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1.  Cohesin loss alters adult hematopoietic stem cell homeostasis, leading to myeloproliferative neoplasms 
The Journal of Experimental Medicine  2015;212(11):1833-1850.
Mullenders et al. report that loss of cohesin alters stem cell homeostasis and myelopoiesis, facilitating the development of a myeloid malignancy.
The cohesin complex (consisting of Rad21, Smc1a, Smc3, and Stag2 proteins) is critically important for proper sister chromatid separation during mitosis. Mutations in the cohesin complex were recently identified in a variety of human malignancies including acute myeloid leukemia (AML). To address the potential tumor-suppressive function of cohesin in vivo, we generated a series of shRNA mouse models in which endogenous cohesin can be silenced inducibly. Notably, silencing of cohesin complex members did not have a deleterious effect on cell viability. Furthermore, knockdown of cohesin led to gain of replating capacity of mouse hematopoietic progenitor cells. However, cohesin silencing in vivo rapidly altered stem cells homeostasis and myelopoiesis. Likewise, we found widespread changes in chromatin accessibility and expression of genes involved in myelomonocytic maturation and differentiation. Finally, aged cohesin knockdown mice developed a clinical picture closely resembling myeloproliferative disorders/neoplasms (MPNs), including varying degrees of extramedullary hematopoiesis (myeloid metaplasia) and splenomegaly. Our results represent the first successful demonstration of a tumor suppressor function for the cohesin complex, while also confirming that cohesin mutations occur as an early event in leukemogenesis, facilitating the potential development of a myeloid malignancy.
PMCID: PMC4612095  PMID: 26438359
2.  Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway 
Autophagy  2015;11(2):344-354.
Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.
PMCID: PMC4502731  PMID: 25831014
autophagic cell death; autophagy; E. coli O157:H7; ER stress; Shiga toxins; 3-MA, 3-methyladenine; Δ, knockout; AO, acridine orange; ATF4, activating transcription factor 4; ATG, autophagy-related; Baf A1, bafilomycin A1; BECN1, Beclin 1, autophagy-related; CASP3, caspase 3, apoptosis-related cysteine peptidase; DDIT3, DNA-damage-inducible transcript 3; EHEC O157, Escherichia coli O157:H7; FACS, fluorescence activated cell sorting; MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; MAPK, mitogen-activated protein kinase; MDC, monodansylcadaverine; NUPR1, nuclear protein, transcriptional regulator, 1; PBS, phosphate-buffered saline; PARP1, poly (ADP-ribose) polymerase 1; PI, propidium iodide; Stxs, Shiga toxins; Thap, thapsigargin; TEM, transmission electron microscopy; TRIB3, tribbles pseudokinase 3; WT, wild type; Z-VAD, Z-VAD-FMK
3.  Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria 
Aging Cell  2015;15(2):279-290.
Hutchinson–Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single‐nucleotide mutation in the LMNA gene. Previous reports have focused on nuclear phenotypes in HGPS cells, yet the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high‐resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC‐1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial‐targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic approach for HGPS.
PMCID: PMC4783354  PMID: 26663466
aging; methylene blue; mitochondria; PGC1‐α; progeria
4.  Colorectal Cancer Genetic Heterogeneity Delineated by Multi-Region Sequencing 
PLoS ONE  2016;11(3):e0152673.
Intratumor heterogeneity (ITH) leads to an underestimation of the mutational landscape portrayed by a single needle biopsy and consequently affects treatment precision. The extent of colorectal cancer (CRC) genetic ITH is not well understood in Chinese patients. Thus, we conducted deep sequencing by using the OncoGxOne™ Plus panel, targeting 333 cancer-specific genes in multi-region biopsies of primary and liver metastatic tumors from three Chinese CRC patients. We determined that the extent of ITH varied among the three cases. On average, 65% of all the mutations detected were common within individual tumors. KMT2C aberrations and the NCOR1 mutation were the only ubiquitous events. Subsequent phylogenetic analysis showed that the tumors evolved in a branched manner. Comparison of the primary and metastatic tumors revealed that PPP2R1A (E370X), SETD2 (I1608V), SMAD4 (G382T), and AR splicing site mutations may be specific to liver metastatic cancer. These mutations might contribute to the initiation and progression of distant metastasis. Collectively, our analysis identified a substantial level of genetic ITH in CRC, which should be considered for personalized therapeutic strategies.
PMCID: PMC4811559  PMID: 27023146
5.  In Vitro Assessment Reveals Parameters-Dependent Modulation on Excitability and Functional Connectivity of Cerebellar Slice by Repetitive Transcranial Magnetic Stimulation 
Scientific Reports  2016;6:23420.
Repetitive transcranial magnetic stimulation (rTMS) is an increasingly common technique used to selectively modify neural excitability and plasticity. There is still controversy concerning the cortical response to rTMS of different frequencies. In this study, a novel in vitro paradigm utilizing the Multi-Electrodes Array (MEA) system and acute cerebellar slicing is described. In a controllable environment that comprises perfusion, incubation, recording and stimulation modules, the spontaneous single-unit spiking activity in response to rTMS of different frequencies and powers was directly measured and analyzed. Investigation using this in vitro paradigm revealed frequency-dependent modulation upon the excitability and functional connectivity of cerebellar slices. The 1-Hz rTMS sessions induced short-term inhibition or lagged inhibition, whereas 20-Hz sessions induced excitation. The level of modulation is influenced by the value of power. However the long-term response fluctuated without persistent direction. The choice of evaluation method may also interfere with the interpretation of modulation direction. Furthermore, both short-term and long-term functional connectivity was strengthened by 1-Hz rTMS and weakened by 20-Hz rTMS.
PMCID: PMC4802318  PMID: 27000527
6.  Preliminary analysis of salivary microbiome and their potential roles in oral lichen planus 
Scientific Reports  2016;6:22943.
Several studies have explored the origin and development mechanism of oral lichen planus (OLP) with limited attention to the role of bacteria in the progression of this common oral disease. Here we utilized MiSeq sequencing of 16S rRNA gene amplicons to identify complex oral microbiota associated with OLP from saliva samples of two subtypes (reticular and erosive) of OLP patients and healthy controls. Our analyses indicated that the overall structure of the salivary microbiome was not significantly affected by disease status. However, we did observe evident variations in abundance for several taxonomic groups in OLP. Porphyromonas and Solobacterium showed significantly higher relative abundances, whereas Haemophilus, Corynebacterium, Cellulosimicrobium and Campylobacter showed lower abundances in OLP patients, as compared with healthy controls. In addition, we explored specific microbial co-occurrence patterns in OLP, and revealed significantly fewer linkers of Streptococcus comprising species in erosive OLP. Furthermore, the disease severity and immune dysregulation were also genus-associated, including with Porphyromonas that correlated to disease scores and salivary levels of interleukin (IL)-17 and IL-23. Overall, this study provides a general description of oral microbiome in OLP, and it will be useful for further investigation of their potential roles in the initiation and immune modulation of OLP.
PMCID: PMC4785528  PMID: 26961389
7.  Hepatic resection provided long-term survival for patients with intermediate and advanced-stage resectable hepatocellular carcinoma 
Hepatic resection has the highest local controllability that results in long-term survival for hepatocellular carcinoma (HCC). This study aimed to investigate the role of hepatic resection in selected patients of intermediate and advanced stage.
Clinical, pathological, and outcome data of 542 consecutive patients were retrospectively analyzed from a single center. The Kaplan-Meier method was used to estimate survival. Postoperative prognostic factors were evaluated using univariate and multivariate analyses.
The 1-, 3-, and 5-year overall survival rates were 89.0, 64.3, and 53.0 %, respectively. The 1-, 3-, and 5-year disease-free survival rates were 72.2, 44.5, and 34.2 %, respectively. Preoperative α-fetoprotein level >400 ng/mL, macroscopic vascular invasion, microscopic portal vein thrombosis, multiple tumor nodules, and the largest tumor size >5 cm were significantly correlated with overall survival. When these clinical risk factors were used in a postoperative staging system, assigning one point for each factor, the total score was precisely predictive of long-term survival. For patients with surgery plus adjuvant TACE (transarterial chemoembolization), the median overall survival was 56 months (range 1–110 months) and the 5-year OS rate was 48.5 %.
Hepatic resection is efficient and safe for HCC patients of intermediate and advanced stage. The adjuvant TACE should be recommended for HCC patients with poor risk factors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12957-016-0811-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4776356  PMID: 26936459
Hepatocellular; Hepatic resection; Prognosis; Large; Multinodular
8.  TERT Promoter Mutations and TERT mRNA but Not FGFR3 Mutations Are Urinary Biomarkers in Han Chinese Patients With Urothelial Bladder Cancer 
The Oncologist  2015;20(3):263-269.
Assays for mutations in the TERT promoter and the FGFR3 gene have been shown to be promising biomarkers for UBC diagnosis and surveillance in Western patients. Surprisingly, results of this study showed that the frequency of FGFR3 mutations was very low in Han Chinese patients with UBC and was unlikely to be a diagnostic marker for them. These patients also had a relatively low rate of TERT promoter mutations.
The TERT promoter and FGFR3 gene mutations are two of the most common genetic events in urothelial bladder cancer (UBC), and these mutation assays in patient urine have been shown to be promising biomarkers for UBC diagnosis and surveillance. These results were obtained mainly from studies of patients with UBC in Western countries, and little is known about such information in Han Chinese patients with UBC. In the present study, we addressed this issue by analyzing tumors from 182 Han Chinese patients with UBC and urine samples from 102 patients for mutations in the TERT promoter and FGFR3 and TERT mRNA expression in tumors and/or urine. TERT promoter and FGFR3 mutations were identified in 87 of 182 (47.8%) and 7 of 102 (6.7%) UBC cases, respectively. In 46 urine samples from patients with TERT promoter mutation-carrying tumors, the mutant promoter was detected in 24 (52%) prior to operation and disappeared in most examined urine samples (80%) taken 1 week after operation. TERT mRNA was detected in urine derived from 46 of 49 patients (94%) that was analyzed before operation independently of the presence of TERT promoter mutations. Collectively, FGFR3 mutations occur at a very low rate in Han Chinese UBC and cannot serve as diagnostic markers for Chinese patients. Han Chinese patients with UBC have relatively low TERT promoter mutation frequency compared with patients in Western countries, and simultaneous detection of both mutant TERT promoter and TERT mRNA improves sensitivity and specificity of urine-based diagnosis.
PMCID: PMC4350803  PMID: 25657201
FGFR3; Telomerase; TERT; Urinary markers; Urothelial bladder cancer
9.  Successful Salvage of the Upper Limb After Crush Injury Requiring Nine Operations: A Case Report 
International Surgery  2015;100(3):540-546.
Emergency treatment of amputation is one of the most frequently used therapeutic methods for patients with severe upper limb crush injury with a mangled extremity severity score (MESS) of more than 7. With the development of advanced surgical repair techniques and reconstructive technology, cases that once required amputation can now be salvaged with appropriate management, and some limb functions may also be reserved. A patient with a severe upper limb crush injury with a MESS score of 10 was treated in our hospital. The limb was salvaged after 9 surgeries over 10 months. The follow-up visits over the next 18 months post-injury showed that the shoulder joint functions were rated as “excellent” (90) according to the Neer score, the Harris hip evaluation (HHS) for elbow joint functions was “good” (80), and the patient was very satisfied with the overall therapeutic outcome. We conclude from the successful outcome of this extreme injury that salvage attempts should be the first management choice for upper limbs with complex injuries to save as much function as possible. Amputation should only be adopted when the injury is life-threatening or no more function can be saved. The level of evidence was V.
PMCID: PMC4370549  PMID: 25785341
Trauma; Upper limb; Crush injury
10.  Intraoperative Identification of Liver Cancer Microfoci Using a Targeted Near-Infrared Fluorescent Probe for Imaging-Guided Surgery 
Scientific Reports  2016;6:21959.
Difficulties in the highly sensitive detection of tumour microfoci represent a critical obstacle toward improved surgical intervention in liver cancer. Conventional preoperative imaging methods and surgeons’ subjective experience are limited by their inability to effectively detect tumour lesions measuring less than 2 mm; however, intraoperative fluorescence molecular imaging may overcome this limitation. Here, we synthesised an arginine-glycine-aspartic acid (RGD)-conjugated mesoporous silica nanoparticle (MSN) highly loaded with indocyanine green (ICG) dye that could accurately delineate liver cancer margins and provide excellent tumour-to-normal tissue contrast intraoperatively. The increased ICG loading capacity and tumour specificity enabled the identification of residual microtumours and satellite lesions measuring less than 1 mm in living mice. Histological analysis validated the sensitivity and accuracy of this approach. We believe this technique utilising a new fluorescent nanoprobe with intraoperative optical imaging may offer a more sensitive and accurate method for liver cancer resection guidance, resulting in better surgical outcomes.
PMCID: PMC4770417  PMID: 26923919
11.  Papaverine inhibits lipopolysaccharide-induced microglial activation by suppressing NF-κB signaling pathway 
To investigate the effects of papaverine (PAP) on lipopolysaccharide (LPS)-induced microglial activation and its possible mechanisms.
Materials and methods
BV2 microglial cells were first pretreated with PAP (0, 0.4, 2, 10, and 50 μg/mL) and then received LPS stimulation. Transcription and production of proinflammatory factors (IL1β, TNFα, iNOS, and COX-2) were used to evaluate microglial activation. The transcriptional changes undergone by M1/M2a/M2b markers were used to evaluate phenotype transformation of BV2 cells. Immunofluorescent staining and Western blot were used to detect the location and expression of P65 and p-IKK in the presence or absence of PAP pretreatment.
Pretreatment with PAP significantly inhibited the expression of IL1β and TNFα, and suppressed the transcription of M1/M2b markers Il1rn, Socs3, Nos2 and Ptgs2, but upregulated the transcription of M2a markers (Arg1 and Mrc1) in a dose-dependent manner. In addition, PAP pretreatment significantly decreased the expression of p-IKK and inhibited the nuclear translocation of P65 after LPS stimulation.
PAP not only suppressed the LPS-induced microglial activity by inhibiting transcription/production of proinflammatory factors, but also promoted the transformation of activated BV2 cells from cytotoxic phenotypes (M1/M2b) to a neuroprotective phenotype (M2a). These effects were probably mediated by NF-κB signaling pathway. Thus, it would be a promising candidate for the treatment of neurodegenerative diseases.
PMCID: PMC4777259  PMID: 27013863
papaverine; microglia; neuroprotection; neuroinflammation
12.  Role of a liver-first approach for synchronous colorectal liver metastases 
World Journal of Gastroenterology  2016;22(6):2126-2132.
AIM: To evaluate the feasibility and survival outcomes of a liver-first approach.
METHODS: Between January 2009 and April 2013, 18 synchronous colorectal liver metastases (sCRLMs) patients with a planned liver-first approach in the Hepatopancreatobiliary Surgery Department I of the Beijing Cancer Hospital were enrolled in this study. Clinical data, surgical outcomes, morbidity and mortality rates were collected. The feasibility and long-term outcomes of the approach were retrospectively analyzed.
RESULTS: Sixteen patients (88.9%) completed the treatment protocol for primary and liver tumors. The main reason for treatment failure was liver disease recurrence. The 1 and 3 year overall survival rates were 94.4% and 44.8%, respectively. The median survival time was 30 mo. The postoperative morbidity and mortality were 22.2% and 0%, respectively, following a hepatic resection, and were 18.8% and 0%, respectively, after a colorectal surgery.
CONCLUSION: The liver-first approach appeared to be feasible and safe. It can be performed with a comparable mortality and morbidity to the traditional treatment paradigm. This approach might offer a curative opportunity for sCRLM patients with a high liver disease burden.
PMCID: PMC4726685  PMID: 26877617
Liver metastases; Resection; Colorectal cancer; Synchronous
13.  Illuminating necrosis: From mechanistic exploration to preclinical application using fluorescence molecular imaging with indocyanine green 
Scientific Reports  2016;6:21013.
Tissue necrosis commonly accompanies the development of a wide range of serious diseases. Therefore, highly sensitive detection and precise boundary delineation of necrotic tissue via effective imaging techniques are crucial for clinical treatments; however, no imaging modalities have achieved satisfactory results to date. Although fluorescence molecular imaging (FMI) shows potential in this regard, no effective necrosis-avid fluorescent probe has been developed for clinical applications. Here, we demonstrate that indocyanine green (ICG) can achieve high avidity of necrotic tissue owing to its interaction with lipoprotein (LP) and phospholipids. The mechanism was explored at the cellular and molecular levels through a series of in vitro studies. Detection of necrotic tissue and real-time image-guided surgery were successfully achieved in different organs of different animal models with the help of FMI using in house-designed imaging devices. The results indicated that necrotic tissue with a 0.6 mm diameter could be effectively detected with precise boundary definition. We believe that the new discovery and the associated imaging techniques will improve personalized and precise surgery in the near future.
PMCID: PMC4749996  PMID: 26864116
14.  Covered versus Uncovered Self-Expandable Metal Stents for Managing Malignant Distal Biliary Obstruction: A Meta-Analysis 
PLoS ONE  2016;11(2):e0149066.
To compare the efficacy of using covered self-expandable metal stents (CSEMSs) and uncovered self-expandable metal stents (UCSEMSs) to treat objective jaundice caused by an unresectable malignant tumor.
We performed a comprehensive electronic search from 1980 to May 2015. All randomized controlled trials comparing the use of CSEMSs and UCSEMSs to treat malignant distal biliary obstruction were included.
The analysis included 1417 patients enrolled in 14 trials. We did not detect significant differences between the UCSEMS group and the CSEMS group in terms of cumulative stent patency (hazard ratio (HR) 0.93, 95% confidence interval (CI) 0.19–4.53; p = 0.93, I2 = 0%), patient survival (HR 0.77, 95% CI 0.05–10.87; p = 0.85, I2 = 0%), overall stent dysfunction (relative ratio (RR) 0.85, M-H, random, 95% CI 0.57–1.25; p = 0.83, I2 = 63%), the overall complication rate (RR 1.26, M-H, fixed, 95% CI 0.94–1.68; p = 0.12, I2 = 0%) or the change in serum bilirubin (weighted mean difference (WMD) -0.13, IV fixed, 95% CI 0.56–0.3; p = 0.55, I2 = 0%). However, we did detect a significant difference in the main causes of stent dysfunction between the two groups. In particular, the CSEMS group exhibited a lower rate of tumor ingrowth (RR 0.25, M-H, random, 95% CI 0.12–0.52; p = 0.002, I2 = 40%) but a higher rate of tumor overgrowth (RR 1.76, M-H, fixed, 95% CI 1.03–3.02; p = 0.04, I2 = 0%). Patients with CSEMSs also exhibited a higher migration rate (RR 9.33, M-H, fixed, 95% CI 2.54–34.24; p = 0.008, I2 = 0%) and a higher rate of sludge formation (RR 2.47, M-H, fixed, 95% CI 1.36–4.50; p = 0.003, I2 = 0%).
Our meta-analysis indicates that there is no significant difference in primary stent patency and stent dysfunction between CSEMSs and UCSEMSs during the period from primary stent insertion to primary stent dysfunction or patient death. However, when taking further management for occluded stents into consideration, CSEMSs is a better choice for patients with malignant biliary obstruction due to their removability.
PMCID: PMC4747571  PMID: 26859673
15.  PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance 
The Journal of Cell Biology  2015;210(3):485-502.
A coincidence detection mechanism regulates phagosomal sealing and couples it with phosphoinositide conversion from PtdIns(4,5)P2 enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.
Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P2 to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P2, PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P2 enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.
PMCID: PMC4523610  PMID: 26240185
16.  Angle-Polarization Estimation for Coherent Sources with Linear Tripole Sensor Arrays 
We propose a parallel factor (PARAFAC) analysis-based angle and polarization estimation algorithm for multiple coherent sources using a uniformly-spaced linear tripole sensor array. By forming a PARAFAC model using the spatial signature of the tripole array, the new algorithm requires neither spatial smoothing nor vector-field smoothing to decorrelate the signal coherency. We also establish that the angle-polarization parameters of K coherent signals can be uniquely determined by PARAFAC analysis, as long as the number of tripoles L≥2K−1. In addition, the proposed algorithm can offer enhanced angle and polarization estimation accuracy by extending the interspacing of the tripoles beyond a half wavelength.
PMCID: PMC4801624  PMID: 26907273
parallel factor; tripole sensor; angle estimation; polarization estimation
17.  EEG oscillatory patterns and classification of sequential compound limb motor imagery 
A number of studies have been done on movement imagination of motor sequences with a single limb. However, brain oscillatory patterns induced by movement imagination of motor sequences involving multiple limbs have not been reported in recent years. The goal of the present study was to verify the feasibility of application of motor sequences involving multiple limbs to brain-computer interface (BCI) systems based on motor imagery (MI). The changes of EEG patterns and the inter-influence between movements associated with the imagination of motor sequences were also investigated.
The experiment, where 12 healthy subjects participated, involved one motor sequence with a single limb and three kinds of motor sequences with two or three limbs. The activity involved mental simulation, imagining playing drums with two conditions (60 and 30 beats per minute for the first and second conditions, respectively).
Movement imagination of different limbs in the sequence contributed to time-variant event-related desynchronization (ERD) patterns within both mu and beta rhythms, which was more obvious for the second condition compared with the first condition. The ERD values of left/right hand imagery with prior hand imagery were significantly larger than those with prior foot imagery, while the phase locking values (PLVs) between central electrodes and the mesial frontocentral electrode of non-initial movement were significantly larger than those of the initial movement during imagination of motor sequences for both conditions. Classification results showed that the power spectral density (PSD) based method outperformed the multi-class common spatial patterns (multi-CSP) based method: The highest accuracies were 82.86 % and 91.43 %, and the mean values were 65 % and 74.14 % for the first and second conditions, respectively.
This work implies that motor sequences involving multiple limbs can be utilized to build a multimodal classification paradigm in MI-based BCI systems, and that prior movement imagination can result in the changes of neural activities in motor areas during subsequent movement imagination in the process of limb switching.
PMCID: PMC4731999  PMID: 26822435
Sequential compound limb motor imagery; Event-related desynchronization; Phase locking value; Brain-computer interface; Support vector machine
18.  MiR-548c impairs migration and invasion of endometrial and ovarian cancer cells via downregulation of Twist 
MicroRNAs (miRNAs) are a class of small non-coding RNAs, which post-transcriptionally repress the expression of genes involved in cancer initiation and progression. Although some miRNAs that target many signaling pathways (also called universe miRNAs) are supposed to play a global role in diverse human tumors, their regulatory functions in gynecological cancers remain largely unknown. We investigated the biological role and underlying mechanism of miR-548c (one universe miRNA) in endometrial and ovarian cancer.
The effects of miR-548c overexpression on cell proliferation, migration and invasion were studied in endometrial and ovarian cancer cells. TWIST1 (Twist) was identified as a direct miR-548c target by western blot analysis and luciferase activity assay. The expression of miR-548c and Twist were examined by qRT-PCR in endometrial and ovarian cancer tissues.
Here, we report that miR-548c is down-regulated in endometrial and ovarian cancer tissues when compared to normal tissues, and our meta-analysis reveal that decreased miR-548c expression correlates with poor prognosis in endometrial cancer patients. We show that in endometrial and ovarian cancer cells, ectopic expression of miR-548c significantly inhibits whereas knockdown of miR-548c dramatically induces cancer cell proliferation, migration and invasion. By using luciferase reporter assay, we demonstrate that Twist, a known oncogene in endometrial and ovarian cancers, is a direct target of miR-548c. Furthermore, the expression of Twist partially abrogates the tumor suppressive effects of miR-548c on cell migration and invasion.
These findings suggest that miR-548c directly downregulates Twist, and provide a novel mechanism for Twist upregulation in both endometrial and ovarian cancers. The use of miR-548c may hold therapeutic potential for the treatment of Twist-overexpressing tumors.
PMCID: PMC4712560  PMID: 26762267
microRNA; miR-548c; EMT; Endometrial cancer; Ovarian cancer
19.  Intracoronary Transplantation of Mesenchymal Stem Cells with Overexpressed Integrin-Linked Kinase Improves Cardiac Function in Porcine Myocardial Infarction 
Scientific Reports  2016;6:19155.
The effect of mesenchymal stem cell (MSCs)-based therapy on treating acute myocardial infarction (MI) is limited due to poor engraftment and limited regenerative potential. Here we engineered MSCs with integrin-linked kinase (ILK), a pleiotropic protein critically regulating cell survival, proliferation, differentiation, and angiogenesis. We firstly combined ferumoxytol with poly-L-lysine (PLL), and found this combination promisingly enabled MRI visualization of MSCs in vitro and in vivo with good safety. We provided visually direct evidence that intracoronary ILK-MSCs had substantially enhanced homing capacity to infarct myocardium in porcine following cardiac catheterization induced MI. Intracoronary transplantation of allogeneic ILK-MSCs, but not vector-MSCs, significantly enhanced global left ventricular ejection fraction (LVEF) by 7.8% compared with baseline, by 10.3% compared with vehicles, and inhibited myocardial remodeling compared with vehicles at 15-day follow-up. Compared with vector-MSCs, ILK-MSCs significantly improved regional LV contractile function, reduced scar size, fibrosis, cell apoptosis, and increased regional myocardial perfusion and cell proliferation. This preclinical study indicates that ILK-engineered MSCs might promote the clinical translation of MSC-based therapy in post-MI patients, and provides evidence that ferumoxytol labeling of cells combined with PLL is feasible in in vivo cell tracking.
PMCID: PMC4707493  PMID: 26750752
20.  Yak whole-genome resequencing reveals domestication signatures and prehistoric population expansions 
Nature Communications  2015;6:10283.
Yak domestication represents an important episode in the early human occupation of the high-altitude Qinghai-Tibet Plateau (QTP). The precise timing of domestication is debated and little is known about the underlying genetic changes that occurred during the process. Here we investigate genome variation of wild and domestic yaks. We detect signals of selection in 209 genes of domestic yaks, several of which relate to behaviour and tameness. We date yak domestication to 7,300 years before present (yr BP), most likely by nomadic people, and an estimated sixfold increase in yak population size by 3,600 yr BP. These dates coincide with two early human population expansions on the QTP during the early-Neolithic age and the late-Holocene, respectively. Our findings add to an understanding of yak domestication and its importance in the early human occupation of the QTP.
Based on whole genome resequencing of more than 80 wild and domestic yaks from high-altitude Qinghai-Tibet Plateau, this study recovers demographic and genetic processes underlying the domestication of this animal. Qiu et al. also identify genes and associated pathways as candidates for selection during the domestication process.
PMCID: PMC4703879  PMID: 26691338
21.  Preliminary X-ray diffraction analysis of thermostable β-1,4-xylanase from Streptomyces sp. S9 
Recombinant XynAS9, a thermophilic GH10 xylanase from Streptomyces sp. S9, was crystallized and X-ray diffraction data were collected to 2.08 Å resolution.
Xylanase, which catalyzes the random hydrolysis of internal xylosidic linkages, is a critical enzyme participating in xylan decomposition and has been widely applied in industrial utilizations. Xylanase isolated from the extremophilic Streptomyces sp. S9 (XynAS9) possesses broad adaptability to temperature and pH and thus is an attractive candidate in industrial applications. In particular, the major products of XynAS9 are xylose and xylobiose, which enable the subsequent bioconversion to be carried out with higher efficiency. Therefore, the three-dimensional structure of XynAS9 and its catalytic machinery are of great interest. Here, recombinant XynAS9 protein was expressed in Pichia pastoris, purified and crystallized. Crystals belonging to the hexagonal space group P6522, with unit-cell parameters a = b = 80.9, c = 289.3 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.08 Å resolution. Initial phase determination using molecular replacement indicated that the crystal contains one molecule in an asymmetric unit. Further model building and structural refinement are in progress.
PMCID: PMC3943089  PMID: 24419629
xylanase; Streptomyces; thermotolerance; industrial enzymes
22.  EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway 
Journal of Virology  2015;89(13):6608-6618.
The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.
IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.
PMCID: PMC4468487  PMID: 25878102
23.  Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay 
Still, there is need for significant improvements in reliable and accurate diagnosis for Alzheimer's disease (AD) at early stages. It is widely accepted that changes in the concentration and conformation of amyloid-β (Aβ) appear several years before the onset of first symptoms of cognitive impairment in AD patients. Because Aβ oligomers are possibly the major toxic species in AD, they are a promising biomarker candidate for the early diagnosis of the disease. To date, a variety of oligomer-specific assays have been developed, many of them ELISAs. Here, we demonstrate the sFIDA assay, a technology highly specific for Aβ oligomers developed toward single particle sensitivity. By spiking stabilized Aβ oligomers to buffer and to body fluids from control donors, we show that the sFIDA readout correlates with the applied concentration of stabilized oligomers diluted in buffer, cerebrospinal fluid (CSF), and blood plasma over several orders of magnitude. The lower limit of detection was calculated to be 22 fM of stabilized oligomers diluted in PBS, 18 fM in CSF, and 14 fM in blood plasma.
PMCID: PMC4731524  PMID: 26858588
Alzheimer's disease; amyloid-β peptide; diagnostic biomarker; early diagnosis; sFIDA; surface-based fluorescence intensity distribution analysis; stabilized oligomers; standard molecule
24.  Genome-Wide Dissection of the MicroRNA Expression Profile in Rice Embryo during Early Stages of Seed Germination 
PLoS ONE  2015;10(12):e0145424.
The first 24 hours after imbibition (HAI) is pivotal for rice seed germination, during which embryo cells switch from a quiescent state to a metabolically active state rapidly. MicroRNAs (miRNAs) have increasingly been shown to play important roles in rice development. Nevertheless, limited knowledge about miRNA regulation has been obtained in the early stages of rice seed germination. In this study, the small RNAs (sRNAs) from embryos of 0, 12, and 24 HAI rice seeds were sequenced to investigate the composition and expression patterns of miRNAs. The bioinformatics analysis identified 289 miRNA loci, including 59 known and 230 novel miRNAs, and 35 selected miRNAs were confirmed by stem-loop real-time RT-PCR. Expression analysis revealed that the dry and imbibed seeds have unique miRNA expression patterns compared with other tissues, particularly for the dry seeds. Using three methods, Mireap, psRNATarget and degradome analyses, 1197 potential target genes of identified miRNAs involved in various molecular functions were predicted. Among these target genes, 39 had significantly negative correlations with their corresponding miRNAs as inferred from published transcriptome data, and 6 inversely expressed miRNA-target pairs were confirmed by 5ʹ-RACE assay. Our work provides an inventory of miRNA expression profiles and miRNA-target interactions in rice embryos, and lays a foundation for further studies of miRNA-mediated regulation in initial seed germination.
PMCID: PMC4683037  PMID: 26681181
25.  Retrospective analysis of high intensity focused ultrasound combined with S-1 in the treatment of metastatic pancreatic cancer after failure of gemcitabine 
The purpose of this study was to verify the efficacy and safety of high intensity focused ultrasound therapy (HIFU) combined with S-1 in the treatment of metastatic pancreatic cancer after failure of gemcitabine (GEM). In total, 120 patients with GEM-refractory PC who received HIFU and S-1 between Aug 2012 and December 2014 were randomly assigned to 2 groups. The patients in group A (n = 61) received HIFU in combination with S-1 and those in group B (n = 59) were given S-1 alone. S-1 was administered orally twice a day on days 1-14. Cycles were repeated every 21 days. The follow up time was 3~19 months in both groups. The median overall survival (OS) time and progression free survival (PFS) were analysed by Kaplan-Meier method and Logrank test. The pain remission rate of the two groups was compared by χ2 test. Patient characteristics and prognostic factors were compared. Patient characteristics did not significantly differ between the 2 groups. Median OS was significantly longer in group A (10.3 months) than in group B (6.6 months, P = 0.000). Median PFS was also significantly longer in group A than in group B (5.1 months vs 2.3 months, P = 0.000). Meanwhile, the pain remission rate was markedly higher in group A than in group B (57% vs. 20%, P = 0.000). There were mild side effects and no significant difference was observed between the two groups. The treatment effect was independently associated with a good outcome. HIFU in combination with S-1 might be effective and well tolerated as salvage chemotherapy in the treatment for metastatic pancreatic cancer.
PMCID: PMC4759399  PMID: 27073725
Pancreatic neoplasm; high intensity focused ultrasound therapy; S-1

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