting indicated that the expression of JAK-2, p-STAT3, MMP-2 and VEGF at protein levels were decreased (P < 0.01), while that of STAT-3 protein had no difference among each group (P > 0.05). Immunofluorescence staining demonstrated that the expression of eNOS was down-regulated (P < 0.01). Curcumin and AG490 significantly inhibits invasion and vasculogenic mimicry of laryngeal squamous cell carcinoma in vitro, and JAK-2/STAT-3 signaling pathway promotes above processes.
PMCID: PMC4300723  PMID: 25628937
Laryngeal squamous cell carcinoma; curcumin; JAK-2/STAT-3 signaling pathway; vasculogenic mimicry
12.  The metabolite alpha-ketoglutarate extends lifespan by inhibiting the ATP synthase and TOR 
Nature  2014;510(7505):397-401.
Metabolism and ageing are intimately linked. Compared to ad libitum feeding, dietary restriction (DR) or calorie restriction (CR) consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms1,2. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits3,4. Recently, several metabolites have been identified that modulate ageing5,6 with largely undefined molecular mechanisms. Here we show that the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (α-KG) extends the lifespan of adult C. elegans. ATP synthase subunit beta is identified as a novel binding protein of α-KG using a small-molecule target identification strategy called DARTS (drug affinity responsive target stability)7. The ATP synthase, also known as Complex V of the mitochondrial electron transport chain (ETC), is the main cellular energy-generating machinery and is highly conserved throughout evolution8,9. Although complete loss of mitochondrial function is detrimental, partial suppression of the ETC has been shown to extend C. elegans lifespan10–13. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit beta and is dependent on the target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased upon starvation and α-KG does not extend the lifespan of DR animals, indicating that α-KG is a key metabolite that mediates longevity by DR. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator, and DR in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
doi:10.1038/nature13264
PMCID: PMC4263271  PMID: 24828042
13.  Force Field for Peptides and Proteins based on the Classical Drude Oscillator 
Presented is a polarizable force field based on a classical Drude oscillator framework, currently implemented in the programs CHARMM and NAMD, for modeling and molecular dynamics (MD) simulation studies of peptides and proteins. Building upon parameters for model compounds representative of the functional groups in proteins, the development of the force field focused on the optimization of the parameters for the polypeptide backbone and the connectivity between the backbone and side chains. Optimization of the backbone electrostatic parameters targeted quantum mechanical conformational energies, interactions with water, molecular dipole moments and polarizabilities and experimental condensed phase data for short polypeptides such as (Ala)5. Additional optimization of the backbone φ, ψ conformational preferences included adjustments of the tabulated two-dimensional spline function through the CMAP term. Validation of the model included simulations of a collection of peptides and proteins. This 1st generation polarizable model is shown to maintain the folded state of the studied systems on the 100 ns timescale in explicit solvent MD simulations. The Drude model typically yields larger RMS differences as compared to the additive CHARMM36 force field (C36) and shows additional flexibility as compared to the additive model. Comparison with NMR chemical shift data shows a small degradation of the polarizable model with respect to the additive, though the level of agreement may be considered satisfactory, while for residues shown to have significantly underestimated S2 order parameters in the additive model, improvements are calculated with the polarizable model. Analysis of dipole moments associated with the peptide backbone and tryptophan side chains show the Drude model to have significantly larger values than those present in C36, with the dipole moments of the peptide backbone enhanced to a greater extent in sheets versus helices and the dipoles of individual moieties observed to undergo significant variations during the MD simulations. Although there are still some limitations, the presented model, termed Drude-2013, is anticipated to yield a molecular picture of peptide and protein structure and function that will be of increased physical validity and internal consistency in a computationally accessible fashion.
doi:10.1021/ct400781b
PMCID: PMC3896220  PMID: 24459460
14.  Vibration-Induced Nystagmus in Patients with Unilateral Peripheral Vestibular Disorders 
This study was done to clarify the clinical significance of vibration-induced nystagmus (VIN) and to calculate the sensitivity and the specificity of the vibration test. One hundred and twelve patients with unilateral peripheral vestibular disorders and thirty normal subjects were enrolled into this study. However, patients with spontaneous nystagmus were excluded. Vibration stimuli (approximately 100 Hz) were presented to the mastoids and the forehead. Patients and normal subjects also underwent head shaking nystagmus (HSN) test and caloric testing. Among the 112 patients, 91(81 %) showed VIN which were mainly horizontal. VIN was more frequently elicited on the mastoids than on the forehead. In the majority of patients (76 cases), the direction of VIN was toward the healthy side, whereas patients with Meniere’s disease (15 cases), showed nystagmus toward the affected side. None of 30 normal subjects showed VIN. Whereas HSN was found in 70 (63 %) patients and 9 (30 %) in normal subjects. Among 112 patients, 10 showed a canal paresis (CP) value of caloric testing less than 25 %, while 32 with a CP value between 25 and 40 %, 48 with a CP value between 40 and 70 %, and 22 with a CP value no less than 70 %. It is notable that with increasing CP value on caloric testing, VIN was more likely to be elicited. So VIN test is a simple, non-invasive and well-tolerated clinical test that indicates unilateral peripheral vestibular dysfunction. VIN test had greater sensitivity and specificity than HSN test in the diagnosis of unilateral peripheral vestibular disorders.
doi:10.1007/s12070-013-0638-6
PMCID: PMC3851498  PMID: 24427594
Vibration-induced nystagmus (VIN); Caloric testing; Head shaking nystagmus (HSN); Unilateral vestibular dysfunction
15.  The immunoglobulin heavy chain VH6-1 promoter regulates Ig transcription in non-B cells 
Cancer Cell International  2014;14(1):114.
Background
Non-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.
Methods
The promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.
Results
Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.
Conclusion
The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.
doi:10.1186/s12935-014-0114-8
PMCID: PMC4260249  PMID: 25493072
VH6-1; Promoter activity; Oct-1; Transcriptional regulation; Non-B cells
16.  Angiogenesis effect of therapeutic ultrasound on ischemic hind limb in mice 
Although significant progress in bypass surgery and catheter intervention against peripheral artery disease, the number of severe critical limb ischemia (CLI) patients is increasing. Thus, it is crucial to develop new, non-invasive therapeutic strategies. The purpose of this study was to determine the mechanism of therapeutic ultrasound (TUS) on ischemic angiogenesis using mouse model of hindlimb ischemia and the cellular/molecular mechanisms underlying TUS-related neovascularization. The hindlimb ischemic mice were exposed to extracorporeal TUS for 3, 6, 9 minute per day (1 MHz, 0.3 W/cm2) until day 14 after left femoral artery ligation. Increased blood perfusion and capillary density were determined following 9 min of TUS compared with ischemic group. Moreover, TUS treatment increased the protein levels of vascular endothelial growth factor (VEGF), hypoxic inducible factor-1α (HIF-1α), endothelial nitric oxide synthase (eNOS) and p-Akt in vivo. TUS promoted capillary-like tube formation, migration and motility of human umbilical venous endothelial cells (HUVECs). Furthermore, the protein expressions of VEGF, eNOS and p-Akt were increased after TUS treatment. In conclusion, TUS therapy promotes postnatal neovascularization through multiple angiogenic pathways in mice model of ischemic hindlimb.
PMCID: PMC4297338  PMID: 25628781
Therapeutic ultrasound; angiogenesis; critical limb ischemia; VEGF
17.  Palm-Vein Classification Based on Principal Orientation Features 
PLoS ONE  2014;9(11):e112429.
Personal recognition using palm–vein patterns has emerged as a promising alternative for human recognition because of its uniqueness, stability, live body identification, flexibility, and difficulty to cheat. With the expanding application of palm–vein pattern recognition, the corresponding growth of the database has resulted in a long response time. To shorten the response time of identification, this paper proposes a simple and useful classification for palm–vein identification based on principal direction features. In the registration process, the Gaussian-Radon transform is adopted to extract the orientation matrix and then compute the principal direction of a palm–vein image based on the orientation matrix. The database can be classified into six bins based on the value of the principal direction. In the identification process, the principal direction of the test sample is first extracted to ascertain the corresponding bin. One-by-one matching with the training samples is then performed in the bin. To improve recognition efficiency while maintaining better recognition accuracy, two neighborhood bins of the corresponding bin are continuously searched to identify the input palm–vein image. Evaluation experiments are conducted on three different databases, namely, PolyU, CASIA, and the database of this study. Experimental results show that the searching range of one test sample in PolyU, CASIA and our database by the proposed method for palm–vein identification can be reduced to 14.29%, 14.50%, and 14.28%, with retrieval accuracy of 96.67%, 96.00%, and 97.71%, respectively. With 10,000 training samples in the database, the execution time of the identification process by the traditional method is 18.56 s, while that by the proposed approach is 3.16 s. The experimental results confirm that the proposed approach is more efficient than the traditional method, especially for a large database.
doi:10.1371/journal.pone.0112429
PMCID: PMC4226569  PMID: 25383715
18.  The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks 
Molecular cell  2013;52(3):10.1016/j.molcel.2013.09.021.
SUMMARY
The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single molecule technique for visualizing encounters of replication forks with ICLs, as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs, and that these lesions are not always absolute blocks to replication.
doi:10.1016/j.molcel.2013.09.021
PMCID: PMC3880019  PMID: 24207054
19.  Low-Molecular-Weight Heparin and Unfractionated Heparin Decrease Th-1, 2, and 17 Expressions 
PLoS ONE  2014;9(11):e109996.
Background
We evaluated the effects of T helper cell differentiation in a mite-allergic animal model treated with inhaled heparins of different molecular weight.
Method
BALB/c mice were divided into four groups: 1. Control, 2. Mite intratracheal (mIT), 3. Inhaled heparin (hIN), 4. Inhaled low-molecular-weight heparin (lmwhIN). Groups 2, 3, and 4 were sensitized twice with Der p allergen subcutaneously on day 1 and day 8. Der p allergen was administered intratracheally on day 15. Groups 3 and 4 were treated with heparin or low-molecular-weight (lmw) heparin intranasally from day 1 to 22. Splenocytes from sacrificed mice stimulated with 16 µg/ml of Der p were cultured for 72 hours. Supernatants of splenocyte were collected to analyze the effect of Interleukin (IL)17-A/F, Interferon(IFN)-γ, IL-4, IL-13, and IL-10. Serum was also collected for Der P-specific IgE level on day 23. Total RNA was extracted from spleen tissue for mRNA expression. Gene expression of Foxp3, IL-10 IFN-γ, GATA3, IL-5, and RORγt were analyzed.
Results
Both hIN and lmwhIN groups had lower serum IgE level than that of the mIT group (both p<0.0001). Both hIN and lmwhIN groups showed significantly decreased transcripts of GATA-3, IFN-γ, IL-5, and RORγt mRNA in their spleen. Regarding the supernatant of splenocyte culture stimulated with Der p, compared with the mIT group, there were significant decreases in IL-17A/F, IFN-γ, IL-4, IL-13, and IL-10 secretion in inhaled hIN and lmwhIN groups.
Conclusions
From this balb/c mice study, the analyses of mRNA and cytokines revealed that both intranasal heparin and lmw heparin treatment decreased the expression of Th1, Th2, and Th17 in spleen. The underlying mechanism(s) warrant further studies.
doi:10.1371/journal.pone.0109996
PMCID: PMC4217718  PMID: 25364825
20.  Multiple climate cooling prior to Sturtian glaciations: Evidence from chemical index of alteration of sediments in South China 
Scientific Reports  2014;4:6868.
Investigation of climatic conditions prior to the Sturtian glaciations is critical to understanding the trigger mechanism for the series of Neoproterozoic global glaciations. In this study, we report high-resolution chemical index of alteration (CIA) records in the sediments of South China prior to the Sturtian glaciation (820~720 Ma). Our results showed there occurred multiple climate cooling before the Sturtian glaciations in South China: (1) a series of episodic and possibly global climate cooling periods from ca. 750 Ma to 725 Ma, which also caused some diachronous regional glaciations; (2) a permanent climate cooling period between ca. 800 Ma and 770 Ma, probably contemporaneous to the global “Bitter Springs stage” δ13C negative excursion; (3) a climate cooling period between ca. 815 Ma and 810 Ma. The three stages of climate cooling are also supported by their correspondence to previously reported extremely low δ18O records of igneous/metamorphic minerals from South China. These climate cooling periods also coincide with the magmatism and rifting events in South China. We argue that tectonic movements were the primary control on the climate cooling before the Neoproterozoic global glaciations.
doi:10.1038/srep06868
PMCID: PMC4215299  PMID: 25359610
21.  cDNA-AFLP analysis reveals the adaptive responses of citrus to long-term boron-toxicity 
BMC Plant Biology  2014;14(1):284.
Background
Boron (B)-toxicity is an important disorder in agricultural regions across the world. Seedlings of ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) were fertigated every other day until drip with 10 μM (control) or 400 μM (B-toxic) H3BO3 in a complete nutrient solution for 15 weeks. The aims of this study were to elucidate the adaptive mechanisms of citrus plants to B-toxicity and to identify B-tolerant genes.
Results
B-toxicity-induced changes in seedlings growth, leaf CO2 assimilation, pigments, total soluble protein, malondialdehyde (MDA) and phosphorus were less pronounced in C. sinensis than in C. grandis. B concentration was higher in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. Here we successfully used cDNA-AFLP to isolate 67 up-regulated and 65 down-regulated transcript-derived fragments (TDFs) from B-toxic C. grandis leaves, whilst only 31 up-regulated and 37 down-regulated TDFs from B-toxic C. sinensis ones, demonstrating that gene expression is less affected in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. These differentially expressed TDFs were related to signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein and amino acid metabolism, lipid metabolism, cell wall and cytoskeleton modification, stress responses and cell transport. The higher B-tolerance of C. sinensis might be related to the findings that B-toxic C. sinensis leaves had higher expression levels of genes involved in photosynthesis, which might contribute to the higher photosyntheis and light utilization and less excess light energy, and in reactive oxygen species (ROS) scavenging compared to B-toxic C. grandis leaves, thus preventing them from photo-oxidative damage. In addition, B-toxicity-induced alteration in the expression levels of genes encoding inorganic pyrophosphatase 1, AT4G01850 and methionine synthase differed between the two species, which might play a role in the B-tolerance of C. sinensis.
Conclusions
C. sinensis leaves could tolerate higher level of B than C. grandis ones, thus improving the B-tolerance of C. sinensis plants. Our findings reveal some novel mechanisms on the tolerance of plants to B-toxicity at the gene expression level.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0284-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0284-5
PMCID: PMC4219002  PMID: 25348611
Boron-tolerance; Boron-toxicity; cDNA-AFLP; Citrus grandis; Citrus sinensis; Photosynthesis
22.  Adoptive Transfer of Heme Oxygenase-1 (HO-1)–Modified Macrophages Rescues the Nuclear Factor Erythroid 2–Related Factor (Nrf2) Antiinflammatory Phenotype in Liver Ischemia/Reperfusion Injury 
Molecular Medicine  2014;20(1):448-455.
Macrophages are instrumental in the pathophysiology of liver ischemia/reperfusion injury (IRI). Although Nrf2 regulates macrophage-specific heme oxygenase-1 (HO-1) antioxidant defense, it remains unknown whether HO-1 induction might rescue macrophage Nrf2-dependent antiinflammatory functions. This study explores the mechanisms by which the Nrf2–HO-1 axis regulates sterile hepatic inflammation responses after adoptive transfer of ex vivo modified HO-1 overexpressing bone marrow–derived macrophages (BMMs). Livers in Nrf2-deficient mice preconditioned with Ad-HO-1 BMMs, but not Ad-β-Gal-BMMs, ameliorated liver IRI (at 6 h of reperfusion after 90 min of warm ischemia), evidenced by improved hepatocellular function (serum alanine aminotransferase [sALT] levels) and preserved hepatic architecture (Suzuki histological score). Treatment with Ad-HO-1 BMMs decreased neutrophil accumulation, proinflammatory mediators and hepatocellular necrosis/apoptosis in ischemic livers. Moreover, Ad-HO-1 transfection of Nrf2-deficient BMMs suppressed M1 (Nos2+) while promoting the M2 (Mrc-1/Arg-1+) phenotype. Unlike in controls, Ad-HO-1 BMMs increased the expression of Notch1, Hes1, phosphorylation of Stat3 and Akt in IR-stressed Nrf2-deficient livers as well as in lipopolysaccharide (LPS)-stimulated BMMs. Thus, adoptive transfer of ex vivo generated Ad-HO-1 BMMs rescued Nrf2-dependent antiinflammatory phenotype by promoting Notch1/Hes1/Stat3 signaling and reprogramming macrophages toward the M2 phenotype. These findings provide the rationale for a novel clinically attractive strategy to manage IR liver inflammation/damage.
doi:10.2119/molmed.2014.00103
PMCID: PMC4212016  PMID: 25014792
23.  miR-21 and miR-375 microRNAs as candidate diagnostic biomarkers in squamous cell carcinoma of the larynx: association with patient survival 
The dismal outcome of laryngeal squamous cell carcinoma (SCC) patients highlights the need for novel prognostic biomarkers. The involvement of microRNAs in cancer and their potential as biomarkers of diagnosis and prognosis are becoming increasingly appreciated. We sought to identify microRNAs that exhibit altered expression in laryngeal SCC and to determine whether microRNA (miRNA) expression is predictive of disease progression and/or patient survival. The expression of two miRNAs, miR-21 and miR-375, was evaluated using total RNA isolated from freshly-frozen primary tumors and non-cancerous laryngeal squamous epithelial tissues and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. We further analyzed the association between the expression of miRNAs and the clinicopathological features. A marked difference in the microRNA expression pattern was observed between tumors and non-cancerous tissue. MiR-21 and miR-375 were expressed at higher and lower levels, respectively, in the laryngeal SCC samples, compared to the normal samples (p < 0.01 and p < 0.001, respectively). There was no correlation between characteristics such as age, sex, clinical stage, and alcohol use, and the expression level of mir-21. The relative expression of mir-375 in laryngeal SCC was shown to be associated with localization of the tumor in these patients (p = 0.037) and with alcohol use (p < 0.05). Patients with high miR-21 or low miR-375 expression in tumor tissues had poorer prognoses compared to patients with lower miR-21 or higher miR-375 expression. Furthermore, the miR-21/miR-375 expression ratio was highly sensitive (0.94) and specific (0.94) for disease prediction. These data suggest that the pattern of microRNA expression in primary laryngeal SCC tissues is reflective of the disease status and that miR-21 and miR-375 expression levels, in particular, may serve as potential biomarkers with applications in the clinical setting.
PMCID: PMC4212934  PMID: 25360224
Laryngeal squamous cell carcinoma; microRNA; biomarker; prognosis
24.  Transport and Metabolism Behavior of Brazilein during Its Entrance into Neural Cells 
PLoS ONE  2014;9(10):e108000.
Brazilein, a natural small molecule, shows a variety of pharmacological activities, especially on nervous system and immune system. As a potential multifunctional drug, we studied the distribution and the transport behavior and metabolic behavior of brazilein in vivo and in vitro. Brazilein was found to be able to distribute in the mouse brain and transport into neural cells. A metabolite was found in the brain and in the cells. Positive and negative mode-MS/MS and Q-TOF were used to identify the metabolite. MS/MS fragmentation mechanisms showed the methylation occurred at the10-hydroxyl of brazilein (10-O-methylbrazilein). Further, catechol-O- methyltransferase (COMT) was confirmed as a crucial enzyme correlated with the methylated metabolite generation by molecular docking and pharmacological experiment.
doi:10.1371/journal.pone.0108000
PMCID: PMC4183444  PMID: 25275506
25.  Iterative Reconstruction for X-Ray Computed Tomography using Prior-Image Induced Nonlocal Regularization 
Repeated x-ray computed tomography (CT) scans are often required in several specific applications such as perfusion imaging, image-guided biopsy needle, image-guided intervention, and radiotherapy with noticeable benefits. However, the associated cumulative radiation dose significantly increases as comparison with that used in the conventional CT scan, which has raised major concerns in patients. In this study, to realize radiation dose reduction by reducing the x-ray tube current and exposure time (mAs) in repeated CT scans, we propose a prior-image induced nonlocal (PINL) regularization for statistical iterative reconstruction via the penalized weighted least-squares (PWLS) criteria, which we refer to as “PWLS-PINL”. Specifically, the PINL regularization utilizes the redundant information in the prior image and the weighted least-squares term considers a data-dependent variance estimation, aiming to improve current low-dose image quality. Subsequently, a modified iterative successive over-relaxation algorithm is adopted to optimize the associative objective function. Experimental results on both phantom and patient data show that the present PWLS-PINL method can achieve promising gains over the other existing methods in terms of the noise reduction, low-contrast object detection and edge detail preservation.
doi:10.1109/TBME.2013.2287244
PMCID: PMC3999310  PMID: 24235272
X-ray computed tomography; prior image; statistical iterative reconstruction; penalized weighted least-squares; regularization

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