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1.  Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris 
PLoS ONE  2015;10(7):e0131757.
The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.
doi:10.1371/journal.pone.0131757
PMCID: PMC4489709  PMID: 26134129
4.  Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4 signalling pathway to alleviate microglial activation 
Scientific Reports  2015;5:11445.
Excessive microglial activation often contributes to inflammation-mediated neurotoxicity in the ischemic penumbra during the acute stage of ischemic stroke. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation via the NF-κB pathway. Isoflurane preconditioning (IP) can provide neuroprotection and inhibit microglial activation. In this study, we investigated the roles of the TLR4 signalling pathway in IP to exert neuroprotection following ischemic stroke in vivo and in vitro. The results showed that 2% IP alleviated neurological deficits, reduced the infarct volume, attenuated apoptosis and weakened microglial activation in the ischemic penumbra. Furthermore, IP down-regulated the expression of HSP 60, TLR4 and MyD88 and up-regulated inhibitor of IκB-α expression compared with I/R group in vivo. In vitro, 2% IP and a specific inhibitor of TLR4, CLI-095, down-regulated the expression of TLR4, MyD88, IL-1β, TNF-α and Bax, and up-regulated IκB-α and Bcl-2 expression compared with OGD group. Moreover, IP and CLI-095 attenuated microglial activation-induced neuronal apoptosis, and overexpression of the TLR4 gene reversed the neuroprotective effects of IP. In conclusion, IP provided neuroprotection by regulating TLR4 expression directly, alleviating microglial activation and neuroinflammation. Thus, inhibiting the activation of microglial activation via TLR4 may be a new avenue for stroke treatment.
doi:10.1038/srep11445
PMCID: PMC4471883  PMID: 26086415
5.  Atrophic Patterns of the Frontal-Subcortical Circuits in Patients with Mild Cognitive Impairment and Alzheimer’s Disease 
PLoS ONE  2015;10(6):e0130017.
Atrophy of the cortical thickness and gray matter volume are regarded as sensitive markers for the early clinical diagnosis of Alzheimer’s disease (AD). This study aimed to investigate differences in atrophy patterns in the frontal-subcortical circuits between MCI and AD, assess whether these differences were essential for the pathologic basis of cognitive impairment. A total of 131 individuals were recruited, including 45 with cognitively normal controls (CN), 46 with MCI, and 40 with AD. FreeSurfer software was used to perform volumetric measurements of the frontal-subcortical circuits from 3.0T magnetic resonance (MR) scans. Data revealed that both MCI and AD subjects had a thinner cortex in the left caudal middle frontal gyrus and the left lateral orbitofrontal gyrus compared with CN individuals. The left lateral orbitofrontal gyrus was also thinner in AD compared with MCI patients. There were no statistically significant differences in the cortical mean curvature among the three groups. Both MCI and AD subjects exhibited smaller bilateral hippocampus volumes compared with CN individuals. The volumes of the bilateral hippocampus and the right putamen were also smaller in AD compared with MCI patients. Logistic regression analyses revealed that the left lateral orbitofrontal gyrus and bilateral hippocampus were risk factors for cognitive impairment. These current results suggest that atrophy was heterogeneous in subregions of the frontal-subcortical circuits in MCI and AD patients. Among these subregions, the reduced thickness of the left lateral orbitofrontal and the smaller volume of the bilateral hippocampus seemed to be markers for predicting cognitive impairment.
doi:10.1371/journal.pone.0130017
PMCID: PMC4466229  PMID: 26066658
6.  Chemical synthesis of a two-photon-activatable chemokine and photon-guided lymphocyte migration in vivo 
Nature Communications  2015;6:7220.
Chemokine-guided lymphocyte positioning in tissues is crucial for normal operation of the immune system. Direct, real-time manipulation and measurement of single-cell responses to chemokines is highly desired for investigating the cell biology of lymphocyte migration in vivo. Here we report the development of the first two-photon-activatable chemokine CCL5 through efficient one-pot total chemical synthesis in milligram scale. By spatiotemporally controlled photoactivation, we show at the single-cell level that T cells perceive the directional cue without relying on PI3K activities, which are nonetheless required for persistent migration over an extended period of time. By intravital imaging, we demonstrate artificial T-cell positioning in cutaneous tissues and lymph nodes. This work establishes a general strategy to develop high-quality photo-activatable protein agents through tailor-designed caging of multiple residues and highlights the potential of photo-activatable chemokines for understanding and potential therapeutic manipulation of cell positioning and position-controlled cell behaviours in vivo.
The precise spatiotemporal control of chemokine exposure would be an advantageous tool for immune cell research. Here, Chen et al. develop a two-photon-activatable chemokine CCL5 and use it to direct lymphocyte migration in vivo and to show that PI3-kinase is not required to sense a gradient in vitro.
doi:10.1038/ncomms8220
PMCID: PMC4455097  PMID: 26008852
7.  Exome sequencing reveals HINT1 mutations as a cause of distal hereditary motor neuropathy 
Distal hereditary motor neuropathies (dHMNs) are a heterogenous group of genetic disorders with length-dependent degeneration of motor axons. Obtaining a genetic diagnosis in patients with dHMN remains challenging. We performed exome sequencing in a diagnostic setting in 12 patients with a clinical diagnosis of dHMN. Potential disease-causing variants in genes associated with dHMN and other forms of inherited neuropathies/motor neuron diseases were validated using Sequenom. The coverage in the genes studied was >95% with an average coverage of >50 times. In none of the patients a mutations was found in genes previously reported to be associated with dHMN. However, in 2/12 patients a recessive mutation in histidine triad nucleotide binding protein 1 (HINT1, recently discovered as a cause of axonal neuropathy with neuromyotonia) was identified. Our results demonstrate the diagnostic value of exome sequencing for patients with inherited neuropathies. The phenotypic spectrum of recessive mutations in HINT1 includes dHMN. HINT1 should be added to the list of genes to check for in dHMN.
doi:10.1038/ejhg.2013.231
PMCID: PMC4023208  PMID: 24105373
distal hereditary motor neuropathies; exome sequencing; HINT1
8.  Associations Between Serum-Intact Parathyroid Hormone, Serum 25-Hydroxyvitamin D, Oral Vitamin D Analogs and Metabolic Syndrome in Peritoneal Dialysis Patients: A Multi-Center Cross-Sectional Study 
♦ Introduction: Although previous studies have suggested associations between serum intact parathyroid hormone (iPTH), 25-hydroxyvitamin D (25(OH)D) and metabolic syndrome (MS) in the general population, these associations are still uncharacterized in peritoneal dialysis (PD) patients.
♦ Methods: In total, 837 prevalent PD patients from 5 centers in China were enrolled between April 1, 2011 and November 1, 2011. The demographic data, biochemical parameters and medical records were collected, except for serum 25(OH)D which was measured in 347 of 837 patients. The definition of MS was modified from National Cholesterol Education Program Third Adult Treatment Panel (NCEP-ATPIII).
♦ Results: 55.4% of 837 patients were found to have MS. The median concentration of iPTH, 25(OH)D and doses of oral vitamin D analogs for participants with MS was significantly lower than those without MS. The iPTH, 25(OH)D values and doses of vitamin D analogs were all associated with one or more components of MS. After multivariate adjustment, low serum iPTH values and oral vitamin D analogs, rather than serum 25(OH)D, were significantly associated with the presence of MS, abnormal fasting blood glucose (FBG) and high-density lipoprotein cholesterol (HDL-C). Compared to iPTH < 130pg/mL, iPTH 130-585 pg/mL and > 585pg/mL were associated with a lower risk of MS with adjusted odds ratio (OR) of 0.59 and 0.33, respectively. Taking vitamin D analogs was also associated with a lower risk of MS with adjusted OR of 0.55.
♦ Conclusions: Serum iPTH and the use of active vitamin D supplements rather than serum 25(OH)D were independently associated with the presence of MS in patients on PD.
doi:10.3747/pdi.2013.00001
PMCID: PMC4079492  PMID: 24497582
Metabolic syndrome; parathyroid hormone; peritoneal dialysis; vitamin D
9.  A novel lead compound CM-118 
Cancer Biology & Therapy  2014;15(6):721-734.
The anaplastic lymphoma kinase (ALK) and the c-Met receptor tyrosine kinase play essential roles in the pathogenesis in multiple human cancers and present emerging targets for cancer treatment. Here, we describe CM-118, a novel lead compound displaying low nanomolar biochemical potency against both ALK and c-Met with selectivity over >90 human kinases. CM-118 potently abrogated hepatocyte growth factor (HGF)-induced c-Met phosphorylation and cell migration, phosphorylation of ALK, EML4-ALK, and ALK resistance mutants in transfected cells. CM-118 inhibited proliferation and/or induced apoptosis in multiple c-Met- and ALK-addicted cancer lines with dose response profile correlating target blockade. We show that the CM-118-induced apoptosis in c-Met-amplified H1993 NSCLC cells involved a rapid suppression of c-Met activity and c-Met-to-EGFR cross-talk, and was profoundly potentiated by EGFR inhibitors as shown by the increased levels of apoptotic proteins cleaved-PARP and Bim as well as reduction of the survival protein Mcl-1. Bim-knockdown or Mcl-1 overexpression each significantly attenuated apoptosis. We also revealed a key role by mTOR in mediating CM-118 action against the EML4-ALK-dependent NSCLC cells. Abrogation of EML4-ALK in H2228 cells profoundly reduced signaling capacity of the rapamycin-sensitive mTOR pathway leading to G1 cell cycle arrest and mitochondrial hyperpolarization, a metabolic perturbation linked to mTOR inhibition. Depletion of mTOR or mTORC1 inhibited H2228 cell growth, and mTOR inhibitors potentiated CM-118’s antitumor activity in vitro and in vivo. Oral administration of CM-118 at a wide range of well tolerated dosages diminished c-Met- and ALK phosphorylation in vivo, and caused tumor regression or growth inhibition in multiple c-Met- and ALK-dependent tumor xenografts in mice. CM-118 exhibits favorable pharmacokinetic and drug metabolism properties hence presents a candidate for clinical evaluation.
doi:10.4161/cbt.28409
PMCID: PMC4049788  PMID: 24618813
c-Met; ALK; EGFR; mTOR; targeted cancer therapy; combination therapy
10.  Molecular cloning, characterization and expression of the energy homeostasis-associated gene in piglet*  
The energy homeostasis-associated (Enho) gene encodes a secreted protein, adropin, which regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor γ, a major regulator of lipogenesis. In the present study, the porcine (Sus scrofa) homologue of the Enho gene, which was named pEnho, was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The gene sequence was submitted into the GenBank of NCBI, and the access number is GQ414763. The pEnho encodes a protein of 76 amino acids which shows 75% similarity to Homo sapiens adropin. The expression profile of pEnho in tissues (liver, muscle, anterior jejunum, posterior jejunum, and ileum) was determined by quantitative real-time RT-PCR. pEnho was localized on porcine chromosome 10 and no introns were found. In conclusion, pEnho was cloned and analysed with the aim of increasing knowledge about glucose and lipid metabolism in piglets and helping to promote the health and growth of piglets through adropin regulation.
doi:10.1631/jzus.B1400260
PMCID: PMC4471604  PMID: 26055914
Adropin; Energy homeostasis-associated (Enho) gene; Gene coloning; Piglets; RT-PCR
11.  Ranitidine reduced levodopa-induced dyskinesia by remodeling neurochemical changes in hemiparkinsonian model of rats 
Background
Levodopa (l-dopa) remains the best drug in the treatment of Parkinson’s disease (PD). Unfortunately, long-term l-dopa caused motor complications, one of which is l-dopa-induced dyskinesia (LID). The precise mechanisms of LID are not fully understood. We have previously reported that ranitidine could reduce LID by inhibiting the activity of protein kinase A pathway in a rat model of PD. It is demonstrated that neurotransmitters such as γ-aminobutyric-acid (GABA) and glutamate (Glu) are also involved in the expression of LID. But whether ranitidine could reduce LID by remodeling the neurochemical changes is unknown.
Methods
In the present study, we produced PD rats by injection of 6-hydroxydopamine. Then PD rats were treated with vehicle, l-dopa (6 mg/kg, plus benserazide 12 mg/kg, intraperitoneal [ip]) or l-dopa (6 mg/kg, plus benserazide 12 mg/kg, ip) plus ranitidine (10 mg/kg, oral). Abnormal voluntary movements were adopted to measure the antidyskinetic effect of ranitidine in PD rats. Rotarod tests were used to observe whether ranitidine treatment affects the antiparkinsonian effect of l-dopa. In vivo microdialysis was used to measure nigral GABA and striatal Glu in PD rats.
Results
We found that ranitidine pretreatment reduced abnormal voluntary movements in l-dopa-primed PD rats without affecting the antiparkinsonian effect of l-dopa. In parallel with behavioral improvement, ranitidine pretreatment reduced protein kinase A activity and suppressed the surge of nigral GABA and striatal Glu.
Conclusion
These data indicated that ranitidine could reduce LID by modeling neurochemical changes induced by l-dopa, suggesting a novel mechanism of ranitidine in the treatment of LID.
doi:10.2147/NDT.S80174
PMCID: PMC4455849  PMID: 26064051
ranitidine; Parkinson’s disease; levodopa-induced dyskinesia; PKA; γ-aminobutyric-acid; glutamate
12.  Clinical Values of Elevated Serum Cytokeratin-18 Levels in Hepatitis: A Meta-Analysis 
Hepatitis Monthly  2015;15(5):e25328.
Background:
As an important intermediate filament protein within liver cells, cytokeratin-18 (CK-18) has been confirmed as a potential indicator in various hepatitis progressions.
Objectives:
We sought to clarify the connection between serum CK-18 levels and hepatitis pathogenesis in the present meta-analysis.
Materials and Methods:
With the application of various computerized databases, including PubMed, Embase, Cochrane Library, Google Scholar, Web of Science, China BioMedicine (CBM), China National Knowledge Infrastructure (CNKI), published papers that assessed the relationship between serum CK-18 levels and hepatitis were obtained. The main key words used are “Hepatitis”, “hepatitides”, “Cytokeratin-18”, “Keratin-18” and “CK-18”. Statistical analysis was conducted using the STATA software (version 12.0).
Results:
Eight case-control studies published between 2010 and 2014 were confirmed eligible, according to our selection criteria. The results of the meta-analysis showed that serum levels of CK-18 in hepatitis patients were higher compared to healthy controls (standardized mean difference (SMD) = 3.71, 95%CI: 2.27-5.14, P < 0.001). Subgroup analysis by ethnicity and disease implicated that high serum CK-18 levels might be a risk factor for non-alcoholic steatohepatitis (NASH), chronic hepatitis C (CHC), and chronic hepatitis B (CHB) (all P < 0.05) among Asians (SMD = 2.89, 95%CI: 2.35-3.43, P < 0.001), Africans (SMD = 0.69, 95%CI: 0.12-1.26, P = 0.017), and Caucasians (SMD = 4.86, 95%CI: 1.82-7.89, P = 0.002).
Conclusions:
Serum CK-18 levels in hepatitis patients were higher, compared with healthy controls. Our results revealed the clinical values of CK-18, in combination with other apoptosis markers, in identifying the development of hepatitis.
doi:10.5812/hepatmon.15(5)2015.25328
PMCID: PMC4451272  PMID: 26045704
Cytokeratin-18; Fatty Liver; Hepatitis; Meta-Analysis
13.  Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis 
PLoS ONE  2015;10(5):e0126623.
During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.
doi:10.1371/journal.pone.0126623
PMCID: PMC4438001  PMID: 25993499
14.  Sunitinib for patients with locally advanced or distantly metastatic dermatofibrosarcoma protuberans but resistant to imatinib 
Purpose: This study evaluated the efficacy and adverse effects of Imatinib therapy to advanced Dermatofibrosarcoma protuberan (DFSP) and Sunitinib therapy to advanced Dermatofibrosarcoma protuberan (DFSP) after Imatinib resistance. Methods: We analyzed the efficacy, adverse effects and survival of 95 patients with locally advanced or metastatic DFSP treated by Imatinib between January 2003 and December 2009, and also analyzed the efficacy and adverse effects of 30 patients after Imatinib failure between January 2008 and December 2011. Results: In all 95 patients treated by Imatinib, 16 had complete response (CR, 16.8%), 44 had partial response (PR, 46.3%), 23 had stable disease (SD, 24.2%) and 12 had progressive disease (PD, 12.6%). The DCR (CR+PR+SD) was 87.4%. The median PFS was 23 months and the OS was 40 months. For 30 patients had Sunitinib treatment after Imatinib failure, 2 had CR (6.7%), 10 had PR (33.3%), 12 had SD (40%) and 6 had PD (20%). The disease control rate (DCR=CR+PR+SD) was 73.3%. The progression free survival (PFS) of CR and PR patients were 22 months and 20 months respectively. The PFS of 12 SD was 18 months, and overall survival (OS) was 28 months. And the median PFS and OS of all 30 patients were 19 and 27 months respectively after Sunitinib treatment. Most of the Imatinib-induced adverse effects are of grade 1-2, including nausea, water retention/edema, fatigue, etc. Conclusion: Imatinib has been proven to be effective and well-tolerated in the treatment of locally advanced or inoperable patients with DFSP. After Imatinib failure, Sunitinib therapy showed good clinical efficacy and tolerated adverse effects as a new treatment option for such patients.
PMCID: PMC4509357  PMID: 26221412
Dermatofibrosarcoma protuberan; imatinib; resistance; Sunitinib; efficacy
15.  Low bone mineral density is associated with increased arterial stiffness in participants of a health records based study 
Journal of Thoracic Disease  2015;7(5):790-798.
Aims
Many epidemiological studies have shown that low bone mineral density (BMD) and atherosclerosis appear to be related. However, their precise correlation is not completely understood after full adjustment the shared confounders of atherosclerosis and bone metabolism. The aim of this cross-sectional study was to investigate the relationship between BMD and subclinical atherosclerosis in a healthy Chinese population and the difference in gender.
Methods
The study population consisted of 2,487 subjects (1,467 men, 1,020 women) who participated in health check-up programs and were selected to be free of major diseases which might affect atherosclerosis and bone metabolism. Bone status was assessed by BMD in lumbar spine. The brachial-ankle PWV (baPWV) was assessed as a functional marker of atherosclerosis. The ankle-brachial index (ABI), carotid artery intima-media thickness (CIMT), estimated glomerular filtration rate (eGRF) and microalbuminuria were evaluated as indexes of structural markers of atherosclerosis.
Results
After adjustment for risk factors, significant association was shown between baPWV and BMD in both genders (male: r=−0.084, P=0.035; female: r=−0.088, P=0.014). The correlation was stronger in females than in males, and in females, the correlation was stronger after menopause. Similarly, mean baPWV differed significantly according to the decreased BMD (normal BMD, Osteopenia, Osteoporosis). In contrast, no significant differences were observed for ABI, CIMT, eGFR or microalbuminuria with BMD.
Conclusions
Independent of confounding factors, low BMD is associated with the functional marker of subclinical atherosclerosis (increased baPWV), but not with structural markers (ABI, CIMT, eGFR or microalbuminuria) among healthy females and males.
doi:10.3978/j.issn.2072-1439.2015.04.47
PMCID: PMC4454857  PMID: 26101634
Low bone mineral density (BMD); atherosclerosis; health check-up; arterial stiffness
16.  Immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in healthy Chinese girls and women aged 9 to 45 years 
Human Vaccines & Immunotherapeutics  2014;10(7):1795-1806.
Immunogenicity and safety of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine were evaluated in healthy Chinese females aged 9–45 years in 2 phase IIIB, randomized, controlled trials. Girls aged 9–17 years (ClinicalTrials.gov, NCT00996125) received vaccine (n = 374) or control (n = 376) and women aged 26–45 years (NCT01277042) received vaccine (n = 606) or control (n = 606) at months 0, 1, and 6. The primary objective was to show non-inferiority of anti-HPV-16 and -18 immune responses in initially seronegative subjects at month 7, compared with Chinese women aged 18–25 years enrolled in a separate phase II/III trial (NCT00779766). Secondary objectives were to describe the anti-HPV-16 and -18 immune response, reactogenicity and safety. At month 7, immune responses were non-inferior for girls (9–17 years) vs. young women (18–25 years): the upper limit of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio (women/girls) was below the limit of 2 for both anti-HPV-16 (0.37 [95% CI: 0.32, 0.43]) and anti-HPV-18 (0.42 [0.36, 0.49]). Immune responses at month 7 were also non-inferior for 26–45 year-old women vs. 18–25 year-old women: the upper limit of the 95% CI for the difference in seroconversion (18–25 minus 26–45) was below the limit of 5% for both anti-HPV-16 (0.00% [–1.53, 1.10]) and anti-HPV-18 (0.21% [–1.36, 1.68]). GMTs were 2- to 3-fold higher in girls (9–17 years) as compared with young women (18–25 years). The HPV-16/18 AS04-adjuvanted vaccine had an acceptable safety profile when administered to healthy Chinese females aged 9–45 years.
doi:10.4161/hv.28702
PMCID: PMC4186032  PMID: 25424785
China; HPV-16/18 AS04-adjuvanted vaccine; cervical cancer; female; human papillomavirus; immunogenicity; safety
17.  Transcriptome-Wide Identification of miRNAs and Their Targets from Typha angustifolia by RNA-Seq and Their Response to Cadmium Stress 
PLoS ONE  2015;10(4):e0125462.
MicroRNAs (miRNAs) play important roles in plant responses to environmental stress. In this work, we used high-throughput sequencing to analyze transcriptome and small RNAs (sRNAs) in Typha angustifolia under cadmium (Cd) stress. 57,608,230 raw reads were obtained from deep sequencing of a pooled cDNA library. Sequence assembly and analysis yielded 102,473 unigenes. We subsequently sequenced two sRNA libraries from T. angustifolia with or without Cd exposure respectively. Based on transcriptome data of T. angustifolia, we catalogued and analyzed the sRNAs, resulting in the identification of 114 conserved miRNAs and 41 novel candidate miRNAs in both small RNA libraries. In silico analysis revealed 764 targets for 89 conserved miRNAs and 21 novel miRNAs. Statistical analysis on sequencing reads abundance and experimental validation revealed that 4 conserved and 6 novel miRNAs showed specific expression. Combined with function of target genes, these results suggested that miRNAs might play a role in plant Cd stress response. This study provided the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in T. angustifolia, which provide a framework for further analysis of miRNAs and their role in regulating plant responses to Cd stress.
doi:10.1371/journal.pone.0125462
PMCID: PMC4414455  PMID: 25923807
18.  Heme oxygenase and the immune system in normal and pathological pregnancies 
Normal pregnancy is an immunotolerant state. Many factors, including environmental, socioeconomic, genetic, and immunologic changes by infection and/or other causes of inflammation, may contribute to inter-individual differences resulting in a normal or pathologic pregnancy. In particular, imbalances in the immune system can cause many pregnancy-related diseases, such as infertility, abortions, pre-eclampsia, and preterm labor, which result in maternal/fetal death, prematurity, or small-for-gestational age newborns. New findings imply that myeloid regulatory cells and regulatory T cells (Tregs) may mediate immunotolerance during normal pregnancy. Effector T cells (Teffs) have, in contrast, been implicated to cause adverse pregnancy outcomes. Furthermore, feto-maternal tolerance affects the developing fetus. It has been shown that the Treg/Teff balance affects litter size and adoptive transfer of pregnancy-induced Tregs can prevent fetal rejection in the mouse. Heme oxygenase-1 (HO-1) has a protective role in many conditions through its anti-inflammatory, anti-apoptotic, antioxidative, and anti-proliferative actions. HO-1 is highly expressed in the placenta and plays a role in angiogenesis and placental vascular development and in regulating vascular tone in pregnancy. In addition, HO-1 is a major regulator of immune homeostasis by mediating crosstalk between innate and adaptive immune systems. Moreover, HO-1 can inhibit inflammation-induced phenotypic maturation of immune effector cells and pro-inflammatory cytokine secretion and promote anti-inflammatory cytokine production. HO-1 may also be associated with T-cell activation and can limit immune-based tissue injury by promoting Treg suppression of effector responses. Thus, HO-1 and its byproducts may protect against pregnancy complications by its immunomodulatory effects, and the regulation of HO-1 or its downstream effects has the potential to prevent or treat pregnancy complications and prematurity.
doi:10.3389/fphar.2015.00084
PMCID: PMC4408852  PMID: 25964759
fetus; HO-1; immunomodulation; immunotolerance; newborn; placenta; pregnancy
19.  Mechanistic Insights into Elastin Degradation by Pseudolysin, the Major Virulence Factor of the Opportunistic Pathogen Pseudomonas aeruginosa 
Scientific Reports  2015;5:9936.
Pseudolysin is the most abundant protease secreted by Pseudomonas aeruginosa and is the major extracellular virulence factor of this opportunistic human pathogen. Pseudolysin destroys human tissues by solubilizing elastin. However, the mechanisms by which pseudolysin binds to and degrades elastin remain elusive. In this study, we investigated the mechanism of action of pseudolysin on elastin binding and degradation by biochemical assay, microscopy and site-directed mutagenesis. Pseudolysin bound to bovine elastin fibers and preferred to attack peptide bonds with hydrophobic residues at the P1 and P1’ positions in the hydrophobic domains of elastin. The time-course degradation processes of both bovine elastin fibers and cross-linked human tropoelastin by pseudolysin were further investigated by microscopy. Altogether, the results indicate that elastin degradation by pseudolysin began with the hydrophobic domains on the fiber surface, followed by the progressive disassembly of macroscopic elastin fibers into primary structural elements. Moreover, our site-directed mutational results indicate that five hydrophobic residues in the S1-S1’ sub-sites played key roles in the binding of pseudolysin to elastin. This study sheds lights on the pathogenesis of P. aeruginosa infection.
doi:10.1038/srep09936
PMCID: PMC4407726  PMID: 25905792
20.  Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models 
Oncotarget  2015;6(11):8900-8913.
We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis.
PMCID: PMC4496191  PMID: 25823926
Macrophages; malignant transformation; NF-κB; STAT3; microfluidic chip
21.  Assessment of Renal Function in Patients with Unilateral Ureteral Obstruction Using Whole-Organ Perfusion Imaging with 320-Detector Row Computed Tomography 
PLoS ONE  2015;10(4):e0122454.
Background
Obstructed nephropathy is a common complication of several disease processes. Accurate evaluation of the functional status of the obstructed kidney is important to achieve a good outcome. The purpose of this study was to investigate renal cortical and medullary perfusion changes associated with unilateral ureteral obstruction (UUO) using whole-organ perfusion imaging with 320-detector row computed tomography (CT).
Methodology/Principle Findings
Sixty-four patients with UUO underwent whole-organ CT perfusion imaging. Patients were divided into 3 groups, mild, moderate, and severe, based on hydronephrosis severity. Twenty sex- and age-matched patients without renal disease, who referred to abdominal CT, were chosen as control subjects. Mean cortical and medullary perfusion parameters of obstructed and contralateral kidneys were compared, and mean perfusion ratios between obstructed and contralateral kidneys were calculated and compared. Mean cortical or medullary blood flow (BF) and blood volume (BV) of the obstructed kidneys in the moderate UUO and BF, BV, and clearance (CL) in the severe UUO were significantly lower than those of the contralateral kidneys (p < 0.05). The mean cortical or medullary BF of the obstructed kidney in the moderate UUO, and BF, BV, and CL in the severe UUO were significantly lower than those of the kidneys in control subjects (p < 0.05). Mean cortical or medullary BF of the non-obstructed kidneys in the severe UUO were statistically greater than that of normal kidneys in control subjects (p < 0.05). An inverse correlation was observed between cortical and medullary perfusion ratios and grades of hydronephosis (p < 0.01).
Conclusions/Significance
Perfusion measurements of the whole kidney can be obtained with 320-detector row CT, and estimated perfusion ratios have potential for quantitatively evaluating UUO renal injury grades.
doi:10.1371/journal.pone.0122454
PMCID: PMC4398441  PMID: 25874690
22.  Physiological and Comparative Proteomic Analysis Reveals Different Drought Responses in Roots and Leaves of Drought-Tolerant Wild Wheat (Triticum boeoticum) 
PLoS ONE  2015;10(4):e0121852.
To determine the proteomic-level responses of drought tolerant wild wheat (Triticum boeoticum), physiological and comparative proteomic analyses were conducted using the roots and the leaves of control and short term drought-stressed plants. Drought stress was imposed by transferring hydroponically grown seedlings at the 3-leaf stage into 1/2 Hoagland solution containing 20% PEG-6000 for 48 h. Root and leaf samples were separately collected at 0 (control), 24, and 48 h of drought treatment for analysis. Physiological analysis indicated that abscisic acid (ABA) level was greatly increased in the drought-treated plants, but the increase was greater and more rapid in the leaves than in the roots. The net photosynthetic rate of the wild wheat leaves was significantly decreased under short-term drought stress. The deleterious effects of drought on the studied traits mainly targeted photosynthesis. Comparative proteomic analysis identified 98 and 85 differently changed protein spots (DEPs) (corresponding to 87 and 80 unique proteins, respectively) in the leaves and the roots, respectively, with only 6 mutual unique proteins in the both organs. An impressive 86% of the DEPs were implicated in detoxification and defense, carbon metabolism, amino acid and nitrogen metabolism, proteins metabolism, chaperones, transcription and translation, photosynthesis, nucleotide metabolism, and signal transduction. Further analysis revealed some mutual and tissue-specific responses to short-term drought in the leaves and the roots. The differences of drought-response between the roots and the leaves mainly included that signal sensing and transduction-associated proteins were greatly up-regulated in the roots. Photosynthesis and carbon fixation ability were decreased in the leaves. Glycolysis was down-regulated but PPP pathway enhanced in the roots, resulting in occurrence of complex changes in energy metabolism and establishment of a new homeostasis. Protein metabolism was down-regulated in the roots, but enhanced in the leaves. These results will contribute to the existing knowledge on the complexity of root and leaf protein changes that occur in response to drought, and also provide a framework for further functional studies on the identified proteins.
doi:10.1371/journal.pone.0121852
PMCID: PMC4393031  PMID: 25859656
23.  Association of High Expression of Groβ with Clinical and Pathological Characteristics of Unfavorable Prognosis in Gastrointestinal Stromal Tumors 
Disease Markers  2015;2015:171035.
GROβ (CXCL2) is a chemokine produced by endotoxin-treated macrophages that mediates inflammation and tumor development. However, little is known about GROβ expression in gastrointestinal stromal tumors (GIST) or the relationship between GROβ expression and clinical attributes of GIST. GROβ expression was examined via immunohistochemical staining of 173 GIST samples using tissue microarray. The relationship between GROβ expression and relevant patient and tumor characteristics was assessed, using chi-square tests. Univariate and multivariate analysis was carried out using the Cox regression method. High GROβ cytoplasm staining was detected in 56 (32.4%) specimens; high GROβ nuclear staining was detected in 64 (37.0%) specimens. High GROβ cytoplasm staining was significantly associated with patients' age (P = 0.043) and tumor location (P = 0.014), while high GROβ nucleus staining was significantly associated with mitotic index (P = 0.034), tumor location (P = 0.049), and AFIP-Miettinen risk classification (P = 0.048). Kaplan-Meier survival curves showed GIST patients with low GROβ cytoplasm expression (P = 0.023) and mitotic index < 6 per 50 HPFs (P = 0.026) to have a more favorable prognosis. These findings indicate that GROβ expression correlates with malignant GIST phenotypes and could be an unfavorable prognostic marker in patients with GIST.
doi:10.1155/2015/171035
PMCID: PMC4405288  PMID: 25944970
24.  Material Properties and Constitutive Modeling of Infant Porcine Cerebellum Tissue in Tension at High Strain Rate 
PLoS ONE  2015;10(4):e0123506.
Background
The mechanical characterization of infant porcine cerebellum tissue in tension at high strain rate is crucial for modeling traumatic cerebellum injury, which is in turn helpful for understanding the biomechanics of such injuries suffered in traffic accidents.
Material and Method
In this study, the infant porcine cerebellum tissue was given three loading velocities, ie, 2s-1, 20s-1 and 100s-1 with up to 30% strain to investigate the tensile properties. At least six tensile tests for each strain rate were validly performed. Fung, Gent, Ogden and exponential models were applied to fit the constitutive equations, so as to obtain material parameters from the experimental data.
Results
The Lagrange stress of infant porcine cerebellum tissue in tension appeared to be no more than 3000Pa at each loading velocity. More specifically, the Lagrange stress at 30% strain was (393.7±84.4)Pa, (928.3±56.3)Pa and (2582.4±282.2)Pa at strain rates of 2s-1, 20s-1 and 100s-1, respectively. Fung (0.833≤R2≤0.924), Gent (0.797≤R2≤0.875), Ogden (0.859≤R2≤0.944) and exponential (0.930≤R2≤0.972) models provided excellent fitting to experimental data up to 30% strain.
Conclusions
The infant cerebellum tissue shows a stiffer response with increase of the loading speed, indicating a strong strain-rate sensitivity. This study will enrich the knowledge on the material properties of infant brain tissue, which may augment the biofidelity of finite element model of human pediatric cerebellum.
doi:10.1371/journal.pone.0123506
PMCID: PMC4382295  PMID: 25830545
25.  A Phylogeographic Investigation of African Monkeypox 
Viruses  2015;7(4):2168-2184.
Monkeypox is a zoonotic disease caused by a virus member of the genus Orthopoxvirus and is endemic to Central and Western African countries. Previous work has identified two geographically disjuct clades of monkeypox virus based on the analysis of a few genomes coupled with epidemiological and clinical analyses; however, environmental and geographic causes of this differentiation have not been explored. Here, we expand previous phylogenetic studies by analyzing a larger set of monkeypox virus genomes originating throughout Sub-Saharan Africa to identify possible biogeographic barriers associated with genetic differentiation; and projected ecological niche models onto environmental conditions at three periods in the past to explore the potential role of climate oscillations in the evolution of the two primary clades. Analyses supported the separation of the Congo Basin and West Africa clades; the Congo Basin clade shows much shorter branches, which likely indicate a more recent diversification of isolates within this clade. The area between the Sanaga and Cross Rivers divides the two clades and the Dahomey Gap seems to have also served as a barrier within the West African clade. Contraction of areas with suitable environments for monkeypox virus during the Last Glacial Maximum, suggests that the Congo Basin clade of monkeypox virus experienced a severe bottleneck and has since expanded its geographic range.
doi:10.3390/v7042168
PMCID: PMC4411695  PMID: 25912718
monkeypox; orthopoxvirus; Phylogenetics; Pleistocene; ecological niche model; evolution

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