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1.  Thermo-sensitive hydrogels combined with decellularised matrix deliver bFGF for the functional recovery of rats after a spinal cord injury 
Scientific Reports  2016;6:38332.
Because of the short half-life, either systemic or local administration of bFGF shows significant drawbacks to spinal injury. In this study, an acellular spinal cord scaffold (ASC) was encapsulated in a thermo-sensitive hydrogel to overcome these limitations. The ASC was firstly prepared from the spinal cord of healthy rats and characterized by scanning electronic microscopy and immunohistochemical staining. bFGF could specifically complex with the ASC scaffold via electrostatic or receptor-mediated interactions. The bFGF-ASC complex was further encapsulated into a heparin modified poloxamer (HP) solution to prepare atemperature-sensitive hydrogel (bFGF-ASC-HP). bFGF release from the ASC-HP hydrogel was more slower than that from the bFGF-ASC complex alone. An in vitro cell survival study showed that the bFGF-ASC-HP hydrogel could more effectively promote the proliferation of PC12 cells than a bFGF solution, with an approximate 50% increase in the cell survival rate within 24 h (P < 0.05). Compared with the bFGF solution, bFGF-ASC-HP hydrogel displayed enhanced inhibition of glial scars and obviously improved the functional recovery of the SCI model rat through regeneration of nerve axons and the differentiation of the neural stem cells. In summary, an ASC-HP hydrogel might be a promising carrier to deliver bFGF to an injured spinal cord.
doi:10.1038/srep38332
PMCID: PMC5138609  PMID: 27922061
2.  Different outcomes of never-treated and treated patients with schizophrenia: 14-year follow-up study in rural China 
The British Journal of Psychiatry  2015;207(6):495-500.
Background
The long-term outcome of never-treated patients with schizophrenia is unclear.
Aims
To compare the 14-year outcomes of never-treated and treated patients with schizophrenia and to establish predictors for never being treated.
Method
All participants with schizophrenia (n = 510) in Xinjin, Chengdu, China were identified in an epidemiological investigation of 123 572 people and followed up from 1994 to 2008.
Results
The results showed that there were 30.6%, 25.0% and 20.4% of patients who received no antipsychotic medication in 1994, 2004 and 2008 respectively. Compared with treated patients, those who were never treated in 2008 were significantly older, had significantly fewer family members, had higher rates of homelessness, death from other causes, being unmarried, living alone, being without a caregiver and poor family attitudes. Partial and complete remission in treated patients (57.3%) was significantly higher than that in the never-treated group (29.8%). Predictors of being in the never-treated group in 2008 encompassed baseline never-treated status, being without a caregiver and poor mental health status in 1994.
Conclusions
Many patients with schizophrenia still do not receive antipsychotic medication in rural areas of China. The 14-year follow-up showed that outcomes for the untreated group were worse. Community-based mental healthcare, health insurance and family intervention are crucial for earlier diagnosis, treatment and rehabilitation in the community.
doi:10.1192/bjp.bp.114.157685
PMCID: PMC4664855  PMID: 26382951
3.  Myeloid-derived Suppressor Cells Contribute to Bone Erosion in Collagen-induced Arthritis by Differentiating to Osteoclasts 
Journal of autoimmunity  2015;65:82-89.
Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/J1 mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP+ osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.
doi:10.1016/j.jaut.2015.08.010
PMCID: PMC4679657  PMID: 26318644
MDSC; osteoclast precursor; osteoclast; bone destruction; collagen-induced arthritis and rheumatoid arthritis
4.  Minimally Invasive Cochlear Implantation Assisted by Bi-planar Device: An Exploratory Feasibility Study in vitro 
Chinese Medical Journal  2016;129(20):2476-2483.
Background:
A single drilled tunnel from the lateral mastoid cortex to the cochlea via the facial recess is essential for minimally invasive cochlear implant surgery. This study aimed to explore the safety profile of this kind of new image-guided and bi-planar device-assisted surgery procedure in vitro.
Methods:
Image-guided minimally invasive cochlear implantations were performed on eight cadaveric temporal bone specimens. The main procedures were: (1) temporal bone specimens were prepared for surgery and fiducial markers were registered. (2) computed tomography (CT) scans were performed for future reference. (3) CT scan images were processed and drill path was planned to minimize cochlear damage. (4) bi-planar device-assisted drilling was performed on the specimens using the registration. (5) surgical safety was evaluated by calculating the deviation between the drill and the planned paths, and by measuring the closest distance between the drilled path and critical anatomic structures.
Results:
Eight cases were operated successfully to the basal turn of the cochlear with intact facial nerves (FNs). The deviations from target points and entrance points were 0.86 mm (0.68–1.00 mm) and 0.44 mm (0.30–0.96 mm), respectively. The angular error between the planned and the drilled trajectory was 1.74° (1.26–2.41°). The mean distance from the edge of the drilled path to the FN and to the external canal was 0.60 mm (0.35–0.83 mm) and 1.60 mm (1.30–2.05 mm), respectively. In five specimens, the chorda tympani nerves were well preserved. In all cases, no injury happened to auditory ossicles.
Conclusions:
This exploratory study demonstrated the safety of the newly developed image-guided minimally invasive cochlear implantation assisted by the bi-planar device and established the operational procedures. Further, more in vitro experiments are needed to improve the system operation and its safety.
doi:10.4103/0366-6999.191787
PMCID: PMC5072261  PMID: 27748341
Cochlear Implantation; Minimally Invasive Surgical Procedures; Navigation
5.  Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) Enhances Multidrug Resistance of Hepatocellular Carcinoma Cells 
PLoS ONE  2016;11(10):e0164752.
China accounts for almost half of the total number of liver cancer cases and deaths worldwide, and hepatocellular carcinoma (HCC) is the most primary liver cancer. Snail family transcriptional repressor 2 (SNAI2) is known as an epithelial to mesenchymal transition-inducing transcription factor that drives neoplastic epithelial cells into mesenchymal phenotype. However, the roles of endogenous SNAI2 remain controversial in different types of malignant tumors. Herein, we surprisingly identify that anchorage-independent growth, including the formation of tumor sphere and soft agar colony, is significantly increased when SNAI2 expression is inhibited by shRNAs in HCC cells. Suppression of SNAI2 suffices to up-regulate several cancer stem genes. Although unrelated to the metastatic ability, SNAI2 inhibition does increase the efflux of Hoechst 33342 and enhance multidrug resistance in vitro and in vivo. In agreement with this data, we demonstrate for the first time that decreasing SNAI2 level can transcriptionally upregulate several ATP binding cassette (ABC) transporter genes such as ABCB1. Moreover, ABC transporters’ inhibitor verapamil can rescue the multidrug resistance induced by SNAI2 inhibition. Our results implicate that SNAI2 behaves as a tumor suppressor by inhibiting multidrug resistance via suppressing ABC transporter genes in HCC cells.
doi:10.1371/journal.pone.0164752
PMCID: PMC5070735  PMID: 27760172
6.  Th17 Cells Exhibit Antitumor Effects in MDS Possibly through Augmenting Functions of CD8+ T Cells 
Journal of Immunology Research  2016;2016:9404705.
Th17 cells are a newly found subset of distinct CD4+ Th effector cells' family and are found to play an important role in cancers. Myelodysplastic syndromes (MDS) are a common malignant hematological disease. Here, we showed that both the percentage and the function of Th17 cells were elevated in low-risk MDS while being decreased in high-risk MDS. Levels of upstream molecules of Th17 cells, IL-6 and IL-23, were higher in low-risk MDS but lower in high-risk MDS patients. The abnormal percentage of Th17 cells was closely related to clinical parameters including karyotype, morphologic blast percentage of bone marrow, peripheral absolute neutrophil count, and hemoglobin concentration. Furthermore, expression rates of perforin and granzyme B in BM CD3+CD8+ cells (cytotoxic T lymphocyte, CTL) positively correlated with levels of IL-17 but negatively correlated with BM blast percentage and could be significantly increased after stimulation with human recombinant IL-17 (rhIL-17). Our results suggested that Th17 cells might play an antitumor effect in the pathogenesis of MDS through IL-17/CTL pathway.
doi:10.1155/2016/9404705
PMCID: PMC5046048  PMID: 27722177
7.  HAPLESS13-Mediated Trafficking of STRUBBELIG Is Critical for Ovule Development in Arabidopsis 
PLoS Genetics  2016;12(8):e1006269.
Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis.
Author Summary
Ovules, being the progenitors of seeds, hold an important role in crop production. Ovule is a complex organ connecting two generations, i.e. diploid sporophytic cells and haploid female gametophytes (FG). Fertilization of female gametes leads to the development of embryo and endosperm while the sporophytic cells of ovules become seed coats. Because of its importance for plant reproduction, the development of ovules is tightly controlled. The integument cells are divided in a polar way so that integument cell layers fully enclose a mature FG, leaving an opening only for pollen tube entrance. The integuments also instruct FG development, synchronizing the two generations. Receptor-like kinases, especially STRUBBELIG (SUB), are crucial both for the establishment of ovular polarity and the intercellular communication between sporophytic and gametophytic cells. However, how SUB is post-translationally regulated, as would be expected for cell-surface proteins, is unclear. We report here that the dynamic trafficking of SUB via a subpopulation of endosomes is crucial for its functionality during ovule development. The membrane association of SUB was impaired when the recycling route from the trans-Golgi network/early endosome to the plasma membrane was disrupted in hap13-1, a mutant of HAPLESS13. We further demonstrate that defects of SUB targeting in the outer integuments compromised FG development, suggesting an intriguing intercellular communication. Our results highlight the importance of vesicular trafficking in the establishment of ovular polarity and in the communications between spatially separated cells.
doi:10.1371/journal.pgen.1006269
PMCID: PMC4991792  PMID: 27541731
8.  Comparison of Postoperative Events between Spinal Anesthesia and General Anesthesia in Laparoscopic Cholecystectomy: A Systemic Review and Meta-Analysis of Randomized Controlled Trials 
BioMed Research International  2016;2016:9480539.
Background. Laparoscopic cholecystectomy is usually carried out under general anesthesia. There were a few studies which have found spinal anesthesia as a safe alternative. We aimed to evaluate the postoperative events between spinal anesthesia and general anesthesia in patients undergoing laparoscopic cholecystectomy. Methods. We searched PubMed, Embase, and Cochrane Library (from inception to January 2016) for eligible studies. The primary outcome was the visual analogue scale score. Secondary outcomes included postoperative nausea and vomiting and urine retention 24 hours postoperatively. We calculated pooled risk ratios and 95% confidence interval using random- or fixed-effects models. Results. Eight trials involving 723 patients were listed. Meta-analysis showed that patients in spinal anesthesia groups have lower visual analogue scale score 24 hours postoperatively. There were significant decreases in the occurrence of postoperative nausea and vomiting in spinal anesthesia group when compared with general anesthesia group (odds ratios: 0.38, 95% confidence interval: 0.19–0.76; P = 0.006) with heterogeneity accepted (I2 = 13%; P = 0.33), while urine retention rate was increased in patients with spinal anesthesia (odds ratios: 4.95, 95% confidence interval: 1.24–19.71; P = 0.02) without any heterogeneity (I2 = 0%; P = 0.98). Conclusions. Spinal anesthesia may be associated with less postoperative pain and postoperative nausea and vomiting compared with general anesthesia.
doi:10.1155/2016/9480539
PMCID: PMC4976158  PMID: 27525282
9.  The Relationship between C-Reactive Protein Level and Discharge Outcome in Patients with Acute Ischemic Stroke 
Previous studies showed that C-reactive protein (CRP), an inflammatory marker, was associated with stroke severity and long-term outcome. However, the relationship between the acute-phase CRP level and discharge outcome has received little attention. We prospectively studied 301 patients with acute ischemic stroke (over a period of two weeks) from two hospital stroke wards and one rehabilitation department in Henan, China. Patients’ demographic and clinical data were collected and evaluated at admission. Poor discharge outcome was assessed in patients at discharge using the Modified Rankin Scale (MRS > 2). Multivariate logistic regression analysis was performed to determine the risk factors of poor discharge outcome after adjusting for potential confounders. Poor discharge outcome was observed in 78 patients (25.9%). Univariate analyses showed that factors significantly influencing poor discharge outcome were age, residence, recurrent acute ischemic stroke, coronary heart disease, the National Institutes of Health Stroke Scale (NIHSS) score at admission, non-lacunar stroke, time from onset of stroke to admission, CRP, TBIL (total bilirubin), direct bilirubin (DBIL), ALB (albumin), FIB (fibrinogen) and D-dimer (p < 0.05). After adjusting for age, residence, recurrent ischemic stroke, coronary heart disease, NIHSS score at admission, lacunar stroke, time from onset of stroke to admission, CRP, TBIL, DBIL, ALB, FIB and D-dimer, multivariate logistic regression analyses revealed that poor outcome at discharge was associated with recurrent acute ischemic stroke (OR, 2.115; 95% CI, 1.094–4.087), non-lacunar stroke (OR, 2.943; 95% CI, 1.436–6.032), DBIL (OR, 1.795; 95% CI, 1.311–2.458), and CRP (OR, 4.890; 95% CI, 3.063–7.808). In conclusion, the CRP level measured at admission was found to be an independent predictor of poor outcome at discharge. Recurrent acute ischemic stroke, non-lacunar stroke and DBIL were also significantly associated with discharge outcome in acute ischemic stroke.
doi:10.3390/ijerph13070636
PMCID: PMC4962177  PMID: 27355961
acute ischemic stroke; C-reactive protein; recurrence; discharge outcome
10.  Hollow Cone Electron Imaging for Single Particle 3D Reconstruction of Proteins 
Scientific Reports  2016;6:27701.
The main bottlenecks for high-resolution biological imaging in electron microscopy are radiation sensitivity and low contrast. The phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Recently, phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Single particle cryo-electron microscopy is mostly used to minimize the radiation damage and to enhance the resolution of the 3D reconstructions but it requires averaging images of a massive number of individual particles. Here we present a new route to achieve the same goals by hollow cone dark field imaging using thermal diffuse scattered electrons giving about a 4 times contrast increase as compared to bright field imaging. We demonstrate the 3D reconstruction of a stained GroEL particle can yield about 13.5 Å resolution but using a strongly reduced number of images.
doi:10.1038/srep27701
PMCID: PMC4904375  PMID: 27292544
11.  Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system 
Background
The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis.
Results
Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils.
Conclusions
The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.
doi:10.1186/s12934-016-0503-x
PMCID: PMC4901475  PMID: 27287016
Zymomonas mobilis; Salt stress; Tn5-based transposon mutagenesis; Genome walking; himA
12.  Different Phenotypes Among Lesch-Nyhan Variants: Clinical Reality or Limitation of Ascertainment? 
Archives of neurology  2011;68(2):270-271.
doi:10.1001/archneurol.2010.360
PMCID: PMC4891938  PMID: 21321001
13.  Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages 
Background
NLRP3 inflammasome has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The activation of NLRP3 inflammasome results in the production of IL-1β and the subsequent inflammation. Anti-dsDNA antibodies (anti-dsDNA Abs) play critical roles in the development and progression of SLE. However, the mechanism of NLRP3 inflammasome activation in SLE is still not known. This study investigated the activation of NLRP3 inflammasome stimulated by anti-dsDNA Abs in monocytes/macrophages from SLE patients.
Methods
Monocytes/macrophages from SLE patients or healthy controls were stimulated with anti-dsDNA Ab-positive serum or purified anti-dsDNA Abs. Activation of inflammasome was measured by flow cytometry or Western blot. Anti-dsDNA Abs isolated from active SLE patients were injected into female (NZB × NZW) F1 mice and the activation of NLRP3 inflammasome and the frequencies of Th17 and Treg were examined.
Results
The activity of caspase-1 was significantly increased in active SLE patients and was correlated with serum levels of anti-dsDNA Abs and disease activities. The concentrations of IL-1β and IL-17A were also significantly higher in SLE patients compared to healthy controls. Anti-dsDNA Ab-positive serum rather than healthy serum or RF (rheumatoid factor)-positive serum stimulated the activation of caspase-1 in monocytes. Anti-dsDNA Abs bound to TLR4 on macrophages and induced the production of ROS. Mitochondria-targeting antioxidant Mito-TEMPO, IκB kinase inhibitor peptide or TLR4 siRNA inhibited the activation of NLRP3 inflammasome and the secretion of IL-1β induced by anti-dsDNA Abs. Injection of anti-dsDNA Abs into (NZB × NZW) F1 mice resulted in increased caspase-1 activation and production of IL-1β and IL-17A. The Th17/Treg cell ratio also significantly increased following anti-dsDNA Ab injection.
Conclusions
Anti-dsDNA Abs activated NLRP3 inflammasome in monocytes/macrophages from SLE patients by binding to TLR4 and inducing the production of mitochondrial ROS.
doi:10.1186/s12967-016-0911-z
PMCID: PMC4888250  PMID: 27250627
SLE; Anti-dsDNA antibodies; NLRP3 inflammasome; TLR4; Mitochondrial ROS
14.  Advances of wogonin, an extract from Scutellaria baicalensis, for the treatment of multiple tumors 
OncoTargets and therapy  2016;9:2935-2943.
As the major bioactive compound of Scutellaria baicalensis that has been approved to be effective as an anti-inflammatory and antiviral inhibitor in cardiovascular diseases, wogonin (WG) showed potent and promising antitumor effects both in vitro and in vivo. It has been proved that WG has the ability to inhibit the growth of tumor cells, induce apoptosis, and suppress angiogenesis. The molecular mechanisms involve reactive oxygen species, Ca2+, NF-κB, tumor necrosis factor-related apoptosis-inducing ligand, and tumor necrosis factor-alpha. Furthermore, the synergistic effect of WG with 5-fluorouracil, etoposide, and adriamycin to enhance chemotherapy and reverse drug resistance has also been confirmed. In this review, we summarize the advances in recent years on the antitumor effect of WG on multiple tumors; in addition, we also present information regarding the synergistic and chemosensitizing effects of WG with other drugs to illustrate its potential use in the clinic.
doi:10.2147/OTT.S105586
PMCID: PMC4876109  PMID: 27274287
antitumor effect; drug resistance; mechanisms; synergistic effect
15.  H-Type Hypertension and C Reactive Protein in Recurrence of Ischemic Stroke 
Hypertension with high homocysteine (HHcy) (H-type hypertension) and C reactive protein (CRP) can increase the incidence of ischemic stroke. However, it is not clear whether recurrent ischemic stroke (RIS) is related to H-type hypertension and CRP. The present study investigated the correlation of H-type hypertension and CRP level with RIS. Totally, 987 consecutive patients with acute ischemic stroke were recruited in a teaching hospital in Henan province, China during March 2014 to March 2015. The demographic and clinical characteristics and blood biochemical parameters of patients were analyzed. Elevated levels of CRP and homocysteine (Hcy) were defined as >8.2 mg/L and 10 μmol/L, respectively. Among the 987 patients, 234 were RIS. Thirty-eight percent of RIS patients had elevated CRP level and 91.5% of RIS patients had HHcy. In multivariate analysis, adjusted odds ratio (OR) of RIS in patients aged ≥60 years was 1.576 (95% CI: 1.125–2.207), in male patients 1.935 (95% CI: 1.385–2.704), in patients with diabetes 1.463 (95% CI: 1.037–2.064), CRP levels 1.013 (95% CI: 1.006–1.019), simple hypertension 3.370 (95% CI: 1.15–10.183), and H-type hypertension 2.990 (95% CI: 1.176–7.600). RIS was associated with older age, male, diabetes, H-type hypertension and CRP. Controlling H-type hypertension and CRP level may reduce the risk of RIS.
doi:10.3390/ijerph13050477
PMCID: PMC4881102  PMID: 27164124
C reactive protein; H-type hypertension; recurrence ischemic stroke
16.  Loss of Dopamine Phenotype Among Midbrain Neurons in Lesch–Nyhan Disease 
Annals of neurology  2014;76(1):95-107.
Objective
Lesch–Nyhan disease (LND) is caused by congenital deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Affected patients have a peculiar neurobehavioral syndrome linked with reductions of dopamine in the basal ganglia. The purpose of the current studies was to determine the anatomical basis for the reduced dopamine in human brain specimens collected at autopsy.
Methods
Histopathological studies were conducted using autopsy tissue from 5 LND cases and 6 controls. Specific findings were replicated in brain tissue from an HGprt-deficient knockout mouse using immunoblots, and in a cell model of HGprt deficiency by flow-activated cell sorting (FACS).
Results
Extensive histological studies of the LND brains revealed no signs suggestive of a degenerative process or other consistent abnormalities in any brain region. However, neurons of the substantia nigra from the LND cases showed reduced melanization and reduced immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. In the HGprt-deficient mouse model, immunohistochemical stains for TH revealed no obvious loss of midbrain dopamine neurons, but quantitative immunoblots revealed reduced TH expression in the striatum. Finally, 10 independent HGprt-deficient mouse MN9D neuroblastoma lines showed no signs of impaired viability, but FACS revealed significantly reduced TH immunoreactivity compared to the control parent line.
Interpretation
These results reveal an unusual phenomenon in which the neurochemical phenotype of dopaminergic neurons is not linked with a degenerative process. They suggest an important relationship between purine recycling pathways and the neurochemical integrity of the dopaminergic phenotype.
doi:10.1002/ana.24191
PMCID: PMC4827147  PMID: 24891139
17.  Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase 
Biochemistry  2016;55(10):1485-1493.
Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.
doi:10.1021/acs.biochem.5b01364
PMCID: PMC4806677  PMID: 26901612
18.  Effect of HMGCR genetic variation on neuroimaging biomarkers in healthy, mild cognitive impairment and Alzheimer's disease cohorts 
Oncotarget  2016;7(12):13319-13327.
Alzheimer's disease (AD) has become a considerable public health issue. The mechanisms underlying AD onset and progression remain largely unclear. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is a strong functional AD candidate gene because it encodes part of the statin-binding domain of the enzyme, which serves as the rate-limiting step in cholesterol synthesis in all mammalian cells. Here, we evaluated the potential role of HMGCR (rs3846662) in AD-related pathology by assessing neuroimaging biomarkers. We enrolled in 812 subjects from the Alzheimer's disease Neuroimaging Initiative dataset. In general, it is possible that HMGCR (rs3846662) could be involved in preventing the atrophy of right entorhinal (P=0.03385) and left hippocampus (P=0.01839) in the follow-up research of two years. What's more, it lowered the drop rate of glucose metabolism in right temporal. We then further validated them in the AD, mild cognitive impairment (MCI), normal control (NC) sub-groups. All the results in the MCI groups confirmed the association. The results of our study indicated that HMGCR (rs3846662) plays a vital role in AD pathology mainly by influencing brain structure and glucose metabolism during AD progression.
doi:10.18632/oncotarget.7797
PMCID: PMC4924644  PMID: 26950278
Alzheimer's disease; brain structure; HMGCR; neuroimaging; glucose metabolism; Gerotarget
19.  Expression and function of hematopoiesis-stimulating factor receptors on the GPI− and GPI+ hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome 
Paroxysmal nocturnal hemoglobinuria/aplastic anemia (PNH/AA) syndrome presents a markedly increased population of cells deficient in glycophosphatidylinositol (GPI− cells) and signs of bone marrow failure, which requires treatment with hematopoiesis-stimulating factors, such as granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). However, little is known about the effects of these stimulating factors on GPI− cells. In order to explore the effects of stimulating factors in PNH/AA, G-CSF receptor (CD114) and SCF receptor (CD117) expression levels on GPI+ and GPI− hematopoietic stem cells (HSCs) were measured by flow cytometry (FCM). The mean fluorescence intensity (MFI) values of signal transducer and activator of transcription 5 (STAT5) and phosphorylated (P)-STAT5 were measured in GPI+ and GPI− HSCs by FCM following stimulation with G-CSF or SCF in vitro. The expression levels of CD114 and CD117 on GPI− HSCs were significantly lower (P<0.01) than those on GPI+ HSCs in PNH/AA patients and normal controls. The MFI values of STAT5 in the GPI− and GPI+ HSCs of PNH/AA patients and normal controls were not significantly different. However, the MFI values of P-STAT5 in the GPI− HSCs of PNH/AA patients were significantly lower than those in the GPI+ HSCs of PNH/AA patients and normal controls prior to and following stimulation with G-CSF or SCF (P<0.01). The GPI− HSCs of PNH/AA patients responded poorly to stimulation by hematopoiesis-stimulating factors, which indicates that these factors can be used safely in patients with PNH/AA.
doi:10.3892/etm.2016.3163
PMCID: PMC4840540  PMID: 27168787
hemoglobinuria; paroxysmal; hematopoietic stem cell; anemia; aplastic; CD114; CD117; STAT5
20.  New role and molecular mechanism of Gadd45a in hepatic fibrosis 
World Journal of Gastroenterology  2016;22(9):2779-2788.
AIM: To investigate the role of Gadd45a in hepatic fibrosis and the transforming growth factor (TGF)-β/Smad signaling pathway.
METHODS: Wild-type male BALB/c mice were treated with CCl4 to induce a model of chronic liver injury. Hepatic stellate cells (HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs (siRNAs) targeting Gadd45a or the pcDNA3.1-Gadd45a recombinant plasmid. Cellular α-smooth muscle actin (α-SMA), β-actin, type I collagen, phospho-Smad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The mRNA levels of α-SMA, β-actin, and type I collagen were determined by quantitative real-time (qRT)-PCR analyses. Reactive oxygen species production was monitored by flow cytometry using 2,7-dichlorodihydrofluorescein diacetate. Gadd45a, Gadd45b, anti-Gadd45g, type I collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses.
RESULTS: Significant downregulation of Gadd45a, but not Gadd45b or Gadd45g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl4 treatment. Overexpression of Gadd45a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45a with siRNA reversed this process. Gadd45a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and (CAGA)9 MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes.
CONCLUSION: Gadd45a may counteract hepatic fibrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.
doi:10.3748/wjg.v22.i9.2779
PMCID: PMC4778000  PMID: 26973416
Antioxidant; Gadd45a; Hepatic fibrosis; Hepatic stellate cells; Transforming growth factor-β/Smad signaling
21.  Association study of the PLXNA4 gene with the risk of Alzheimer’s disease 
Background
The Plexin-A 4 (PLXNA4) gene has recently been recognized as a functional candidate gene of late-onset Alzheimer’s disease (LOAD). The single nucleotide polymorphism (SNP) rs13232207 of PLXNA4 gene has been reported to be associated with Alzheimer’s disease (AD) in Japanese cohorts. We sought to clarify whether this novel locus gains the same effect in northern Han Chinese.
Methods
To investigate the relationship between SNP rs13232207 and AD sufferers, a case-control study of unrelated individuals was conducted with a total sample size of 2,318 subjects (978 cases and 1,340 age and gender matched healthy controls) in a Northern Han Chinese population. SPSS 22.0 was applied for the statistical process.
Results
No significant difference in polymorphic distribution of rs13232207 was observed on LOAD risk independently under dominant (P=0.057), additive (P=0.233) or recessive model (P=0.392). In terms of interaction with apolipoprotein E (APOE), there is also no positive interaction in dominant (P=0.438), additive (P=0.055) or recessive model (P=0.095).
Conclusions
Replication of association between the PLXNA4 rs13232207 and AD in a Han ethnic group indicates that this link is not the result of chance.
doi:10.21037/atm.2016.03.23
PMCID: PMC4828739  PMID: 27127761
Plexin-A 4 (PLXNA4); Alzheimer’s disease (AD); polymorphism
22.  Effects of decitabine on megakaryocyte maturation in patients with myelodysplastic syndromes 
Oncology Letters  2016;11(4):2347-2352.
Thrombocytopenia is a common, often fatal complication experienced by patients with myelodysplastic syndromes (MDS). 5-aza-2′-deoxycytidine (decitabine) has been used to treat MDS patients with thrombocytopenia with a response rate of 45–50%. However, the mechanism of its effects on megakaryocytes remains unclear. In the present study, the effect of decitabine on megakaryocyte maturation was investigated. A total of 20 MDS patients diagnosed with thrombocytopenia were enrolled, including 16 refractory anemia with excess blasts (RAEB)-1 patients and 4 RAEB-2 patients], in addition to 20 leukemia patients that had achieved complete remission and 20 healthy donors. Overall, 65% of MDS patients exhibited a response to decitabine, with an increase in platelet count identified in 80% of patients. In the MDS group, the mean platelet count was significantly increased following one cycle of decitabine chemotherapy (36.85±24.54 vs. 84.90±61; P=0.001); however, no significant difference in megakaryocyte number was identified prior to and following treatment. Additionally, bone marrow mononuclear cells of the MDS patients were cultured in vitro with various concentrations of decitabine (0.0, 2.0, 2.5, 3.0 µM), and cluster of differentiation (CD)41 levels were examined via flow cytometry. The MDS and normal control groups exhibited the highest levels of CD41 expression following treatment with 2.0 µM decitabine (mean fluorescence intensity, 294.07±47.34 and 258.95±28.05, respectively). In conclusion, these results indicate that the DNA-hypomethylating agent, decitabine, may induce the differentiation and maturation of myelodysplastic megakaryocytes in MDS patients, even at low concentrations. Thus, the repeated administration of decitabine at lower doses in MDS patients may be useful in clinical practice, and may lead to the development of alternative treatments for other diseases of abnormal megakaryocyte differentiation, such as idiopathic thrombocytopenic purpura, however, future studies are required to investigate this.
doi:10.3892/ol.2016.4259
PMCID: PMC4812569  PMID: 27073478
myelodysplastic syndromes; decitabine; thrombocytopenia
23.  Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats 
PLoS ONE  2016;11(2):e0148848.
Purpose
Intranasal administration of phospholipid-based gelatin nanoparticles (GNP) was prepared to investigate the neuro-recovery effects of neuropeptide Substance P (SP) on hemiparkinsonian rats.
Methods
The SP-loaded gelatin nanoparticles (SP-GNP) were prepared by a water-in-water emulsion method and possessed high stability, encapsulating efficiency and loading capacity. PC-12 cells were used to examine the growth enhancement of SP-GNP in vitro by MTT assays and flow cytometry (FCM). The therapeutic effects of SP-GNP on 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats were assessed by quantifying rotational behavior and the levels of tyrosine hydroxylase (TH), phosphorylated c-Jun protein (p-c-Jun) and Caspase-3 (Cas-3) expressed in substantia nigra (SN) region of hemiparkinsonian rats.
Results
PC-12 cells under SP-GNP treatment showed better cell viability and lower degree of apoptosis than those under SP solution treatment. Hemiparkinsonian rats under intranasal SP-GNP administration demonstrated better behavioral improvement, higher level of TH in SN along with much lower extent of p-c-Jun and Cas-3 than those under intranasal SP solution administration and intravenous SP-GNP administration.
Conclusions
With the advantages of GNP and nose-to-brain pathway, SP can be effectively delivered into the damaged SN region and exhibit its neuro-recovery function through the inhibition on JNK pathway and dopaminergic neuron apoptosis.
doi:10.1371/journal.pone.0148848
PMCID: PMC4760767  PMID: 26894626
24.  Ameliorative effects of glycine in an experimental nonalcoholic steatohepatitis and its correlation between TREM-1 and TREM-2 
Inflammation plays an important role in Nonalcoholic Steatohepatitis (NASH), triggering receptor expressed on myeloid cells-1 and 2 (TREM-1 and TREM-2) modulates inflammatory and innate immune, they have been investigated in various inflammatory diseases, but not in NASH. Meanwhile we added glycine in HFO (HFOG) to investigate if the liver pathologic relief is related with TREM-1 and TREM-2. Liver tissue staining and serum indexes showed that the NASH was successful from the 4th weekend and glycine can improved many features of NASH. Results from Q-PCR and ELISA study showed that compareaded with control, TREM-1 is upregulated and TREM-2 is downregulated respectively in 4 and 8-week NASH model (TREM-1: p < 0.001; TREM-2: p < 0.001).Compared with HFO group, HFOG group with an extra 5% Glycine into the diet of NASH, we found that all model liver pathologic and serum indexes ameliorated in this group. Furthermore, Results from Q-PCR and ELISA study showed that compareaded with HFO group, TREM-2 of this group is upregulated and TREM-1 is downregulated respectively from the 4th weekend, which is more significant at the 8th weekend (TREM-1: p <0.001; TREM-2: p =0.048). Pearson correlation showed that TREM-1 and TREM-2 were closely associated with serum ET, TNF-α, TLR-4 and PC III. Besides, using multiple-stepwise regression analysis, we found that the ameliorative effects of glycine in HFOG was mainly related to its counteraction of PC III, TREM-1 and upregulation of TREM-2. Furthermore, we detected the expression of TREM-1 and TREM-2 in gall stone patients without drinking excessively before undergoing cholecystectomy, and found that the rise of TREM-1 and reduction of TREM-2 was close associated with the severity of fatty liver. To conclude, our results support the concept that TREM-1 and TREM-2 were close strongly linked to NASH and NALFD. Glycine can relieve NASH by its anti-fibrosis effect, and this ameliorative effect is related to the expression change of TREM-1/2 to some extent.
PMCID: PMC4846883  PMID: 27158326
TREM-1/2; glycine; NASH
25.  Association of Megsin Gene Variants With IgA Nephropathy in Northwest Chinese Population 
Medicine  2016;95(6):e2694.
Abstract
Megsin is a mesangial cell-predominant gene that encodes a serpin family protein which is expressed in the renal mesangium. Overexpression of megsin has been observed in the glomeruli of patients with IgA nephropathy (IgAN). The aim of this study was to evaluate the association of megsin polymorphisms (rs1055901 and rs1055902) with IgAN in a Chinese population.
We examined 351 patients with histologically proven IgAN and compared them with 310 age, sex, and ethnicity-matched healthy subjects. Two single nucleotide polymorphisms (SNPs) in megsin were genotyped by Sequenom MassARRAY. SPSS 18.0 was used for statistical analyses, and SNP Stats to test for associations between these polymorphisms and IgAN risk. Odds ratios with 95% confidence intervals were used to assess the relationships.
We found that rs1055901 and rs1055902 SNPs were not correlated with susceptibility to IgAN in Northwest Chinese population. Analyses of the relationship between genotypes and clinical variables indicated that in patients with IgAN, rs1055901 was associated with 24-hour proteinuria, an increase in blood pressure, and Lee's grade (P = 0.04, 0.02, and 0.04, respectively), and rs1055902 was associated with 24-hour proteinuria and Lee's grade (P = 0.03 and 0.01, respectively). However, the results showed no association between these gene variants and sex of the patients.
These results indicate that megsin gene variants may play a role in the severity, development, and/or progression of IgAN in Northwest Chinese population.
doi:10.1097/MD.0000000000002694
PMCID: PMC4753896  PMID: 26871801

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