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1.  Meta-analysis of brain iron levels of Parkinson’s disease patients determined by postmortem and MRI measurements 
Scientific Reports  2016;6:36669.
Brain iron levels in patients of Parkinson’s disease (PD) are usually measured in postmortem samples or by MRI imaging including R2* and SWI. In this study we performed a meta-analysis to understand PD-associated iron changes in various brain regions, and to evaluate the accuracy of MRI detections comparing with postmortem results. Databases including Medline, Web of Science, CENTRAL and Embase were searched up to 19th November 2015. Ten brain regions were identified for analysis based on data extracted from thirty-three-articles. An increase in iron levels in substantia nigra of PD patients by postmortem, R2* or SWI measurements was observed. The postmortem and SWI measurements also suggested significant iron accumulation in putamen. Increased iron deposition was found in red nucleus as determined by both R2* and SWI, whereas no data were available in postmortem samples. Based on SWI, iron levels were increased significantly in the nucleus caudatus and globus pallidus. Of note, the analysis might be biased towards advanced disease and that the precise stage at which regions become involved could not be ascertained. Our analysis provides an overview of iron deposition in multiple brain regions of PD patients, and a comparison of outcomes from different methods detecting levels of iron.
doi:10.1038/srep36669
PMCID: PMC5101491  PMID: 27827408
2.  Clinical Characteristics of Patients Who Underwent Surgery for Genital Tract Malformations at Peking Union Medical College Hospital across 31 Years 
Chinese Medical Journal  2016;129(20):2441-2444.
Background:
Female genital malformations represent miscellaneous deviations from normal anatomy. This study aimed to explore the clinical characteristics of patients who underwent surgery for genital tract malformations at Peking Union Medical College Hospital (PUMCH) during a 31-year period.
Methods:
We retrospectively reviewed surgical cases of congenital malformation of the female genital tract at PUMCH for a 31-year period, analyzed the clinical characteristics of 1634 hospitalized patients, and investigated their general condition, diagnosis, and treatment process.
Results:
The average patient age was 27.6 ± 9.9 years. The average ages of patients who underwent surgery for uterine malformation and vaginal malformation were 31.9 ± 8.8 years and 24.7 ± 9.0 years, respectively; these ages differed significantly (P < 0.01). Among patients with genital tract malformation, the percentages of vaginal malformation, uterine malformation, vulva malformation, cervical malformation, and other malformations were 43.9%, 43.5%, 7.4%, 2.3%, and 2.8%, respectively. Among patients with uterine malformation, 34.5% underwent surgery for the genital tract malformation, whereas in patients with vaginal malformation, the proportion is 70.6%; the difference between the two groups was statistically significant (P < 0.01). The percentage of complications of the urinary system in patients with vaginal malformations was 10.2%, which was statistically significantly higher than that (5.3%) in patients with uterine malformations (P < 0.01).
Conclusions:
Compared to patients with uterine malformations, patients with vaginal malformations displayed more severe clinical symptoms, a younger surgical age, and a greater need for attention, early diagnosis, and treatment. Patients with genital tract malformations, particularly vaginal malformations, tend to have more complications of the urinary system and other malformations than patients with uterine malformations.
doi:10.4103/0366-6999.191762
PMCID: PMC5072256  PMID: 27748336
Clinical Characteristics; Female Genital Tract Malformations; Uterine Malformation; Vaginal Malformation
3.  Mitochondrial involvement in propofol-induced cardioprotection: An in vitro study in human myocardium 
Experimental Biology and Medicine  2016;241(5):527-538.
Propofol has been shown to exert cardioprotection, but the underlying mechanisms remain incompletely understood. We examined: (1) whether propofol-induced cardioprotection depended on the time and the dose of administration; (2) the role of mitochondrial adenosine triphosphate-sensitive potassium channels, nitric oxide synthase, and mitochondrial respiratory chain activity in propofol-induced cardioprotection. Human right atrial trabeculae were obtained during cardiopulmonary bypass for coronary artery bypass and aortic valve replacement. Isometric force of contraction of human right atrial trabeculae hanged in an oxygenated Tyrode’s solution was recorded during 30-min hypoxia and 60-min reoxygenation (Control). Propofol 0.1, 1, and 10 µM was administered: (1) 5 min before hypoxia until the end of the experiment; (2) 5 min followed by 5-min washout before hypoxia; (3) during the reoxygenation period, propofol 10 µM was administered in presence of 5-hydroxydecanoate (antagonist of mitochondrial adenosine triphosphate-sensitive potassium channels), and NG-nitro-L-arginine methyl ester (inhibitor of nitric oxide synthase). In addition, mitochondria were isolated from human right atrial at 15 min of reoxygenation. The effect of propofol on activity of the mitochondrial respiratory chain complexes was evaluated by spectrophotometry. The force of contraction (% of baseline) and the complex activity between the different groups were compared with an analysis of variance and post hoc test. Propofol 10 µM administered during the reoxygenation period significantly improved the recovery of force of contraction at the end of reoxygenation (82 ± 6% of baseline value vs. 49 ± 6% in Control; P < 0.001). The beneficial effects of propofol 10 µM were abolished by co-administration with 5-hydroxydecanoate (53 ± 8%) or NG-nitro-L-arginine methyl ester (57 ± 6%). Propofol 10 µM significantly increased enzymatic activities of the mitochondrial respiratory chain complexes, in reoxygenation period, compared to their respective untreated controls. In conclusion, in human myocardium, propofol-induced cardioprotection was mediated by mitochondrial adenosine triphosphate-sensitive potassium channels opening, nitric oxide synthase activation and stimulation of mitochondrial respiratory chain complexes, in early reoxygenation period.
doi:10.1177/1535370215622586
PMCID: PMC4950485  PMID: 26748397
Propofol; cardioprotection; mitochondrial adenosine triphosphate-sensitive potassium channels; mitochondrial respiratory chain complexes
4.  Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury 
eLife  null;5:e12661.
Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury.
DOI: http://dx.doi.org/10.7554/eLife.12661.001
eLife digest
Around 20% of the world’s population experiences long-lasting “chronic” pain, which often results in poor sleep, depression and anxiety. One of the most disabling forms of chronic pain is called neuropathic pain, which results from injuries to sensory nerves. Pain or discomfort is felt in response to touches that are not normally painful. Neuropathic pain is difficult to treat as we do not fully understand the molecular mechanisms that cause it.
Stimulating a nerve causes it to produce action potentials. At a molecular level, these action potentials are generated by ions moving into and out of the neuron through proteins called ion channels. The movement of sodium ions into a neuron triggers an action potential, and the movement of potassium ions out of the neuron returns it to a resting state.
After a sensory nerve is cut or otherwise damaged it becomes hyperactive and produces spontaneous electrical activity that the brain interprets as pain signals. However, it is not fully understood how cutting a nerve affects the ion channels in a way that generates this hyperactivity.
Different types of ion channel are found in different regions of the nerve cell; for example, type 1 potassium channels are normally found in a region called the juxtaparanode at the axon of the neuron. Calvo et al. have now tracked what happens to type 1 potassium channels after nerve injury in rats. Soon after nerve damage occurred, nearly all of these ion channels disappeared from the juxtaparanode. At the same time, electrical activity in the cut nerve increased, and the recovering animals responded in ways that suggested they were hypersensitive to the nerve being touched.
Three weeks after the injury, most rats lost their hypersensitivity and the electrical activity in the cut nerve returned to near-normal levels. Calvo et al. found that the recovering nerves contained new subtypes of type 1 potassium channels. These potassium channels did not just appear in the juxtaparanode: they also invaded the ‘fence’ region that normally separates potassium channels from sodium channels. The same was observed to happen in the nerves of patients that suffer from neuropathic pain due to a nerve injury.
At this late time point after nerve injury, blocking the activity of potassium channels produced the same abnormal increase in the nerve’s electrical activity as seen immediately after the nerve had been cut. The rats’ hypersensitivity to touch also returned. This suggests that the appearance of the new potassium channel subtypes might be a protective mechanism that reduces the activity of a damaged nerve to decrease pain.
These findings suggest new ways of treating neuropathic pain. Further studies are now needed to investigate whether drugs that can activate the new potassium channel subtypes could stop pain from an injured nerve becoming a long-term problem.
DOI: http://dx.doi.org/10.7554/eLife.12661.002
doi:10.7554/eLife.12661
PMCID: PMC4841771  PMID: 27033551
neuropathic pain; neuropathy; shaker type potassium channels; juxtaparanode; hypersensitivity; Human; Rat
5.  Proteomic Analysis of the Uterosacral Ligament in Postmenopausal Women with and without Pelvic Organ Prolapse 
Chinese Medical Journal  2015;128(23):3191-3196.
Background:
Pelvic organ prolapse (POP) is a major health problem in adult women that involves many factors. No proteomic analysis has been conducted exclusively in POP patients. This study aimed to identify the differential expression of proteins that may be involved in POP by proteomic analysis.
Methods:
Samples of the uterosacral ligament (USL) were collected from five POP patients and five non-POP patients matched according to age, parity, and menopausal status and analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of proteins that showed differential expression in the proteomic analyses.
Results:
Proteins differentially expressed between POP and non-POP patients were detected. Eight proteins that were down-regulated in the POP group were identified by MALDI-TOF-MS. These proteins included electron transfer flavoprotein, apolipoprotein A-I, actin, transgelin, cofilin-1, cyclophilin A, myosin, and galectin-1, and their expression was verified by qRT-PCR.
Conclusion:
Using comparative proteomics, we identified eight differentially expressed proteins (including four cytoskeleton proteins and three proteins related to apoptosis) in the USL that may be involved in apoptosis associated with the tissue effects in POP pathophysiology.
doi:10.4103/0366-6999.170262
PMCID: PMC4794882  PMID: 26612295
Apoptosis; Cytoskeleton; Pelvic Organ Prolapse; Proteomics; Uterosacral Ligament
6.  Disomic Inheritance and Segregation Distortion of SSR Markers in Two Populations of Cynodon dactylon (L.) Pers. var. dactylon 
PLoS ONE  2015;10(8):e0136332.
Common bermudagrass [C. dactylon (L.) Pers. var. dactylon] is economically and environmentally the most important member among Cynodon species because of its extensive use for turf, forage and soil erosion control in the world. However, information regarding the inheritance within the taxon is limited. Accordingly, the objective of this study was to determine qualitative inheritance mode in common bermudagrass. Two tetraploid (2n = 4x = 36), first-generation selfed (S1) populations, 228 progenies of ‘Zebra’ and 273 from A12359, were analyzed for segregation with 21 and 12 simple sequence repeat (SSR) markers, respectively. It is concluded that the inheritance mode of tetraploid bermudagrass was complete or near complete disomic. It is evident that the two bermudagrass parents had an allotetraploid genome with two distinct subgenomes since 33 SSR primer pairs amplified 34 loci, each having two alleles. Severe transmission ratio distortions occurred in the Zebra population while less so in the A12359 population. The findings of disomic inheritance and segregation ratio distortion in common bermudagrass is significant in subsequent linkage map construction, quantitative trait locus mapping and marker-assisted selection in the species.
doi:10.1371/journal.pone.0136332
PMCID: PMC4546580  PMID: 26295707
7.  Glycol chitosan incorporated retinoic acid chlorochalcone (RACC) nanoparticles in the treatment of Osteosarcoma 
Background
Osteosarcoma is the most common of all the bone malignancies and accounts for 30-80 % of the primary skeletal sarcomas. The overall survival rate of patients with osteosarcoma is < 20 % suggesting poor prognosis.
Methods
The present study demonstrates the effect of retinoic acid chlorochalcone (RACC) incorporated glycol chitosan (GC) nanoparticle transfection in osteosarcoma cells. MG-63 and Saos-2 osteosarcoma cells were transfected with various concentrations of RACC-incorporated GC nanoparticle for 24 h. The effect on cell proliferation, Ezh2 expression, apoptosis, cell cycle arrest, cell migration and invasiveness, Akt phosphorylation and local tumour growth and metastases were studied.
Results
MG-63 and Saos-2 osteosarcoma cells on RACC-incorporated GC nanoparticle transfection for 24 h showed a concentration-dependent inhibition of cell proliferation. Of the various concentrations of RACC tested, the effective concentration started from 5 μM with an IC50 of 20 μM. Wound healing assay also showed that RACC-incorporated GC nanoparticles inhibited migration of tumor cells more effectively compared to the parent RA. RACC transfection resulted in inhibition of cell proliferation, Ezh2 expression inhibition, apoptosis through mitochondrial pathway by decrease in membrane potential and release of cytochrome c and cell cycle arrest in the G0/G1 phase. The invasiveness of cells treated with 5 and 20 μM RACC was decreased by 49 and 76 % respectively, compared to the control. RACC-treated mice showed significantly lower number of metastases compared to that in the control mice.
Conclusions
Thus, RACC-incorporated glycol chitosan nanoparticle strategy can be promising for the treatment of osteosarcoma.
doi:10.1186/s12944-015-0068-4
PMCID: PMC4501080  PMID: 26170203
Osteosarcoma; Membrane potential; Migration; Inhibition; Glycol chitosan
8.  Structure of the human P2Y12 receptor in complex with an antithrombotic drug 
Nature  2014;509(7498):115-118.
P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors1. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo2, which limits our under-standing of this receptor family. P2Y12R regulates platelet activation and thrombus formation3,4, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor)5,6 suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors7,8. Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.
doi:10.1038/nature13083
PMCID: PMC4174307  PMID: 24670650
9.  Exome Sequencing Identifies a Novel Gene, WNK1, for Susceptibility to Pelvic Organ Prolapse (POP) 
PLoS ONE  2015;10(3):e0119482.
Pelvic organ prolapse (POP) is a common gynecological disorder; however, the genetic components remain largely unidentified. Exome sequencing has been widely used to identify pathogenic gene mutations of several diseases because of its high chromosomal coverage and accuracy. In this study, we performed whole exome sequencing (WES), for the first time, on 8 peripheral blood DNA samples from representative POP cases. After filtering the sequencing data from the dbSNP database (build 138) and the 1000 Genomes Project, 2 missense variants in WNK1, c.2668G > A (p.G890R) and c.6761C> T (p.P2254L), were identified and further validated via Sanger sequencing. In validation stage, the c.2668G > A (p.G890R) variant and 8 additional variants were detected in 11 out of 161 POP patients. All these variants were absent in 231 healthy controls. Functional experiments showed that fibroblasts from the utero-sacral ligaments of POP with WNK1 mutations exhibited loose and irregular alignment compared with fibroblasts from healthy controls. In sum, our study identified a novel gene, WNK1, for POP susceptibility, expanded the causal mutation spectrums of POP, and provided evidence for the genetic diagnosis and medical management of POP in the future.
doi:10.1371/journal.pone.0119482
PMCID: PMC4349638  PMID: 25739019
10.  Manometric Comparison of Anorectal Function after Posterior Vaginal Compartment Repair with and without Mesh 
Chinese Medical Journal  2015;128(4):438-442.
Background:
Although repair augmented with mesh has been proved its priority in anatomical and functional recovery after anterior compartment reconstruction, the data about posterior compartment are scarce. The aim of this study was to compare bowel functional outcome of posterior vaginal compartment repair with and without mesh in patients with pelvic organ prolapse (POP).
Methods:
This was a prospective, double-blind, clinical pilot study of 22 postmenopausal women with symptomatic POP (overall POP-quantification [POP-Q] Stage III-IV) who underwent total pelvic floor reconstruction. Patients were grouped according to the use of mesh for posterior vaginal compartment repair: A mesh group and a nonmesh group. POP-Q stage, the pelvic floor impact questionnaire short form-7 (PFIQ-7) and anorectal manometry were evaluated before and 3 months after surgery. Anatomical success was defined as POP-Q Stage II or less. A t-test was used to compare preoperative with postoperative data in the two groups.
Results:
Totally, 17 (71%) were available for the follow-up. POP-Q measurements improved significantly compared to baseline (P < 0.05) in both groups. No recurrence was observed. Subjects in both groups reported improvement in pelvic floor symptoms, and there was no significant difference in the PFIQ-7 score between groups at follow-up (P > 0.05). Compared with baseline, the nonmesh group exhibited a statistically significant decrease in anal residual pressure, a significant increase in the anorectal pressure difference during bowel movement, and a reduced rate of dyssynergia defecation pattern (P < 0.05).
Conclusions:
Provided there is sufficient support for the anterior wall and apex of vagina with mesh, posterior compartment repair without mesh may be as effective as repair with mesh for anatomical recovery while providing better anorectal motor function.
doi:10.4103/0366-6999.151065
PMCID: PMC4836243  PMID: 25673442
Anorectal Manometry; Constipation; Mesh; Pelvic Organ Prolapse; Posterior Vaginal Compartment Repair
11.  Tension-free Polypropylene Mesh-related Surgical Repair for Pelvic Organ Prolapse has a Good Anatomic Success Rate but a High Risk of Complications 
Chinese Medical Journal  2015;128(3):295-300.
Background:
Food and Drug Administration announcements have highlighted the standard rate of mesh-related complications. We aimed to report the short-term results and complications of tension-free polypropylene mesh (PROSIMA™) surgical repair of pelvic organ prolapse (POP) using the standard category (C), timing (T), and site (S) classification system.
Methods:
A prospective cohort study of 48 patients who underwent PROSIMA™ mesh kit-related surgical repairs were followed for two years at Peking Union Medical College Hospital. Recurrence was defined as symptomatic POP quantification (POP-Q) Stage II or higher (leading edge ≥ −1 cm). The Patient Global Impression of Change Questionnaire, the Chinese version of the Pelvic Floor Impact Questionnaire short-form-7 and POP/Urinary Incontinence Sexual Questionnaire short-form-12 were used to evaluate the self-perception and sexual function of each patient. Mesh-related complications conformed to the International Urogynecological Association/International Continence Society joint terminology. The paired-sample t-test, one-way analysis of variance, Fisher's exact test, Kaplan-Meier survival analysis and log-rank test were used to analyze data.
Results:
All patients were followed up for ≥12 months; 30 (62.5%) patients completed the 24 months study. We observed a 93.8% (45/48) positive anatomical outcome rate at 12 months and 90.0% (27/30) at 24 months. Recurrence most frequently involved the anterior compartment (P < 0.05). Pelvic symptoms improved significantly from baseline (P < 0.05), although the patients’ impressions of change and sexual function were not satisfying. Vaginal complication was the main complication observed (35.4%, 17/48). The survival analysis did not identify any relationship between vaginal complication and anatomical recurrent prolapse (POP-Q ≥ Stage II) (P = 0.653).
Conclusions:
Tension-free polypropylene mesh (PROSIMA™)-related surgical repair of POP has better short-term anatomical outcomes at the apical and posterior compartments, but a low patient satisfaction rate. The mesh complications were not the definitive cause of recurrence.
doi:10.4103/0366-6999.150088
PMCID: PMC4837857  PMID: 25635422
Pelvic Organ Prolapse; Postoperative Complication; PROSIMA™ Mesh Kit; Quality of Life
12.  New Classification of Herlyn-Werner-Wunderlich Syndrome 
Chinese Medical Journal  2015;128(2):222-225.
Background:
Uterus didelphys and blind hemivagina associated with ipsilateral renal agenesis are collectively known as Herlyn-Werner-Wunderlich syndrome (HWWS). In the literature, the syndrome often appears as a single case report or as a small series. In our study, we reviewed the characteristics of all HWWS patients at Peking Union Medical College Hospital (PUMCH) and suggested a new classification for this syndrome because the clinical characteristics differed significantly between the completely and incompletely obstructed vaginal septum. This new classification allows for earlier diagnosis and treatment.
Methods:
From January 1986 to March 2013, all diagnosed cases of HWWS at PUMCH were reviewed. A retrospective long-term follow-up study of the clinical presentation, surgical prognosis, and pregnancy outcomes was performed. Statistical analyses were performed using SPSS, version 15.0 (IBM, Armonk, NY, USA). Between-group comparisons were performed using the χ2 test, Fisher's exact test, and the t-test. The significance level for all analyses was set at P < 0.05.
Results:
The clinical data from 79 patients with HWWS were analyzed until March 31, 2013. According to our newly identified characteristics, we recommend that the syndrome be classified by the complete or incomplete obstruction of the hemivagina as follows: Classification 1, a completely obstructed hemivagina and Classification 2, an incompletely obstructed hemivagina. The clinical details associated with these two types are distinctly different.
Conclusions:
HWWS patients should be differentiated according to these two classifications. The two classifications could be generalized by gynecologists world-wide.
doi:10.4103/0366-6999.149208
PMCID: PMC4837842  PMID: 25591566
Classification; Diagnosis; Therapy
13.  Three-dimensional reconstruction method for measuring the knee valgus angle of the femur in northern Chinese adults*  
The purpose of this study was to establish a method for measuring the knee valgus angle from the anatomical and mechanical axes on three-dimensional reconstruction imaging models, and to use this method for estimating an average knee valgus angle value for northern Chinese adults. Computed tomographic angiography data in DICOM format for 128 normal femurs from 64 adult subjects were chosen for analysis. After the femur images were subjected to three-dimensional reconstruction, the deepest point in the intercondylar notch (point A), the midpoint of the medullary cavity 20 cm above the knee-joint line (point B), and the landmark of the femoral head rotation center (point C) were identified on each three-dimensional model. The knee valgus angle was defined as the angle enclosed by the distal femoral anatomical axis (line AB) and the femoral mechanical axis (line AC). The average (mean±SD) of knee valgus angle for the 128 femurs was 6.20°±1.20° (range, 3.05° to 10.64°). Significant positive correlations were found between the knee valgus angles of the right and left sides and between the knee valgus angle and age. During total knee arthroplasty, choosing a valgus cut angle of approximately 6° may achieve a good result in reestablishing the natural mechanical alignment of the lower extremity for patients of northern Chinese ethnicity. Larger valgus cut angles should be chosen for older patients.
doi:10.1631/jzus.B1400019
PMCID: PMC4129092  PMID: 25091990
Knee valgus angle; Three-dimensional reconstruction femoral model; Total knee arthroplasty; Northern Chinese adults; Preoperative design
14.  Kv2 dysfunction after peripheral axotomy enhances sensory neuron responsiveness to sustained input☆ 
Experimental Neurology  2014;251(100):115-126.
Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.
Highlights
•Kv2.1 and Kv2.2 are expressed in rat dorsal root ganglion neurons.•Kv2 subunits are most abundant in myelinated sensory neurons.•Kv2.1 and Kv.2 subunits are downregulated in a traumatic nerve injury pain model.•Kv2 inhibition ex vivo allows higher firing rates during sustained stimulation.•We conclude that Kv2 channels contribute to limiting peripheral neuron excitability.
doi:10.1016/j.expneurol.2013.11.011
PMCID: PMC3898477  PMID: 24252178
AP, action potential; APD50, AP half width; AHPD50, after-hyperpolarization half width; ATF3, activating transcription factor 3; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DRG, dorsal root ganglion; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB4, isolectin B4; IHC, immunohistochemistry; IR, input resistance; ISH, in situ hybridization; Kv channel, voltage-gated potassium channel; NF200, neurofilament 200; RP, refractory period; ScTx, stromatoxin-1; SNT, spinal nerve transection; Neuropathic pain; Potassium channel; Dorsal root ganglia
15.  Downstream Signaling Pathways in Mouse Adipose Tissues Following Acute In Vivo Administration of Fibroblast Growth Factor 21 
PLoS ONE  2013;8(9):e73011.
FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation.
doi:10.1371/journal.pone.0073011
PMCID: PMC3765203  PMID: 24039848
16.  Ziyuglycoside II Inhibits the Growth of Human Breast Carcinoma MDA-MB-435 Cells via Cell Cycle Arrest and Induction of Apoptosis through the Mitochondria Dependent Pathway 
Ziyuglycoside II is one of the major active compounds of Sanguisorba officinalis L., which has a wide range of clinical applications including hemostasis, antibiosis, anti-inflammation and anti-oxidation. This study investigated the effect of ziyuglycoside II on the growth of human breast carcinoma MDA-MB-435 cells for the first time. The results showed that ziyuglycoside II could significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression at G0/G1 and S phase as well as via inducing cell apoptosis. Accumulation of reactive oxygen species (ROS) was observed in the progression of cell cycle arrest, which was associated with the increased expression of cell cycle regulating factors, p53 and p21. Subsequent apoptosis induced by ziyuglycoside II was accompanied with the activation of mitochondrial pathway, in particular a decreased mitochondrial membrane potential (MMP) as well as increased Bax/Bcl-2 ratio, cytochrome c release and the activity of caspase-3 and caspase-9. In conclusion, our study was the first to report that ziyuglycoside II has inhibitory effect on the growth of MDA-MB-435 cells, which might become a potential therapeutic approach of breast cancer in the future.
doi:10.3390/ijms140918041
PMCID: PMC3794768  PMID: 24005866
ziyuglycoside II; MDA-MB-435; cell cycle arrest; cell apoptosis
17.  Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury 
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
doi:10.1523/JNEUROSCI.3561-12.2012
PMCID: PMC3713313  PMID: 23197740
18.  Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma 
Background
Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model.
Methods
The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts.
Results
The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed.
Conclusions
These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma.
doi:10.1186/1479-5876-10-256
PMCID: PMC3573957  PMID: 23272637
Gene therapy; Interferon-γ; Nasopharyngeal carcinoma; Adenoviral vector
19.  Body mass index, recreational physical activity and female urinary incontinence in Gansu, China 
Objective
To elucidate the influence of recreational physical activity, body mass index (BMI), and waist circumference on the risk of specific types of urinary incontinence.
Study design
We conducted a population-based cross-sectional survey in Gansu, China among 2603 women aged 20 years or older.
Results
The study found that BMI was positively associated with urinary incontinence (P for trend = 0.008) and the association was mainly observed for stress urinary incontinence (OR = 1.4, 95% CI: 1.1, 1.9 for BMI = 24.0–27.9 kg/m2; OR = 2.3, 95% CI: 1.5, 3.6 for BMI ≥ 28.0 kg/m2; P for trend = 0.0005). A positive association between stress incontinence (OR = 1.7, 95% CI: 1.2, 2.5) and waist circumference was observed for women who had waist circumference between 70 cm and 75 cm compared to waist circumference less than 70 cm. Recreational physical activity was inversely associated with overall and mixed urinary incontinence (P for trend <0.0001 for both). A significant interaction between physical activity and waist circumference was found for overall (P = 0.0007) and stress incontinence (P = 0.001).
Conclusions
The findings that physical activity inversely associated with urinary incontinence and its interaction with waist circumference warrant further investigation, particularly in prospective studies.
doi:10.1016/j.ejogrb.2011.07.016
PMCID: PMC3229870  PMID: 21821342
Recreational physical activity; Body mass index; Waist circumference; Female urinary incontinence
20.  Preliminary study of an intestinal bio-robot system based on nerve stimulation 
Background
Wireless capsule endoscopes for diagnosis and treatment in the gastrointestinal tract face the common problem of active actuation. To tackle this difficulty, a non-invasive intestinal bio-robot system with active actuation based on nerve stimulation was developed.
Methods
This intestinal inspection system utilized a natural organism—the mud eel—to serve as the locomotion mechanism, and it was controlled by a LabVIEW-programmed pulse generator. The exterior control unit was able to actively drive and remotely control the navigation and site-specific anchoring of the organism.
Results
Through in vitro stimulation experiments, a method of controlling the organism’s forward motion was obtained: when the organism was stimulated at the tail, it moved forward at a relatively fast speed and with high repeatability. The stimulator parameters were as follows: amplitude 1.85 μA, frequency 2 Hz, pulse duration 500 μs.
Conclusions
Since this is a preliminary study, considerable work remains to be done. However, the results could provide a solid theoretical basis for further research toward producing a practical intestinal bio-robot for the diagnosis and treatment of the gastrointestinal tract.
doi:10.1186/1743-0003-9-68
PMCID: PMC3546848  PMID: 23020999
Intestinal bio-robot system; Intestinal examination; Nerve stimulation; Locomotion control
21.  Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection 
The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.
doi:10.1590/S0100-879X2012007500130
PMCID: PMC3854230  PMID: 22892829
Hepatitis B virus; B-cell activation; Fc receptor-like 1; Gene expression
22.  Canine Hip Dysplasia is Predictable by Genotyping 
Summary
Objective
To establish a predictive method using whole genome genotyping for early intervention in canine hip dysplasia (CHD) risk management, for the prevention of the progression of secondary osteoarthritis (OA), and for selective breeding.
Design
Two sets of dogs (6 breeds) were genotyped with dense SNPs covering the entire canine genome. The first set contained 359 dogs upon which a predictive formula for genomic breeding value (GBV) was derived by using their estimated breeding value (EBV) of the Norberg angle (a measure of CHD) and their genotypes. To investigate how well the formula would work for an individual dog with genotype only (without using EBV or phenotype), a cross validation was performed by masking the EBV of one dog at a time. The genomic data and the EBV of the remaining dogs were used to predict the GBV for the single dog that was left out. The second set of dogs included 38 new Labrador retriever dogs, which had no pedigree relationship to the dogs in the first set.
Results
The cross validation showed a strong correlation (r>0.7) between the EBV and the GBV. The independent validation showed a strong correlation (r=0.5) between GBV for the Norberg angle and the observed Norberg angle (no EBV was available for the new 38 dogs). Sensitivity, specificity, positive, and negative predictive value of the genomic data were all above 70%.
Conclusions
Prediction of CHD from genomic data is feasible, and can be applied for risk management of CHD and early selection for genetic improvement to reduce the prevalence of CHD in breeding programs. The prediction can be implemented before maturity, at which age current radiographic screening programs are traditionally applied, and as soon as DNA is available.
doi:10.1016/j.joca.2010.12.011
PMCID: PMC3065507  PMID: 21215318
23.  Identification of Autotoxic Compounds in Fibrous Roots of Rehmannia (Rehmannia glutinosa Libosch.) 
PLoS ONE  2012;7(1):e28806.
Rehmannia is a medicinal plant in China. Autotoxicity has been reported to be one of the major problems hindering the consecutive monoculture of Rehmannia. However, potential autotoxins produced by the fibrous roots are less known. In this study, the autotoxicity of these fibrous roots was investigated. Four groups of autotoxic compounds from the aqueous extracts of the fibrous roots were isolated and characterized. The ethyl acetate extracts of these water-soluble compounds were further analyzed and separated into five fractions. Among them, the most autotoxic fraction (Fr 3) was subjected to GC/MS analysis, resulting in 32 identified compounds. Based on literature, nine compounds were selected for testing their autotoxic effects on radicle growth. Seven out of the nine compounds were phenolic, which significantly reduced radicle growth in a concentration-dependent manner. The other two were aliphatic compounds that showed a moderate inhibition effect at three concentrations. Concentration of these compounds in soil samples was determined by HPLC. Furthermore, the autotoxic compounds were also found in the top soil of the commercially cultivated Rehmannia fields. It appears that a close link exists between the autotoxic effects on the seedlings and the compounds extracted from fibrous roots of Rehmannia.
doi:10.1371/journal.pone.0028806
PMCID: PMC3250401  PMID: 22235251
24.  Therapeutic hypertension system based on a microbreathing pressure sensor system 
Background and methods
A novel therapeutic system for the treatment of hypertension was developed on the basis of a slow-breath training mechanism, using a microbreathing pressure sensor device for the detection of human respiratory signals attached to the abdomen. The system utilizes a single-chip AT89C51 microcomputer as a core processor, programmed by Microsoft Visual C++6.0 to communicate with a PC via a full-speed PDIUSBD12 interface chip. The programming is based on a slow-breath guided algorithm in which the respiratory signal serves as a physiological feedback parameter. Inhalation and exhalation by the subject is guided by music signals.
Results and conclusion
Our study indicates that this microbreathing sensor system may assist in slow-breath training and may help to decrease blood pressure.
doi:10.2147/MDER.S19971
PMCID: PMC3417874  PMID: 22915930
hypertension; microbreathing sensor; single-chip microcomputer; slow-pace breathing
25.  Basolateral Amygdala Inactivation Impairs Learning-Induced Long-Term Potentiation in the Cerebellar Cortex 
PLoS ONE  2011;6(1):e16673.
Learning to fear dangerous situations requires the participation of basolateral amygdala (BLA). In the present study, we provide evidence that BLA is necessary for the synaptic strengthening occurring during memory formation in the cerebellum in rats. In the cerebellar vermis the parallel fibers (PF) to Purkinje cell (PC) synapse is potentiated one day following fear learning. Pretraining BLA inactivation impaired such a learning-induced long-term potentiation (LTP). Similarly, cerebellar LTP is affected when BLA is blocked shortly, but not 6 h, after training. The latter result shows that the effects of BLA inactivation on cerebellar plasticity, when present, are specifically related to memory processes and not due to an interference with sensory or motor functions. These data indicate that fear memory induces cerebellar LTP provided that a heterosynaptic input coming from BLA sets the proper local conditions. Therefore, in the cerebellum, learning-induced plasticity is a heterosynaptic phenomenon that requires inputs from other regions. Studies employing the electrically-induced LTP in order to clarify the cellular mechanisms of memory should therefore take into account the inputs arriving from other brain sites, considering them as integrative units. Based on previous and the present findings, we proposed that BLA enables learning-related plasticity to be formed in the cerebellum in order to respond appropriately to new stimuli or situations.
doi:10.1371/journal.pone.0016673
PMCID: PMC3031621  PMID: 21304962

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