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1.  Double CEBPA mutations are prognostically favorable in non-M3 acute myeloid leukemia patients with wild-type NPM1 and FLT3-ITD  
This study is aimed to investigate the pattern of CEBPA mutations and its clinical significance in Chinese non-M3 acute myeloid leukemia (AML) patients. The entire coding region of CEBPA gene was amplified by PCR and then sequenced in samples from 233 non-M3 AML patients. Fifty mutations were identified in 37 (15.8%) patients with eleven (4.7%) double mutated CEBPA (dmCEBPA) and twenty-six (11.1%) single mutated CEBPA (smCEBPA). dmCEBPA was exclusively observed in M1 and M2 subtypes of FAB classification (P = 0.008), whereas smCEBPA occurred in almost all subtypes (P = 0.401). Patients with dmCEBPA had significantly younger age and higher WBC counts than those with wtCEBPA (P = 0.016 and 0.043, respectively). Both dmCEBPA and smCEBPA were mainly present in cytogenetically normal patients. Patients with dmCEBPA achieved higher rate of complete (CR) than wtCEBPA patients (88% vs. 51%, P = 0.037), whereas smCEBPA and wtCEBPA groups are similar (47% vs. 51%, P = 0.810). Patients with dmCEBPA had a superior overall survival (OS) compared with patients with wtCEBPA (P = 0.033), whereas patients with smCEBPA had a similar OS as patients with wtCEBPA (P = 0.976). dmCEBPA but not smCEBPA was also associated with favorable outcome in patients with wild-type NPM1 and FLT3-ITD (NPM1wtFLT3-ITDwt). Our data confirm that dmCEBPA but not smCEBPA is prognostically favorable in NPM1wtFLT3-ITDwt AML, and suggest that the entity AML with mutated CEBPA should be definitely designated as AML with dmCEBPA in WHO classification and smCEBPA should be excluded from the favorable risk of molecular abnormalities.
PMCID: PMC4230151  PMID: 25400766
CEBPA; mutation; prognosis; acute myeloid leukemia
3.  Decreased SFRP2 expression is associated with intermediate and poor karyotypes in de novo acute myeloid leukemia 
Dysregulation of secreted frizzled-related protein 2 (SFRP2) has been found in various cancers. However, it is little known about the pattern of SFRP2 expression in acute myeloid leukemia (AML). This study was aimed to analyze the expression status of SFRP2 gene in AML patients and explore its clinical significance using real-time quantitative PCR (RQ-PCR). The level of SFRP2 expression significantly decreased in AML compared to controls (P<0.001). Receiver operating characteristic curve (ROC) analysis revealed that an area under the ROC curve (AUC) of 0.871 (P<0.001) or 0.902 (P<0.001) in discriminating all patients or cytogenetically normal (CN) patients from controls, respectively. Low level of SFRP2 expression was found more frequently in cytogenetically intermediate and poor groups (72% and 62%, respectively) than in favorable group (42%) (P<0.05). However, there was no significant difference in the rate of complete remission (CR) and overall survival between the groups with low SFRP2 and high expression (P>0.05). SFRP2 expression significantly increased after CR compared to initial diagnosis (P<0.05). These findings suggest that decreased SFRP2 expression is associated with intermediate/poor karyotypes in AML patients and detection of SFRP2 expression may be helpful to the diagnosis and disease monitoring in CN-AML.
PMCID: PMC4152031  PMID: 25197341
SFRP2; acute myeloid leukemia; expression
4.  PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape 
PLoS Computational Biology  2014;10(7):e1003691.
Ubiquitin (Ub) can generate versatile molecular signals and lead to different celluar fates. The functional poly-valence of Ub is believed to be resulted from its ability to form distinct polymerized chains with eight linkage types. To provide a full picture of ubiquitin code, we explore the binding landscape of two free Ub monomers and also the functional landscapes of of all eight linkage types by theoretical modeling. Remarkably, we found that most of the compact structures of covalently connected dimeric Ub chains (diUbs) pre-exist on the binding landscape. These compact functional states were subsequently validated by corresponding linkage models. This leads to the proposal that the folding architecture of Ub monomer has encoded all functional states into its binding landscape, which is further selected by different topologies of polymeric Ub chains. Moreover, our results revealed that covalent linkage leads to symmetry breaking of interfacial interactions. We further propose that topological constraint not only limits the conformational space for effective switching between functional states, but also selects the local interactions for realizing the corresponding biological function. Therefore, the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity. The simulation results also provide several predictions that qualitatively and quantitatively consistent with experiments. Importantly, the K48 linkage model successfully predicted intermediate states. The resulting multi-state energy landscape was further employed to reconcile the seemingly contradictory experimental data on the conformational equilibrium of K48-diUb. Our results further suggest that hydrophobic interactions are dominant in the functional landscapes of K6-, K11-, K33- and K48 diUbs, while electrostatic interactions play a more important role in the functional landscapes of K27, K29, K63 and linear linkages.
Author Summary
Ubiquitination, as an important post-translational modification of proteins, provides a versatile cellular signaling mechanism. This is mostly contributed by the possibility of ubiquitin units to form different polyUb chains through eight different linkages. However, it is still unclear how these linkage types determine the different functions of polyUb chains. In this study, we address this question via the theoretical modeling and molecular dynamics simulation. This allows us to obtain a full picture of topology-function relationship of polyUb chains. The theoretical results led us to propose that topology of polyUb chains selects the functional landscapes from its binding landscape and the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity.
doi:10.1371/journal.pcbi.1003691
PMCID: PMC4081019  PMID: 24992446
5.  Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5 
eLife  2014;3:e02687.
Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity.
DOI: http://dx.doi.org/10.7554/eLife.02687.001
eLife digest
Cells need to allow various molecules to pass through the plasma membrane on their surface. Some molecules have to enter the cell, whereas others have to leave. Cells rely on a process called endocytosis to move large molecules into the cell. This involves part of the membrane engulfing the molecule to form a ‘bubble’ around it. This bubble, which is called an endosome, then moves the molecule to final destination inside the cell.
A protein called Rab5 controls how a new endosome is produced. However, before this can happen, various other molecules—including two proteins called Rabex-5 and Rabaptin-5—must activate the Rab5 protein. Exactly how these three proteins interact with each other was unknown.
Zhang et al. used X-ray crystallography to examine the structures of the complexes formed when Rabex-5 and Rabaptin-5 bind to each other, both when Rab5 is present, and also when it is absent. Biochemical and biophysical experiments confirmed that the Rabex-5/Rabaptin-5 complex is able to activate Rab5.
Zhang et al. also found that Rabex-5, on its own, folds so that the site that normally binds to Rab5 instead binds to a different part of Rabex-5, thus preventing endocytosis. However, when Rabaptin-5 forms a complex with Rabex-5, the Rab5 binding site is freed up.
The Rabex-5/Rabaptin-5 complex can switch between a V shape and a linear structure. Binding to Rab5 stabilizes the linear form of the complex, which then helps activate Rab5, and subsequently the activated Rab5 can interact with other downstream molecules, triggering endocytosis.
DOI: http://dx.doi.org/10.7554/eLife.02687.002
doi:10.7554/eLife.02687
PMCID: PMC4102244  PMID: 24957337
crystal structure; Rab5; rabex-5; Rabaptin-5; GEF activity; molecular mechanism; E. coli; human
6.  Detailed Analysis of Temporal Features on Contrast Enhanced Ultrasound May Help Differentiate Intrahepatic Cholangiocarcinoma from Hepatocellular Carcinoma in Cirrhosis 
PLoS ONE  2014;9(5):e98612.
Aim
To verify if detailed analysis of temporal enhancement patterns on contrast enhanced ultrasound (CEUS) may help differentiate intrahepatic cholangiocarcinoma (ICC) from hepatocellular carcinoma (HCC) in cirrhosis.
Methods
Thirty three ICC and fifty HCC in cirrhosis were enrolled in this study. The contrast kinetics of ICC and HCC was analyzed and compared.
Results
Statistical analysis did not reveal significant difference between ICC and HCC in the time of contrast first appearance and arterial peak maximum time. ICC displayed much earlier washout than that of HCC (47.93±26.45 seconds vs 90.86±31.26 seconds) in the portal phase, and most ICC (87.9%) showed washout before 60 seconds than HCC (16.0%). Much more ICC (78.8%) revealed marked washout than HCC (12.0%) while most HCC (88.0%) showed mild washout or no washout in late part of the portal phase (90–120 seconds). Twenty six out of thirty three ICC (78.8%) demonstrated both early washout(<60seconds) and marked washout in late part of the portal phase, whereas, only six of fifty HCC (12.0%)showed these temporal enhancement features (p = 0.000).When both early washout and marked washout in the portal phase are taken as diagnostic criterion for ICC, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 78.8%,88.0%,81.3%,86.3%,and 84.3% respectively by CEUS.
Conclusions
Analysis of detailed temporal enhancement features on CEUS is helpful differentiate ICC from HCC in cirrhosis.If a nodule in cirrhotic liver displays hyper-enhancement in the arterial phase followed by early and marked washout in the portal phase, the nodule is highly suspicious of ICC rather than HCC.
doi:10.1371/journal.pone.0098612
PMCID: PMC4038593  PMID: 24874413
7.  Regulatory T Cells Protect Fine Particulate Matter-Induced Inflammatory Responses in Human Umbilical Vein Endothelial Cells 
Mediators of Inflammation  2014;2014:869148.
Objective. To investigate the role of CD4+CD25+ T cells (Tregs) in protecting fine particulate matter (PM-) induced inflammatory responses, and its potential mechanisms. Methods. Human umbilical vein endothelial cells (HUVECs) were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm2) of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4+CD25− T cells (Teff), or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined. Results. Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and inflammatory cytokines, such as interleukin (IL-) 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1) to endothelial cells was increased and NF-κB activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF-κB activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors. Conclusions. Tregs could attenuate fine particles-induced inflammatory responses and NF-κB activation in HUVECs.
doi:10.1155/2014/869148
PMCID: PMC4060066  PMID: 24987196
8.  Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease 
Nature genetics  2012;44(8):890-894.
We performed a meta-analysis of 2 genome-wide association studies of coronary artery disease comprising 1,515 cases with coronary artery disease and 5,019 controls, followed by de novo replication studies in 15,460 cases and 11,472 controls, all of Chinese Han descent. We successfully identified four new loci for coronary artery disease reaching genome-wide significance (P < 5 × 10−8), which mapped in or near TTC32-WDR35, GUCY1A3, C6orf10-BTNL2 and ATP2B1. We also replicated four loci previously identified in European populations (PHACTR1, TCF21, CDKN2A/B and C12orf51). These findings provide new insights into biological pathways for the susceptibility of coronary artery disease in Chinese Han population.
doi:10.1038/ng.2337
PMCID: PMC3927410  PMID: 22751097
9.  Cerebral Ischemia Is Exacerbated by Extracellular Nicotinamide Phosphoribosyltransferase via a Non-Enzymatic Mechanism 
PLoS ONE  2013;8(12):e85403.
Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT) is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO) and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD) induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.
doi:10.1371/journal.pone.0085403
PMCID: PMC3877362  PMID: 24392007
10.  Microbial fingerprinting detects intestinal microbiota dysbiosis in Zebrafish models with chemically-induced enterocolitis 
BMC Microbiology  2013;13:289.
Background
Inflammatory bowel disease (IBD) involves a breakdown in interactions between the host immune response and the resident commensal microbiota. Recent studies have suggested gut physiology and pathology relevant to human IBD can be rapidly modeled in zebrafish larvae. The aim of this study was to investigate the dysbiosis of intestinal microbiota in zebrafish models with IBD-like enterocolitis using culture-independent techniques.
Results
IBD-like enterocolitis was induced by exposing larval zebrafish to trinitrobenzenesulfonic acid (TNBS). Pathology was assessed by histology and immunofluorescence. Changes in intestinal microbiota were evaluated by denaturing gradient gel electrophoresis (DGGE) and the predominant bacterial composition was determined with DNA sequencing and BLAST and confirmed by real-time polymerase chain reaction. Larval zebrafish exposed to TNBS displayed intestinal-fold architecture disruption and inflammation reminiscent of human IBD. In this study, we defined a reduced biodiversity of gut bacterial community in TNBS-induced coliitis. The intestinal microbiota dysbiosis in zebrafish larvae with IBD-like colitis was characterized by an increased proportion of Proteobacteria (especially Burkholderia) and a decreased of Firmicutes(Lactobacillus group), which were significantly correlated with enterocolitis severity(Pearson correlation p < 0.01).
Conclusions
This is the first description of intestinal microbiota dysbiosis in zebrafish IBD-like models, and these changes correlate with TNBS-induced enterocolitis. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of human IBD.
doi:10.1186/1471-2180-13-289
PMCID: PMC4029296  PMID: 24325678
Zebrafish; Denaturing gradient gel electrophoresis; Trinitrobenzenesulfonic acid; Microbiota
11.  A novel technique for central hepatectomy: Maintain the blood supply and biliary drainage on one side and the blood supply from the portal vein on the other 
Central hepatectomy is amongst the most difficult surgeries of liver tumors. For the routine local excision of a tumor, if the tumor has invaded the blood vessels or bile duct of the liver, then half of the liver or three lobes of the liver are resected. This results in two major drawbacks, one of which is that the residual hepatic lobe may not compensate for the damage, so it is not possible to perform conventional partial resection. The other is that the volume of normal liver tissue removed may be much more than the volume of tumor removed, causing substantial waste. In the present study, surgery was performed to resect a central liver tumor. In that surgery, the V segment and parts of the IV, VI and VIII segments were resected, and the blood supply and biliary drainage of the left hepatic lobe were kept intact. However, for the remaining VI, VII and VIII segments of the right hepatic lobe, only the blood supply from the portal vein was maintained and no arterial blood supply or biliary drainage was kept so that the patient had the opportunity to undergo radical resection and successful rehabilitation. The reason these opportunities may be possible is that the residual right liver is a temporary replacement therapy in the perioperative period. Therefore, for central hepatic tumors, particularly tumors that have invaded the neighboring bile ducts or blood vessels, if the blood supply and biliary drainage on one side is maintained and the blood supply to the other side from the portal vein is kept intact, then it is possible to perform radical resection. This provides a novel approach to the clinical resection of central liver tumors.
doi:10.3892/etm.2013.1367
PMCID: PMC3861111  PMID: 24348763
middle lobe liver tumor; middle hepatectomy; biliary drainage; blood supply
12.  Analysis of mitochondrial transcription factor A SNPs in alcoholic cirrhosis 
Genetic susceptibility to alcoholic cirrhosis (AC) exists. We previously demonstrated hepatic mitochondrial DNA (mtDNA) damage in patients with AC compared with chronic alcoholics without cirrhosis. Mitochondrial transcription factor A (mtTFA) is central to mtDNA expression regulation and repair; however, it is unclear whether there are specific mtTFA single nucleotide polymorphisms (SNPs) in patients with AC and whether they affect mtDNA repair. In the present study, we screened mtTFA SNPs in patients with AC and analyzed their impact on the copy number of mtDNA in AC. A total of 50 patients with AC, 50 alcoholics without AC and 50 normal subjects were enrolled in the study. SNPs of full-length mtTFA were analyzed using the polymerase chain reaction (PCR) combined with gene sequencing. The hepatic mtTFA mRNA and mtDNA copy numbers were measured using quantitative PCR (qPCR), and mtTFA protein was measured using western blot analysis. A total of 18 mtTFA SNPs specific to patients with AC with frequencies >10% were identified. Two were located in the coding region and 16 were identified in non-coding regions. Conversely, there were five SNPs that were only present in patients with AC and normal subjects and had a frequency >10%. In the AC group, the hepatic mtTFA mRNA and protein levels were significantly lower than those in the other two groups. Moreover, the hepatic mtDNA copy number was significantly lower in the AC group than in the controls and alcoholics without AC. Based on these data, we conclude that AC-specific mtTFA SNPs may be responsible for the observed reductions in mtTFA mRNA, protein levels and mtDNA copy number and they may also increase the susceptibility to AC.
doi:10.3892/etm.2013.1353
PMCID: PMC3861118  PMID: 24348767
alcoholic cirrhosis; mitochondrial transcription factor A; single nucleotide polymorphisms; mitochondrial DNA; susceptibility
13.  Cobalt monoxide-doped porous graphitic carbon microspheres for supercapacitor application 
Scientific Reports  2013;3:2925.
A novel design and facile synthesis process for carbon based hybrid materials, i.e., cobalt monoxide (CoO)-doped graphitic porous carbon microspheres (Co-GPCMs), have been developed. With the synthesis strategy, the mixture of cobalt gluconate, α-cyclodextrin and poly (ethylene oxide)106-poly (propylene oxide)70-poly (ethylene oxide)106 is treated hydrothermally, followed by pyrolysis in argon. The resultant Co-GPCMs exhibits a porous carbon matrix with localized graphitic structure while CoO nanodots are embedded in the carbon frame. Thus, the Co-GPCMs effectively combine the electric double-layer capacitance and pseudo-capacitance when used as the electrode in supercapacitor, which lead to a higher operation voltage (1.6 V) and give rise to a significantly higher energy density. This study provides a new research strategy for electrode materials in high energy density supercapacitors.
doi:10.1038/srep02925
PMCID: PMC3795356  PMID: 24113335
14.  Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB 
AIM: To study the effects of Helicobacter pylori (H. pylori) tumor necrosis factor-α (TNF) inducing protein (Tip-α) on cytokine expression and its mechanism.
METHODS: We cloned Tip-α from the H. pylori strain 26695, transformed Escherichia coli with an expression plasmid, and then confirmed the expression product by Western blotting. Using different concentrations of Tip-α that affected SGC7901 and GES-1 cells at different times, we assessed cytokine levels using enzyme-linked immunosorbent assay. We blocked SGC7901 cells with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor κB (NF-κB). We then detected interleukin (IL)-1β and TNF-α levels in SGC7901 cells.
RESULTS: Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein, which indicated that recombinant Tip-α protein was recombined successfully. The levels of IL-1β, IL-8 and TNF-α were significantly higher following Tip-α interference, whether GES-1 cells or SGC-7901 cells were used (P < 0.05). However, the levels of cytokines (including IL-1β, IL-8 and TNF-α) secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-α at the same concentration and for the same duration (P < 0.05). After blocking NF-κB with PDTC, the cells (GES-1 cells and SGC-7901 cells) underwent interference with Tip-α. We found that IL-1β and TNF-α levels were significantly decreased compared to cells that only underwent Tip-α interference (P < 0.05).
CONCLUSION: Tip-α plays an important role in cytokine expression through NF-κB.
doi:10.3748/wjg.v19.i3.399
PMCID: PMC3554826  PMID: 23372364
Helicobacter pylori; Tumor necrosis factor-α inducing protein; Interleukin-1β; Interleukin-8; Tumor necrosis factor-α; Nuclear factor-κB
15.  Hyperfibrinolysis after parapelvic cyst surgery: A case report 
The present study describes the diagnosis and treatment of hyperfibrinolysis following surgery in a 25-year-old female patient. An examination revealed that the left kidney had been affected by severe hydronephrosis for two weeks prior to hospitalization. The diagnosis of a parapelvic cyst was obtained by preoperative intravenous pyelogram (IVP), computed tomography (CT) and upper left urinary tract retrograde pyelography. Unroofing of the left parapelvic cyst was performed by open surgery. The patient exhibited symptoms of shock 48 h later, and her hemoglobin (Hb) levels dropped to only 62.2 g/l. To treat this, 400 ml erythrocyte suspension transfusion was administered 3 times every other day. The patient’s Hb levels remained between 50 and 60 g/l. The D-dimer assay index rose from 0.3 to 16 mg/l and the fibrin degradation product (FDP) levels progressively increased following the hemorrhage, while the platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (Fg) index were all within normal levels. p-Aminomethylbenzoic acid (PAMBA; 0.5 g) was administered to the patient every day, and as a consequence the Hb levels rose steadily from the next day onwards. After a one week course of PAMBA treatment, the patient’s condition became stable. Blood coagulation and fibrinolytic function measurements were all within the normal ranges in the three months following the surgery. Delayed hemorrhage following surgery should be considered as a possible cause of hyperfibrinolysis. Monitoring FDP and D-dimer levels may aid a rapid and clear diagnosis. Anti-fibrinolytic therapy, such as PAMBA treatment, is safe and effective for use against the type of hemorrhage caused by hyperfibrinolysis.
doi:10.3892/etm.2012.795
PMCID: PMC3524075  PMID: 23251282
parapelvic cyst; hyperfibrinolysis; diagnosis and treatment
16.  Dynamic Alteration of the Colonic Microbiota in Intestinal Ischemia-Reperfusion Injury 
PLoS ONE  2012;7(7):e42027.
Background
Intestinal ischemia-reperfusion (I/R) plays an important role in critical illnesses. Gut flora participate in the pathogenesis of the injury. This study is aimed at unraveling colonic microbiota alteration pattern and identifying specific bacterial species that differ significantly as well as observing colonic epithelium change in the same injury model during the reperfusion time course.
Methodology/Principal Findings
Denaturing gradient gel electrophoresis (DGGE) was used to monitor the colonic microbiota of control rats and experimental rats that underwent 0.5 hour ischemia and 1, 3, 6, 12, 24, and 72 hours following reperfusion respectively. The microbiota similarity, bacterial diversity and species that characterized the dysbiosis were estimated based on the DGGE profiles using a combination of statistical approaches. The interested bacterial species in the gel were cut and sequenced and were subsequently quantified and confirmed with real-time PCR. Meanwhile, the epithelial barrier was checked by microscopy and D-lactate analysis. Colonic flora changed early and differed significantly at 6 hours after reperfusion and then started to recover. The shifts were characterized by the increase of Escherichia coli and Prevotella oralis, and Lactobacilli proliferation together with epithelia healing.
Conclusion/Significance
This study shows for the first time that intestinal ischemia-reperfusion results in colonic flora dysbiosis that follows epithelia damage, and identifies the bacterial species that contribute most.
doi:10.1371/journal.pone.0042027
PMCID: PMC3407053  PMID: 22848694
17.  Nonlinear finite element analysis of three implant–abutment interface designs 
The objective of this study was to investigate the mechanical characteristics of implant–abutment interface design in a dental implant system, using nonlinear finite element analysis (FEA) method. This finite element simulation study was applied on three commonly used commercial dental implant systems: model I, the reduced-diameter 3i implant system (West Palm Beach, FL, USA) with a hex and a 12-point double internal hexagonal connection; model II, the Semados implant system (Bego, Bremen, Germany) with combination of a conical (45° taper) and internal hexagonal connection; and model III, the Brånemark implant system (Nobel Biocare, Gothenburg, Sweden) with external hexagonal connection. In simulation, a force of 170 N with 45° oblique to the longitudinal axis of the implant was loaded to the top surface of the abutment. It has been found from the strength and stiffness analysis that the 3i implant system has the lowest maximum von Mises stress, principal stress and displacement while the Brånemark implant system has the highest. It was concluded from our preliminary study using nonlinear FEA that the reduced-diameter 3i implant system with a hex and a 12-point double internal hexagonal connection had a better stress distribution, and produced a smaller displacement than the other two implant systems.
doi:10.1038/ijos.2012.35
PMCID: PMC3412669  PMID: 22699263
external hexagonal connection; finite element analysis; implant–abutment interface; internal hexagonal connection; nonlinear analysis
18.  Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein 
PLoS Computational Biology  2012;8(4):e1002471.
An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional “backtracking” behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model.
Author Summary
A central goal of biology is to understand the function of the organism and its constituent parts at each of its scales of complexity. Function at the molecular level is often realized by changes in conformation. Unfortunately, experimental explorations of global motions critical for functional conformational changes are still challenging. In the present work, we developed a coarse grained triple-well structure-based model to explore the underlying functional landscape of maltose-binding protein (MBP). By quantitative flux analysis, we uncover the underlying mechanism by which the major induced fit and minor population shift pathways co-exist. Though we have previously lent credence to the assertion that dynamical equilibrium between open and minor closed conformations exist for all the free PBPs, the generality of this rule is still a matter of open debate. We found that the hinge flexibility is favorable to population shift mechanism. This finding provides a theoretical explanation of the mechanism discrepancies in PBP protein family. We also simulated the folding dynamics using this functional multi-basin model which successfully reproduced earlier protein melting experiment. This represents an exciting opportunity to characterize the interplay between folding and function, which is a long-standing question in the community. The theoretical approach employed in this study is general and can be applied to other systems.
doi:10.1371/journal.pcbi.1002471
PMCID: PMC3330084  PMID: 22532792
19.  Molecular-Phylogenetic Characterization of the Microbiota in Ulcerated and Non-Ulcerated Regions in the Patients with Crohn's Disease 
PLoS ONE  2012;7(4):e34939.
Background
The dysbiosis of intestinal microbiota has been established in Crohn's disease (CD), but the molecular characterization of this dysbiosis in Chinese subjects with CD remains unclear. This study aims to investigate the predominant bacterial composition of the faecal and mucosal-associated microbiota in Chinese CD patients using culture-independent techniques.
Methods/Principal Findings
Eighteen patients with CD and 9 healthy controls were included in this study. The faeces and the intestinal mucosal tissues from the ulcerated and nonulcerated sites were subjected to bacterial community fingerprinting using denaturing gradient gel electrophoresis (DGGE). The predominant bacterial composition in the faeces and mucosa was determined with DNA sequencing and BLAST. We showed that the bacterial diversity in the faeces of CD patients was reduced compared with that in healthy controls (p<0.01). The faecal bacterial dysbiosis of the patients was characterized by an elevated abundance of γ-Proteobacteria (especially Escherichia coli and Shigella flexneri) and a reduced proportion of Bacteroidetes and Firmicutes. Five bacterial species defined the microbiota imbalance of the ulcerated mucosa in CD, including an increase in Escherichia coli, a decrease in Faecalibacterium prausnitzii, Lactobacillus coleohominis, Bacteroides sp and Streptococcus gallolyticus in the bacterial community as compared with the nonulcerated (p<0.01).
Conclusions/Significance
This is the first description of intestinal microbiota dysbiosis in Chinese CD patients. These results allow a better understanding of the faecal and mucosal microbiota in CD, showing a predominance of some opportunistic pathogenic bacteria and a decrease in beneficial bacterial species. The findings may provide novel insights into the pathogenesis of CD in Chinese population.
doi:10.1371/journal.pone.0034939
PMCID: PMC3329531  PMID: 22529960
20.  (E)-3-Methyl-N′-(4-nitro­benzyl­idene)benzohydrazide methanol monosolvate 
The title hydrazone compound, C15H13N3O3·CH3OH, crystallized as a methanol solvate. The hydrazone mol­ecule has an E configuration about the C=N bond and is almost planar, with a dihedral angle between the benzene rings of 5.3 (3)°. In the crystal, the hydrazone mol­ecules are linked via the methanol solvent mol­ecule through N—H⋯O and O—H⋯O hydrogen bonds, so forming chains propagating along the a-axis direction.
doi:10.1107/S1600536812003868
PMCID: PMC3297325  PMID: 22412515
21.  (E)-N′-(2-Hy­droxy-3,5-diiodo­benzyl­idene)-3-methyl­benzohydrazide 
In the title compound, C15H12I2N2O2, the dihedral angle between the benzene rings is 26.5 (3)° and the mol­ecule has an E configuration about the C=N bond. An intra­molecular O—H⋯N hydrogen bond is observed in the mol­ecule. In the crystal, mol­ecules are linked by N—H⋯O hydrogen bonds, forming chains along the c axis.
doi:10.1107/S160053681200387X
PMCID: PMC3297326  PMID: 22412516
22.  N′-(2-Chloro­benzyl­idene)-4-nitro­benzohydrazide 
In the title compound, C14H10ClN3O3, the dihedral angle between the benzene rings is 6.64 (13)°. In the crystal, mol­ecules are linked through N—H⋯O hydrogen bonds, forming chains running along the c axis direction.
doi:10.1107/S1600536811055589
PMCID: PMC3275020  PMID: 22346965
23.  N′-(4-Hy­droxy­benzyl­idene)-3-meth­oxy­benzohydrazide 
In the title compound, C15H14N2O3, the dihedral angle between the two benzene rings is 47.9 (3)°. In the crystal, mol­ecules are linked through N—H⋯O, O—H⋯O and O—H⋯N hydrogen bonds, forming layers parallel to the ab plane.
doi:10.1107/S1600536811055565
PMCID: PMC3274989  PMID: 22346934
24.  N′-(5-Bromo-2-hy­droxy­benzyl­idene)-3-nitro­benzohydrazide methanol mono­solvate 
In the title compound, C14H10BrN3O4·CH4O, the dihedral angle between the two benzene rings in the hydrazone mol­ecule is 5.8 (3)° and an intra­molecular O—H⋯N hydrogen bond generates an S(6) ring motif. An O—H⋯O hydrogen bond occurs between the hydrazone mol­ecule and the methanol solvent mol­ecule. In the crystal, the components are linked by inter­molecular N—H⋯O hydrogen bonds, forming chains along the a axis.
doi:10.1107/S1600536811042553
PMCID: PMC3247402  PMID: 22220020
25.  N′-(2-Hy­droxy-4-meth­oxy­benzyl­idene)-3-nitro­benzohydrazide 
In the mol­ecule of the title compound, C15H13N3O3, an intra­molecular O—H⋯N hydrogen bond influences the planarity of the conformation; the dihedral angle between the benzene rings is 11.4 (3)°. In the crystal, mol­ecules are linked by N—H⋯O hydrogen bonds into chains in [101].
doi:10.1107/S160053681104178X
PMCID: PMC3247367  PMID: 22219985

Results 1-25 (46)