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1.  The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis 
The Journal of Cell Biology  2013;203(1):101-114.
Multiple formins cooperate during cytokinesis, but their functions in de novo actin assembly at the division site play the primary role in contractile ring assembly.
Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.
PMCID: PMC3798249  PMID: 24127216
2.  Identification of Thyroid Carcinoma Related Genes with mRMR and Shortest Path Approaches 
PLoS ONE  2014;9(4):e94022.
Thyroid cancer is a malignant neoplasm originated from thyroid cells. It can be classified into papillary carcinomas (PTCs) and anaplastic carcinomas (ATCs). Although ATCs are in an very aggressive status and cause more death than PTCs, their difference is poorly understood at molecular level. In this study, we focus on the transcriptome difference among PTCs, ATCs and normal tissue from a published dataset including 45 normal tissues, 49 PTCs and 11 ATCs, by applying a machine learning method, maximum relevance minimum redundancy, and identified 9 genes (BCL2, MRPS31, ID4, RASAL2, DLG2, MY01B, ZBTB5, PRKCQ and PPP6C) and 1 miscRNA (miscellaneous RNA, LOC646736) as important candidates involved in the progression of thyroid cancer. We further identified the protein-protein interaction (PPI) sub network from the shortest paths among the 9 genes in a PPI network constructed based on STRING database. Our results may provide insights to the molecular mechanism of the progression of thyroid cancer.
PMCID: PMC3981740  PMID: 24718460
3.  Src Is Required for Mechanical Stretch-Induced Cardiomyocyte Hypertrophy through Angiotensin II Type 1 Receptor-Dependent β-Arrestin2 Pathways 
PLoS ONE  2014;9(4):e92926.
Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal) nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP3) is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.
PMCID: PMC3974699  PMID: 24699426
4.  The Stratifying Value of Hangzhou Criteria in Liver Transplantation for Hepatocellular Carcinoma 
PLoS ONE  2014;9(3):e93128.
The selection criteria for patients with hepatocellular carcinoma (HCC) as candidates for deceased donor liver transplantation (DDLT) are well studied. In this era of limited deceased donor organs, the value of living donor liver transplantation (LDLT) for HCC remains controversial. The aim of the present study was to verify the stratification value of the Hangzhou criteria for LDLT.
The data of 47 LDLT recipients and 94 matched DDLT recipients at our center were evaluated. Overall survival and tumor-free survival were calculated. Prognostic factors influencing post-liver transplantation (LT) survival were identified. The stratification values of the Hangzhou criteria and Milan criteria were compared.
LDLT recipients spent much less time on the waiting list. The post-LT survival of recipients fulfilling the Milan criteria and recipients fulfilling the Hangzhou criteria were comparable (P>0.05). The overall and tumor-free survival did not differ statistically between the two groups. In both groups, more recipients not meeting the Milan criteria but with a satisfactory outcome were identified by the Hangzhou criteria. Among recipients who did not meet the Hangzhou criteria, tumor-free survival was better for the LDLT recipients than the DDLT recipients (P = 0.024).
The Hangzhou criteria are reliable for stratifying HCC patients in terms of prognosis. HCC patients fulfilling the Hangzhou criteria gain satisfactory survival from LT. Outcomes after LDLT are better than those after DDLT for HCC patients who do not meet the Hangzhou criteria.
PMCID: PMC3968059  PMID: 24676010
5.  Polymorphism of rs873308 near the transmembrane protein 57 gene is associated with serum lipid levels 
Bioscience Reports  2014;34(2):e00095.
SNP (single-nucleotide polymorphism) of rs10903129 near the TMEM (transmembrane protein) 57 locus has been associated with TC (total cholesterol) in a previous GWAS (genome-wide association study), but the association of TMEM57 rs873308 SNP and serum lipid levels has not been previously reported. The current study was undertaken to detect the association of the TMEM57 rs873308 SNP and several environmental factors with serum lipid profiles in the Han Chinese and Mulao populations. The genotypes of the TMEM57 rs873308 SNP in 865 individuals of Han Chinese and 902 participants of Mulao nationality were determined by PCR and RFLP (restriction-fragment-length polymorphism) combined with gel electrophoresis and then confirmed by direct sequencing. The T allele frequency of TMEM57 rs873308 SNP was not different between Han and Mulao (23.18% versus 25.72%, P>0.05), but different between males and females in the two ethnic groups (P<0.05). The T allele carriers had lower serum TC, Apo (apolipoprotein) B, HDL-C (high-density lipoprotein cholesterol) levels, ApoA1/ApoB ratio in Han; and lower TAG (triacylglycerol), LDL-C (low-density lipoprotein cholesterol), ApoA1 levels and the ApoA1/ApoB ratio and higher HDL-C levels in Mulao than the T allele non-carriers. There was also different association of the TMEM57 rs873308 SNP and serum lipid profiles between males and females in the both ethnic groups. Serum lipid parameters in the two ethnic groups were also associated with several environmental factors. The association of the TMEM57 rs873308 SNP and serum lipid levels was different in the Han Chinese and Mulao populations and between males and females in the both ethnic groups. There may be a sex-specific association of the TMEM57 rs873308 SNP and serum lipid levels in our study populations.
PMCID: PMC3953947  PMID: 24517463
environmental factors; lipids; single-nucleotide polymorphism; transmembrane protein 57; Apo, apolipoprotein; BMI, body mass index; BP, blood pressure; CHD, coronary heart disease; GWAS, genome-wide association study; HDL-C, high-density lipoprotein cholesterol; LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; RFLP, restriction-fragment-length polymorphism; SNP, single-nucleotide polymorphism; TC, total cholesterol; TAG, triacylglycerol; TMEM, transmembrane protein
6.  Orthotopic liver transplantation in situs inversus adult from an ABO-incompatible donor with situs inversus 
BMC Gastroenterology  2014;14:46.
Situs inversus is a rare congenital anomaly characterized by the complete inversion of thoracic and abdominal organs. Liver transplantation in such patients or from donors in situs inversus is technically challenging because of the reversed anatomic structures. A small number of successful liver transplantation cases concerning situs inverus in either recipients or donors have been recently reported with different graft position and orientation. Here we reported an extremely rare case of liver retransplantation from an ABO incompatible situs inversus donor to an adult situs inversus recipient.
Case presentation
A 53-year-old complete situs inversus man developed graft failure due to severe biliary complication after his first liver transplantation from a situs solitus donor. Re-transplantation was performed using a graft liver from a likewise situs inversus donor. Although the blood type between donor and recipient was incompatible, the post-operative outcome was excellent under proper prophylaxis to the antibody-mediated rejection.
To the best of our knowledge, this is the first report of liver transplantation from situs inversus to situs inversus in adult recipient. Liver transplantation using situs matching donor makes the procedure much easier at the surgical point of view, which has a benefit of less potential surgical complications. Furthermore, ABO-incompatibility is acceptable for donor allocation in cases that both donor and recipient are situs inversus.
PMCID: PMC3975226  PMID: 24625305
Liver transplantation; Retransplantation; Situs inversus; Abo incompatible
7.  Quantitative Assessment of the Presence and Severity of Cirrhosis in Patients with Hepatitis B Using Right Liver Lobe Volume and Spleen Size Measured at Magnetic Resonance Imaging 
PLoS ONE  2014;9(3):e89973.
To determine whether right liver lobe volume (RV) and spleen size measured utilizing magnetic resonance (MR) imaging could identify the presence and severity of cirrhosis in patients with hepatitis B.
Two hundred and five consecutive patients with clinically confirmed diagnosis of cirrhosis due to hepatitis B and 40 healthy control individuals were enrolled in this study and underwent abdominal triphasic enhanced scans using MR imaging. Spleen maximal width (W), thickness (T) and length (L), together with RV and spleen volume (SV), were measured utilizing MR imaging. Spleen multidimensional index (SI) was obtained by multiplying previously acquired parameters W×T×L. Then statistical assessment was performed to evaluate the ability of these parameters, including RV, SV, RV/SV and SI, to identify the presence of cirrhosis and define Child-Pugh class of this disease.
SV and SI tended to increase (r = 0.557 and 0.622, respectively; all P<0.001), and RV and RV/SV tended to decrease (r = −0.749 and −0.699, respectively; all P<0.001) with increasing Child-Pugh class of cirrhosis. All the parameters, including RV, SV, RV/SV and SI, might be the indicators used to discriminate the patients with liver cirrhosis from the control group, and to distinguish these patients between Child-Pugh class A and B, between B and C, and between A and C (area under receiver operating characteristic curve [AUC] = 0.609–0.975, all P<0.05). Among these parameters, RV/SV was the best noninvasive factor for the discrimination of liver cirrhosis between Child-Pugh class A and B (AUC = 0.725), between A and C (AUC = 0.975), and between B and C (AUC = 0.876), while SI was the best variable to distinguish the cirrhosis patients from the control group (AUC = 0.960, P<0.05).
RV/SV should be used to identify the severity of cirrhosis, while SI can be recommended to determine the presence of this disease.
PMCID: PMC3942406  PMID: 24594920
8.  Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells 
Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR) is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05). Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38). Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.
PMCID: PMC3975384  PMID: 24663081
RNA binding motif protein 38; HOX transcript antisense RNA; hepatocellular carcinoma; long non-coding RNA
9.  Analysis of the impact of platinum-based combination chemotherapy in small cell cervical carcinoma: a multicenter retrospective study in Chinese patients  
BMC Cancer  2014;14:140.
Small cell cervical carcinoma (SCCC) is a rare, aggressive tumor with a poor prognosis. However, information in relation to its treatment is scarce due to the limited numbers of patients. The aim of this study was to establish whether platinum-based combination chemotherapy may by beneficial in this patient population.
We carried out a multicenter, retrospective study comprising of 72 Chinese patients with SCCC. The patients were treated between 1995 and 2010 at Sun Yat-sen Memorial Hospital or the Cancer Center of Sun Yat-Sen University, and at the First Affiliated Hospital of Shantou University Medical College, China.
Of the 72 patients, 46/72 (63.9%) had Federation of Gynecology and Obstetrics (FIGO) stage Ia–Ib2 and 26/72 (36.1%) had stage IIa–IV disease. Surgery was performed in 63/72 (87.5%) patients, 61/72 (84.7%) patients received chemoradiotherapy and 35/72 (48.6%) received radiotherapy. The 3-year overall survival (OS) and disease-free survival (DFS) rates were as follows: Ia (100%, 100%); Ib1 (62%, 57%); Ib2 (53%, 48%); IIa (36%, 23%); IIb (29%, 21%); IIIb (50%, 50%); and IV (0%, 0%), respectively. The estimated 3-year OS and DFS rates in patients who received platinum-based combination chemotherapy (etoposide + cisplatin [EP], or paclitaxel + cisplatin [TP]) as part of their adjuvant treatment were 64.8% and 63.0%, respectively, compared to 25.2% and 22.0% in those who did not (P = 0.0003; P = 0.0003). Univariate analysis showed that platinum-based combination chemotherapy was associated with improved survival compared to other chemotherapy techniques or no chemotherapy (OS: HR = 0.227; 95% CI, 0.099–0.524; P = 0.001; DFS: HR = 0.210; 95% CI, 0.087–0.506; P = 0.001). Multivariate analysis identified FIGO stage, lymphatic metastasis and platinum-based combination chemotherapy as independent prognostic factors for improved survival in patients with SCCC.
Platinum-based combination chemotherapy (with EP or TP) can improve the 3-year survival outcomes in patients with SCCC. Therefore, it should be considered an important component in a future standardized treatment strategy for SCCC.
PMCID: PMC3939817  PMID: 24575810
Cervical cancer; Small cell carcinoma; Platinum-based combination chemotherapy; Prognosis
10.  Cell-size control 
Cell Cycle  2014;13(5):693-694.
PMCID: PMC3979902  PMID: 24526119
Cdr2 kinase; Pom1 kinase; cell division; cell size; fission yeast; mitotic entry
11.  Salvage Liver Transplantation for Recurrent Hepatocellular Carcinoma after Liver Resection: Retrospective Study of the Milan and Hangzhou Criteria 
PLoS ONE  2014;9(1):e87222.
Salvage liver transplantation (SLT) has recently been proposed for recurrent hepatocellular carcinoma after liver resection; however, criteria for candidate assessment in SLT have not been thoroughly evaluated.
Methods and Findings
We retrospectively analyzed outcomes and factors affecting survival of 53 recipients who received SLT in the Liver Transplantation Center, The First Affiliated Hospital of Zhejiang University between 2004 and 2012. Thirty recipients fulfilled the Hangzhou criteria, of which 16 also fulfilled the Milan criteria, while the remaining 23 exceeded both criteria. The 1-year, 3-year and 5-year overall survival rates and tumor-free survival rates were both superior in patients fulfilling Milan or Hangzhou criteria compared with those exceeding the criteria. For recipients outside Milan criteria but within Hangzhou criteria, the 1-year, 3-year overall survival rates were 70.1%, 70.1%, similar to recipients within Milan criteria, with the 1-year, 3-year and 5-year overall survival of 93.8%%, 62.1% and 62.1% (P = 0.586). The tumor-free survival rates were also similar between these two subgroups, with 51.9% and 51.9% vs. 85.6%, 85.6% and 64.2% during the same time interval, respectively (P = 0.054). Cox regression analysis identified Hangzhou criteria (within vs. outside, hazard ratio (HR) 0.376) and diameter of the largest tumor (HR 3.523) to be independent predictors for overall survival. The only predictor for tumor-free survival was diameter of the largest tumor (HR 22.289).
Hangzhou criteria safely expanded the candidate pool and are feasible in assessment of candidates for SLT. This is helpful in donor liver allocation in transplant practice.
PMCID: PMC3903638  PMID: 24475255
12.  De novo Cancers Following Liver Transplantation: A Single Center Experience in China 
PLoS ONE  2014;9(1):e85651.
De novo cancers are a growing problem that has become one of the leading causes of late mortality after liver transplantation. The incidences and risk factors varied among literatures and fewer concerned the Eastern population.
The aim of this study was to examine the incidence and clinical features of de novo cancers after liver transplantation in a single Chinese center.
569 patients who received liver transplantation and survived for more than 3 months in a single Chinese center were retrospectively reviewed.
A total of 18 de novo cancers were diagnosed in 17 recipients (13 male and 4 female) after a mean of 41±26 months, with an overall incidence of 3.2%, which was lower than that in Western people. Of these, 8 (3.32%) cases were from 241 recipients with malignant liver diseases before transplant, while 10 (3.05%) cases were from 328 recipients with benign diseases. The incidence rates were comparable, p = 0.86. Furthermore, 2 cases developed in 1 year, 5 cases in 3 years and 11 cases over 3 years. The most frequent cancers developed after liver transplantation were similar to those in the general Chinese population but had much higher incidence rates.
Liver transplant recipients were at increased risk for developing de novo cancers. The incidence rates and pattern of de novo cancers in Chinese population are different from Western people due to racial and social factors. Pre-transplant malignant condition had no relationship to de novo cancer. Exact risk factors need further studies.
PMCID: PMC3901656  PMID: 24475047
13.  A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells 
PLoS ONE  2014;9(1):e86260.
Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.
PMCID: PMC3897697  PMID: 24465995
14.  Parecoxib Reduces Systemic Inflammation and Acute Lung Injury in Burned Animals with Delayed Fluid Resuscitation 
Burn injuries result in the release of proinflammatory mediators causing both local and systemic inflammation. Multiple organ dysfunctions secondary to systemic inflammation after severe burn contribute to adverse outcome, with the lungs being the first organ to fail. In this study, we evaluate the anti-inflammatory effects of Parecoxib, a parenteral COX-2 inhibitor, in a delayed fluid resuscitation burned rat model. Anaesthetized Sprague Dawley rats were inflicted with 45% total body surface area full-thickness scald burns and subsequently subjected to delayed resuscitation with Hartmann's solution. Parecoxib (0.1, 1.0, and 10 mg/kg) was delivered intramuscularly 20 min after injury followed by 12 h interval and the rats were sacrificed at 6 h, 24 h, and 48 h. Burn rats developed elevated blood cytokines, transaminase, creatinine, and increased lung MPO levels. Animals treated with 1 mg/kg Parecoxib showed significantly reduced plasma level of CINC-1, IL-6, PGEM, and lung MPO. Treatment of 1 mg/kg Parecoxib is shown to mitigate systemic and lung inflammation without significantly affecting other organs. At present, no specific therapeutic agent is available to attenuate the systemic inflammatory response secondary to burn injury. The results suggest that Parecoxib may have the potential to be used both as an analgesic and ameliorate the effects of lung injury following burn.
PMCID: PMC3918702  PMID: 24579056
15.  One-stage surgical management for tuberculosis of the upper cervical spine by posterior debridement, short-segment fusion, and posterior instrumentation in children 
European Spine Journal  2012;22(1):72-78.
To evaluate the clinical study efficacy and feasibility of 11 children with tuberculosis of the upper cervical spine treated by one-stage posterior debridement, short-segment fusion, and posterior instrumentation.
Eleven children who suffered from tuberculosis of the upper cervical spine were admitted to our hospital between June 2005 and December 2010. All of them were treated by one-stage posterior debridement, short-segment fusion, and posterior instrumentation. Then, the clinical efficacy was evaluated using statistical analysis based on the materials about the visual analogue scale (VAS) scores of pain, JOA scores of nerve function and erythrocyte sedimentation rate (ESR), which were collected at certain time.
The average follow-up period was 28.1 ± 10.5 months (13–42 months). In the 11 cases, no postoperative complications related to instrumentation occurred and neurologic function was improved in various degrees. The average pretreatment ESR was 58.4 ± 4.9 mm/h (53–69 mm/h), which got normal (8.9 ± 6.5 mm/h) within 3 months in all patients. The average preoperative VAS was 7.4 ± 2.2, which decreased to 1.6 ± 1.8 postoperatively. Mean preoperative JOA was 11.2 ± 3.8, and the JOA at the last visit was 16.3 ± 1.0. All patients got bony fusion within 3–8 months after surgery.
One-stage posterior debridement, short-segment fusion, and posterior instrumentation can be an effective treatment method for the treatment of tuberculosis of the upper cervical spine in children.
PMCID: PMC3540302  PMID: 23085858
Tuberculosis; Upper cervical spine; Children; Posterior instrumentation; Short-segment fusion
16.  Human Hepatic Progenitor Cells Express Hematopoietic Cell Markers CD45 and CD109 
Objective: To clarify the precise characteristics of human hepatic progenitor cells (HPCs) for future cytotherapy in liver diseases.
Methods: Hepatic progenitor-like cells were isolated and cultured from the livers of patients who had undergone partial hepatectomy for various pathologies but displayed no sign of hepatic dysfunction. These cells were characterized by transcriptomic profiling, quantitative real-time PCR and immunocyto/histochemistry.
Results:Cultured HPCs contained polygonal, high nucleus/cytoplasm ratio and exhibited a global gene expression profile similar (67.8%) to that of primary hepatocytes. Among the genes with more than 20-fold higher expression in HPCs were a progenitor marker (CD90), a pentraxin-related gene (PTX3), collagen proteins (COL5A2, COL1A1 and COL4A2), cytokines (EGF and PDGFD), metabolic enzymes (CYBRD1, BCAT1, TIMP2 and PAM), a secreted protein (SPARC) and an endothelial protein C receptor (PROCR). Moreover, eight markers (ALB, AFP, CK8, CK18, CK19, CD90, CD117 and Oval-6) previously described as HPC markers were validated by qRT-PCR and/or immunocyto/histochemistry. Interestingly, human HPCs were also positive for the hematopoietic cell markers CD45 and CD109. Finally, we characterized the localization of HPCs in the canals of Hering and periportal areas with six previously described markers (Oval-6, CK8, CK18, CK19, CD90 and CD117) and two potential markers (CD45 and CD109).
Conclusion: The human HPCs are highly similar to primary hepatocytes in their transcriptional profiles. The CD45 and CD109 markers could potentially be utilized to identify and isolate HPCs for further cytotherapy of liver diseases.
PMCID: PMC3880993  PMID: 24396288
Human hepatic progenitor cell; Immunocytochemistry; Transcriptional profile
17.  Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the regulation of fission yeast cytokinesis 
Molecular Biology of the Cell  2013;24(20):3187-3204.
Previous results showed that putative Rho-GEF Gef2 regulates division-site positioning during early cytokinesis in fission yeast. Here Nod1 is identified as a binding partner of Gef2. The two proteins form a complex to regulate division-site positioning and contractile-ring maintenance. In addition, Gef2 binds to GTPases Rho1, Rho4, and Rho5 in vitro.
Cytokinesis is the last step of the cell-division cycle, which requires precise spatial and temporal regulation to ensure genetic stability. Rho guanine nucleotide exchange factors (Rho GEFs) and Rho GTPases are among the key regulators of cytokinesis. We previously found that putative Rho-GEF Gef2 coordinates with Polo kinase Plo1 to control the medial cortical localization of anillin-like protein Mid1 in fission yeast. Here we show that an adaptor protein, Nod1, colocalizes with Gef2 in the contractile ring and its precursor cortical nodes. Like gef2∆, nod1∆ has strong genetic interactions with various cytokinesis mutants involved in division-site positioning, suggesting a role of Nod1 in early cytokinesis. We find that Nod1 and Gef2 interact through the C-termini, which is important for their localization. The contractile-ring localization of Nod1 and Gef2 also depends on the interaction between Nod1 and the F-BAR protein Cdc15, where the Nod1/Gef2 complex plays a role in contractile-ring maintenance and affects the septation initiation network. Moreover, Gef2 binds to purified GTPases Rho1, Rho4, and Rho5 in vitro. Taken together, our data indicate that Nod1 and Gef2 function cooperatively in a protein complex to regulate fission yeast cytokinesis.
PMCID: PMC3806657  PMID: 23966468
18.  Refractive Error, Visual Acuity and Causes of Vision Loss in Children in Shandong, China. The Shandong Children Eye Study 
PLoS ONE  2013;8(12):e82763.
To examine the prevalence of refractive errors and prevalence and causes of vision loss among preschool and school children in East China.
Using a random cluster sampling in a cross-sectional school-based study design, children with an age of 4–18 years were selected from kindergartens, primary schools, and junior and senior high schools in the rural Guanxian County and the city of Weihai. All children underwent a complete ocular examination including measurement of uncorrected (UCVA) and best corrected visual acuity (BCVA) and auto-refractometry under cycloplegia. Myopia was defined as refractive error of ≤−0.5 diopters (D), high myopia as ≤−6.0D, and amblyopia as BCVA ≤20/32 without any obvious reason for vision reduction and with strabismus or refractive errors as potential reasons.
Out of 6364 eligible children, 6026 (94.7%) children participated. Prevalence of myopia (overall: 36.9±0.6%;95% confidence interval (CI):36.0,38.0) increased (P<0.001) from 1.7±1.2% (95%CI:0.0,4.0) in the 4-years olds to 84.6±3.2% (95%CI:78.0,91.0) in 17-years olds. Myopia was associated with older age (OR:1.56;95%CI:1.52,1.60;P<0.001), female gender (OR:1.22;95%CI:1.08,1.39;P = 0.002) and urban region (OR:2.88;95%CI:2.53,3.29;P<0.001). Prevalence of high myopia (2.0±0.2%) increased from 0.7±0.3% (95%CI:0.1,1.3) in 10-years olds to 13.9±3.0 (95%CI:7.8,19.9) in 17-years olds. It was associated with older age (OR:1.50;95%CI:1.41,1.60;P<0.001) and urban region (OR:3.11;95%CI:2.08,4.66);P<0.001). Astigmatism (≥0.75D) (36.3±0.6%;95%CI:35.0,38.0) was associated with older age (P<0.001;OR:1.06;95%CI:1.04,1.09), more myopic refractive error (P<0.001;OR:0.94;95%CI:0.91,0.97) and urban region (P<0.001;OR:1.47;95%CI:1.31,1.64). BCVA was ≤20/40 in the better eye in 19 (0.32%) children. UCVA ≤20/40 in at least one eye was found in 2046 (34.05%) children, with undercorrected refractive error as cause in 1975 (32.9%) children. Amblyopia (BCVA ≤20/32) was detected in 44 (0.7%) children (11 children with bilateral amblyopia).
In coastal East China, about 14% of the 17-years olds were highly myopic, and 80% were myopic. Prevalence of myopia increased with older age, female gender and urban region. About 0.7% of pre-school children and school children were amblyopic.
PMCID: PMC3871613  PMID: 24376575
19.  Differential expression of Notch1 intracellular domain and p21 proteins, and their clinical significance in gastric cancer 
Oncology Letters  2013;7(2):471-478.
Changes in the expression of the Notch1 intracellular domain (NICD) and p21 proteins have been shown to be closely associated with the development and progression of a number of cancers. The present study aimed to investigate the expression levels of the two proteins in gastric carcinoma and precancerous lesions, and to determine the clinical significance of this. A total of 109 gastric cancer, 57 precancerous gastric lesion, 50 chronic superficial gastritis and 17 normal gastric mucosa patients were recruited for immunohistochemical staining of NICD and p21 protein expression. The protein expression levels in the gastric cancer patient samples were associated with the clinicopathological and survival data. NICD protein levels were upregulated gradually from normal gastric mucosae through chronic superficial gastritis and precancerous gastric lesions to gastric cancer tissues (P<0.01), whereas p21 protein levels were downregulated accordingly (P<0.01). Increased NICD and a loss of p21 expression were closely associated with tumor dedifferentiation, depth of tumor invasion, lymph node metastasis, surface morphology and Lauren classification in gastric cancer. Thus, NICD expression was inversely associated with p21 expression. In addition, the overall survival rate was greater in NICD− and P21+ patients than in NICD+ and P21− patients, respectively (P<0.05). The COX regression multivariate analysis revealed that NICD+, p21−, depth of tumor invasion and lymph node metastasis were all independent prognostic factors for patients with gastric cancer. NICD and p21 proteins are differentially expressed in gastric cancer and the aberrant expression of these proteins is associated with an advanced tumor stage, tumor metastasis and overall patient survival. Future studies are required to further evaluate the two proteins as novel prognostic markers for patients with gastric cancer.
PMCID: PMC3881918  PMID: 24396472
Notch1 intracellular doman; p21; gastric cancer; prognosis; biomarkers
20.  Detection of circulating vascular endothelial growth factor and matrix metalloproteinase-9 in non-small cell lung cancer using Luminex multiplex technology 
Oncology Letters  2013;7(2):499-506.
It has been previously reported that vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 are important for the occurrence and development of non-small cell lung cancer (NSCLC). The present study was designed to detect the serum levels of VEGF and MMP-9 in NSCLC, and to explore their diagnostic and prognostic values. A total of 543 cases were involved, of which 332 were NSCLC (272 cases in the pretreatment group and 60 cases in the postoperative group), 91 were patients with benign lung diseases and 120 were healthy controls. The serum levels of VEGF and MMP-9 were determined by Luminex multiplex technology. The serum levels of VEGF and MMP-9 were found to be significantly higher in the pretreatment group than those in the patients with benign lung diseases and healthy controls (VEGF, P<0.0001; MMP-9, P<0.0001). Compared with the pretreatment group, the serum levels of VEGF and MMP-9 in the postoperative group were significantly decreased (VEGF, P=0.005; MMP-9, P=0.002), and the levels of VEGF and MMP-9 in the pretreatment group of patients with stages III and IV were higher than those with stages I and II (VEGF, P<0.0001; MMP-9, P=0.021). In addition, the levels of VEGF and MMP-9 were found to closely correlate with lymph node metastasis (VEGF, P<0.0001; MMP-9, P<0.0001) in the pretreatment group, while being independent of other clinicopathological parameters (P>0.05). Furthermore, a positive correlation was observed between the serum levels of VEGF and MMP-9 (r=0.159; P=0.009). A receiver operating characteristic curve analysis showed that the diagnostic value of MMP-9 was higher than that of VEGF in the pretreatment group. The log-rank test indicated that the inoperable NSCLC patients with low levels of VEGF exhibited a significantly longer overall survival time than those with high VEGF levels (P<0.0001). Additionally, the serum levels of VEGF and lymph node metastasis were identified as independent prognostic factors of the inoperable NSCLC patients in a multivariate Cox regression analysis (P<0.05). These results indicated that VEGF and MMP-9 may be potential biomarkers for the diagnosis and prognosis of NSCLC.
PMCID: PMC3881935  PMID: 24396477
Luminex; vascular endothelial growth factor; matrix metalloproteinase-9; non-small cell lung cancer
21.  Transanal natural orifice specimen extraction for laparoscopic anterior resection in rectal cancer 
AIM: To investigate whether transanal natural orifice specimen extraction (NOSE) is a better technique for rectal cancer resection.
METHODS: A prospectively designed database of a consecutive series of patients undergoing laparoscopic low anterior resection for rectal cancer with various tumor-node-metastasis classifications from March 2011 to February 2012 at the First Affiliated Hospital of Sun Yat-Sen University was analyzed. Patient selection for transanal specimen extraction and intracorporeal anastomosis was made on the basis of tumor size and distance of rectal lesions from the anal verge. Demographic data, operative parameters, and postoperative outcomes were assessed.
RESULTS: None of the patients was converted to laparotomy. Respectively, there were 16 cases in the low anastomosis and five in the ultralow anastomosis groups. Mean age of the patients was 45.4 years, and mean body mass index was 23.1 kg/m2. Mean distance of the lower edge of the lesion from the anal verge was 8.3 cm. Mean operating time was 132 min, and mean intraoperative blood loss was 84 mL. According to the principle of rectal cancer surgery, we performed D2 lymph node dissection in 13 cases and D3 in eight. Mean lymph nodes harvest was 17.8, and the number of positive lymph nodes was 3.4. Median hospital stay was 6.7 d. No serious postoperative complication occurred except for one anastomotic leakage. All patients remained disease free. Mean Wexner score was 3.7 at 11 mo after the operation.
CONCLUSION: Transanal NOSE for total laparoscopic low/ultralow anterior resection is feasible, safe and oncologically sound. Further studies with long-term outcomes are needed to explore its potential advantages.
PMCID: PMC3837275  PMID: 24282364
Transanal specimen extraction; Natural orifice specimen extraction; Laparoscopic anterior resection; Low/ultra-low anastomosis; Total mesorectal excision
22.  Association between PKA gene polymorphism and NTDs in high risk Chinese population in Shanxi 
Objective: This study aimed to investigate the single nucleotide polymorphisms (SNPs) of PKA and neural tube defects (NTDs) in Chinese population. Method: A total of 183 NTDs cases and 200 healthy controls were used in this study. 7 selected single nucleotide polymorphism (SNP) sites in the PKA gene were analyzed with MassArray high-throughput DNA analyzer with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A series of statistical methods were carried out to investigate the correlation between the SNPs and the patient susceptibility to NTDs. Results: Statistical analysis showed a significant correlation between the SNP sites rs12132032 in PRKACB and NTDs. The AA genotype, A-allele and dominant AA in rs12132032 significantly increased the incidence of NTDs especially anencephaly (OR=3.87, 95% CI: 1.80-8.34 with genotype; OR=2.08, 95% CI: 1.43-3.04 with allele; OR=3.10, 95% CI: 1.53-6.26 with dominant). The T-allele of rs594631 in PRKACB was correlative with NTDs in male but not in female. Conclusions: The gene polymorphism loci rs12132032 in PRKACB maybe a potential risk factor for anencephaly in Chinese population from Shanxi, while gender susceptibility may influence the correlation.
PMCID: PMC3843280  PMID: 24294386
Neural tube defects (NTDs); single nucleotide polymorphisms (SNPs); protein kinase A (PKA); Sonic hedgehog pathway; matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)
23.  Liver Lobe Volumes and the Ratios of Liver Lobe Volumes to Spleen Volume on Magnetic Resonance Imaging for Staging Liver Fibrosis in a Minipig Model 
PLoS ONE  2013;8(11):e79681.
To investigate liver lobe volumes and the ratios of liver lobe volumes to spleen volume measured with magnetic resonance imaging (MRI) for quantitatively monitoring and staging liver fibrosis.
Animal study was approved by Institutional Animal Care and Use Committee. Sixteen minipigs were prospectively used to model liver fibrosis, and underwent abdominal gadolinium-enhanced MRI on 0, 5th, 9th, 16th and 21st weekend after modeling this disease staged by biopsy according to METAVIR classification system. On MRI, volume parameters including left lateral liver lobe volume (LLV), left medial liver lobe volume (LMV), right liver lobe volume (RV), caudate lobe volume (CV), and spleen volume (SV) were measured; and LLV/SV, LMV/SV, RV/SV and CV/SV were calculated. Statistical analyses were performed for staging this fibrosis.
LLV and CV increased with increasing stage of fibrosis (r = 0.711, 0.526, respectively; all P < 0.05). RV and LMV increased from stage 0 to 2 and decreased from 2 to 4; and RV/SV decreased from 0 to 1, increased from 1 to 2, and decreased from 3 to 4 (all P > 0.05). LLV/SV, LMV/SV and CV/SV decreased from stage 0 to 4 (r = -0.566, -0.748 and -0.620, respectively; all P < 0.05). LLV, CV, LLV/SV, LMV/SV, RV/SV, and CV/SV could distinguish stage 0–1 from 2–4 and 0–2 from 3–4 (all P < 0.05). Among these parameters, LLV and LMV/SV could best classify stage ≥2 and ≥3, respectively (area under receiver operating characteristic curve = 0.893 and 0.946, respectively).
LLV and LMV/SV complement each other in staging liver fibrosis, and both parameters should be used to stage this disease.
PMCID: PMC3819276  PMID: 24223184
24.  Bioluminescence Assay Platform for Selective and Sensitive Detection of Ub/Ubl Proteases 
Biochimica et biophysica acta  2012;1823(11):2079-2086.
As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z′ =0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases.
PMCID: PMC3465522  PMID: 22705352
Ubiquitin; SUMO; luciferin; deubiquitylation; protease assay; desumoylation
25.  Counting protein molecules using quantitative fluorescence microscopy 
Trends in biochemical sciences  2012;37(11):499-506.
In recent years, quantification of absolute protein numbers in cellular structures using fluorescence microscopy has become a reality. Two popular methods are available to a broad range of researchers with minimal equipment and analysis requirements: stepwise photobleaching to count discrete changes in intensity from a small number of fluorescent fusion proteins, and comparing the fluorescence intensity of a protein to a known in vivo or in vitro standard. This review summarizes the advantages and disadvantages of each method, and gives recent examples of each that answer important questions in their respective fields. We also highlight new counting methods that could become widely available in the future.
PMCID: PMC3482307  PMID: 22948030

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