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1.  Rational Design of Matrix Metalloproteinase-13 (MMP-13) Activatable Probes for Enhanced Specificity 
ACS chemical biology  2013;9(2):510-516.
Due to the important roles of matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities in vitro and in vivo. Our recently developed MMP-13 activatable probe, L-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model (Zhu L et al., Theranostics. 2011;1:18–27). In this study, to further optimize the in vivo behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high resolution LC/MS system. Two major metabolites that contributed to the fluorescence recovery were identified: One was specifically cleaved between Glycine (G4) and Valine (V5) by MMP, while the other one was generated by non-specific cleavage between Glycine (G7) and Lysine (K8). In order to visualize the MMP activity more accurately and specifically, a new probe D-MMP-P12 was designed by replacing the L-lysine with D-lysine in the MMP substrate sequence. The metabolic profile of the new probe, D-MMP-P12, was further characterized by in vitro enzymatic assay and no non-specific metabolite was found by LC/MS. Our in vivo optical imaging also demonstrated that D-MMP-12 had significantly higher tumor-to-background ratio (TBR, 5.55 ± 0.75) compared with L-MMP-P12 (3.73 ± 0.31) at 2 h post-injection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes.
doi:10.1021/cb400698s
PMCID: PMC3944097  PMID: 24266806
Liquid chromatography–mass spectrometry (LC-MS); activatable probe; matrix metalloproteinases (MMPs); metabolite; near-infrared fluorescence imaging
2.  American Ginseng Regulates Gene Expression to Protect against Premature Ovarian Failure in Rats 
BioMed Research International  2015;2015:767124.
Premature ovarian failure (POF) is defined as lost ovarian functions before the age of 40. Three possible molecular markers (PLA2G4A, miR-29a, and miR-144) have been identified in our previous study by integrated analysis of mRNA and miRNA expression profiles. The present study aimed to evaluate American ginseng root's protective potential against POF by studying transcriptional and protein variations between American ginseng treatments and controls in rats. 4-Vinylcyclohexene diepoxide (VCD) was administered to rats for 14 days to induce POF. Additionally, American ginseng was administered to POF rats for one month, and PLA2G4A, miR-29a, and miR-144 expressions were measured in rat ovaries by qRT-PCR. PLA2G4A protein expression was examined by Western Blot, and PGE2, LH, FSH, and E2 serum levels were detected by ELISA. PLA2G4A mRNA and protein were downregulated in American ginseng-treated rats, miR-29a and miR-144 levels increased, and PGE2 serum levels decreased, while LH, FSH, and E2 increased compared to POF induction alone. Analysis of transcriptional and protein variations suggested that American ginseng protects the ovary against POF by regulating prostaglandin biosynthesis, ovulation, and preventing ovarian aging. High hormone levels (PGE2, FSH, and LH) were reduced, and E2 secretion approached normal levels, leading to improved POF symptoms and abnormal ovulation.
doi:10.1155/2015/767124
PMCID: PMC4330957
3.  Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs 
Molecular Microbiology  2014;91(5):918-934.
Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programmes around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline-based drugs is becoming critical. So far only few resistance markers have been identified from which only two transmembrane transporters namely PfMDR1 (an ATP-binding cassette transporter) and PfCRT (a drug-metabolite transporter) have been experimentally verified. Another P. falciparum transporter, the ATP-binding cassette containing multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identified a parasite clone that is derived from the 3D7 P. falciparum strain and shows increased resistance to chloroquine, mefloquine and quinine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5′ upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription and thus increased level of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of genetic polymorphisms within these regions to underlie drug resistance.
doi:10.1111/mmi.12505
PMCID: PMC4286016  PMID: 24372851
4.  Zn(II)-coordination modulated ligand photophysical processes – the development of fluorescent indicators for imaging biological Zn(II) ions 
RSC advances  2014;4(39):20398-20440.
Molecular photophysics and metal coordination chemistry are the two fundamental pillars that support the development of fluorescent cation indicators. In this article, we describe how Zn(II)-coordination alters various ligand-centered photophysical processes that are pertinent to developing Zn(II) indicators. The main aim is to show how small organic Zn(II) indicators work under the constraints of specific requirements, including Zn(II) detection range, photophysical requirements such as excitation energy and emission color, temporal and spatial resolutions in a heterogeneous intracellular environment, and fluorescence response selectivity between similar cations such as Zn(II) and Cd(II). In the last section, the biological questions that fluorescent Zn(II) indicators help to answer are described, which have been motivating and challenging this field of research.
doi:10.1039/C4RA00354C
PMCID: PMC4111279  PMID: 25071933
5.  SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks 
Cell metabolism  2013;18(6):920-933.
Summary
Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.
doi:10.1016/j.cmet.2013.11.013
PMCID: PMC4105152  PMID: 24315375
6.  Genome-wide analysis in Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy during the infectious cycle 
BMC Genomics  2014;15(1):959.
Background
Over the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.
Results
We exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.
Conclusions
The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-959) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-959
PMCID: PMC4232647  PMID: 25373614
7.  Outcome of free digital artery perforator flap transfer for reconstruction of fingertip defects 
Indian Journal of Orthopaedics  2014;48(6):594-598.
Background:
Fingertip defect can be treated with many flaps such as random pattern abdominal flap, retrograde digital artery island flap, V-Y advancement flap, etc. However, swelling in the fingertip, dysfunction of sensation, flexion and extension contracture or injury in the hemi-artery of the finger usually occurs during the recovery phase. Recently, digital artery perforator flaps have been used for fingertip reconstructions. With the development of super microsurgery techniques, free flaps can be more effective for sensory recovery and durability of the fingertip.
Materials and Methods:
Six cases (six fingers) of fingertip defects were treated with free digital artery perforator flaps of appropriate size and shape from the proximal phalanx. During surgery, the superficial veins at the edge of flap were used as reflux vessels and the branches of the intrinsic nerve and dorsal digital nerve toward the flap were used as sensory nerves. The proximal segment of the digital artery (cutaneous branches) towards the flap was cut off to form the pedicled free flap. The fingertips were reconstructed with the free flap by anastomosing the cutaneous branches of digital artery in the flap with the distal branch or trunk of the digital artery, the flap nerve with the nerve stump and the veins of the flap with the digital artery accompanying veins or the superficial veins in the recipient site.
Results:
Six flaps survived with successful skin grafting. Patients were followed up for 6-9 months. The appearance and texture of the flaps was satisfactory. The feeling within the six fingers recovered to S4 level (BMRC scale) and the two point discrimination was 3-8 mm.
Conclusion:
Free digital artery perforator flap is suitable for repairing fingertip defect, with good texture, fine fingertip sensation and without sacrificing the branch of the digital artery or nerve.
doi:10.4103/0019-5413.144228
PMCID: PMC4232829  PMID: 25404772
Fingertip defect; perforator flaps; reconstruction; Surgery; plastic; surgical flaps; finger injuries
8.  Life extension factor klotho enhances cognition 
Cell reports  2014;7(4):1065-1076.
Summary
Aging is the primary risk factor for cognitive decline, an emerging health threat to aging societies worldwide. Whether anti-aging factors such as klotho can counteract cognitive decline is unknown. We show that a life span-extending variant of the human KLOTHO gene, KL-VS, is associated with enhanced cognition in heterozygous carriers. Because this allele increased klotho levels in serum, we analyzed transgenic mice with systemic overexpression of klotho. They performed better than controls in multiple tests of learning and memory. Elevating klotho in mice also enhanced long-term potentiation, a form of synaptic plasticity, and enriched synaptic GluN2B, an NMDA receptor subunit with key functions in learning and memory. Blockade of GluN2B abolished klotho-mediated effects. Surprisingly, klotho effects were evident also in young mice and did not correlate with age in humans, suggesting independence from the aging process. Augmenting klotho or its effects may enhance cognition at different life stages and counteract cognitive decline.
doi:10.1016/j.celrep.2014.03.076
PMCID: PMC4176932  PMID: 24813892
9.  99mTc-3P4-RGD2 Scintimammography in the Assessment of Breast Lesions: Comparative Study with 99mTc-MIBI 
PLoS ONE  2014;9(9):e108349.
Purpose
To compare the potential application of 99mTc-3P-Arg-Gly-Asp (99mTc-3P4-RGD2) scintimammography (SMM) and 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) SMM for the differentiation of malignant from benign breast lesions.
Method
Thirty-six patients with breast masses on physical examination and/or suspicious mammography results that required fine needle aspiration cytology biopsy (FNAB) were included in the study. 99mTc-3P4-RGD2 and 99mTc-MIBI SMM were performed with single photon emission computed tomography (SPECT) at 60 min and 20 min respectively after intravenous injection of 738±86 MBq radiotracers on a separate day. Images were evaluated by the tumor to non-tumor localization ratios (T/NT). Receiver operating characteristic (ROC) curve analysis was performed on each radiotracer to calculate the cut-off values of quantitative indices and to compare the diagnostic performance for the ability to differentiate malignant from benign diseases.
Results
The mean T/NT ratio of 99mTc-3P4-RGD2 in malignant lesions was significantly higher than that in benign lesions (3.54±1.51 vs. 1.83±0.98, p<0.001). The sensitivity, specificity, and accuracy of 99mTc-3P4-RGD2 SMM were 89.3%, 90.9% and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of 99mTc-MIBI in malignant lesions was also significantly higher than that in benign lesions (2.86±0.99 vs. 1.51±0.61, p<0.001). The sensitivity, specificity and accuracy of 99mTc-MIBI SMM were 87.5%, 72.7% and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for 99mTc-3P4-RGD2 SMM (area = 0.851) was higher than that for 99mTc-MIBI SMM (area = 0.781), but the statistical difference was not significant.
Conclusion
99mTc-3P4-RGD2 SMM does not provide any significant advantage over the established 99mTc-MIBI SMM for the detection of primary breast cancer. The T/NT ratio of 99mTc-3P4-RGD2 SMM was significantly higher than that of 99mTc-MIBI SMM. Both tracers could offer an alternative method for elucidating non-diagnostic mammograms.
doi:10.1371/journal.pone.0108349
PMCID: PMC4176966  PMID: 25250628
10.  Gadolinium embedded iron oxide nanoclusters as T1-T2 dual-modal MRI-visible vectors for safe and efficient siRNA delivery 
Nanoscale  2013;5(17):8098-8104.
This report illustrates a new strategy in designing a T1-T2 dual-modal magnetic resonance imaging (MRI)-visible vector for siRNA delivery and MRI. Hydrophobic gadolinium embedded iron oxide (GdIO) nanocrystals are self-assembled into nanoclusters in water phase with the help of stearic acid modified low molecular weight polyethylenimine (stPEI). The resulting water-dispersible GdIO-stPEI nanoclusters possess good stability, monodispersity with narrow size distribution and competitive T1-T2 dual-modal MR imaging properties. The nanocomposite system is capable of binding and delivering siR-NA for knockdown of a gene of interest while maintaining magnetic properties and biocompatibility. This new gadolinium embedded iron oxide nanocluster provides an important platform for safe and efficient gene delivery with non-invasive T1-T2 dual-modal MRI monitoring capability.
doi:10.1039/c3nr02797j
PMCID: PMC3775844  PMID: 23884164
11.  Mixture models for gene expression experiments with two species 
Human Genomics  2014;8(1):12.
Cross-species research in drug development is novel and challenging. A bivariate mixture model utilizing information across two species was proposed to solve the fundamental problem of identifying differentially expressed genes in microarray experiments in order to potentially improve the understanding of translation between preclinical and clinical studies for drug development. The proposed approach models the joint distribution of treatment effects estimated from independent linear models. The mixture model posits up to nine components, four of which include groups in which genes are differentially expressed in both species. A comprehensive simulation to evaluate the model performance and one application on a real world data set, a mouse and human type II diabetes experiment, suggest that the proposed model, though highly structured, can handle various configurations of differential gene expression and is practically useful on identifying differentially expressed genes, especially when the magnitude of differential expression due to different treatment intervention is weak. In the mouse and human application, the proposed mixture model was able to eliminate unimportant genes and identify a list of genes that were differentially expressed in both species and could be potential gene targets for drug development.
doi:10.1186/1479-7364-8-12
PMCID: PMC4135333  PMID: 25085578
Orthology; Drug development; Drug response prediction; Type II diabetes
12.  The Effect of Injection Routes on the Biodistribution, Clearance and Tumor Uptake of Carbon Dots 
ACS nano  2013;7(7):5684-5693.
The emergence of photoluminescent carbon-based nanomaterials has shown exciting potential in the development of benign nanoprobes. However, the in vivo kinetic behaviors of these particles that are necessary for clinical translation are poorly understood to date. In this study, fluorescent carbon dots (C-dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence (NIRF) and positron emission tomography (PET) imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The clearance rate of C-dots is ranked as: intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial system (RES) and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection routes, an important step forward towards safety and efficacy analysis of nanoparticles.
doi:10.1021/nn401911k
PMCID: PMC3725601  PMID: 23731122
Biodistribution; carbon dots; clearance; injection routes; translation; tumor uptake
13.  Development of a new thiol site-specific prosthetic group and its conjugation with [cys40]-exendin-4 for in vivo targeting of insulinomas 
Bioconjugate chemistry  2013;24(7):1191-1200.
A new tracer, N-5-[18F]fluoropentylmaleimide ([18F]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps (18F displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11–17% radiochemical yield (uncorrected), and specific activity of 20–49 GBq/µmol. [18F]FPenM showed comparable labeling efficiency with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys40]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution and metabolic stability of the radiolabeled [18F]FPenM-[cys40]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [18F]FPenM-[cys40]-exendin-4, had high tumor uptake (20.32 ± 4.36 %ID/g at 60 min post-injection) and rapid liver and kidney clearance, which was comparable to the imaging results with [18F]FBEM-[cys40]-exendin-4 reported by our group.
doi:10.1021/bc400084u
PMCID: PMC3779517  PMID: 23750453
14.  Plasminogen Activator Inhibitor-1 Regulates LPS Induced Inflammation in Rat Macrophages through Autophagy Activation 
The Scientific World Journal  2014;2014:189168.
Background. The mechanisms by which plasminogen activator inhibitor-1 (PAI-1) regulates inflammation, especially in acute respiratory distress syndrome (ARDS), are largely unknown. Objective. To assess the relationship between PAI-1 and autophagy in inflammatory reactions induced by LPS in rat NR8383 cells. Methods. ELISA was used to assess the amounts of TNF-α, IL-1β, and PAI-1 in cell culture supernatants; TLR4, MyD88, PAI-1, LC3, Beclin1, and mTOR protein and mRNA levels were determined by western blot and quantitative RT-PCR, respectively; western blot was used to determine NF-κB protein levels. To further evaluate the role of PAI-1, the PAI-1 gene was downregulated and overexpressed using the siRNA transfection technology and the pCDH-PAI-1, respectively. Finally, the GFP Positive Expression Rate Method was used to determine the rate of GFP-LC3 positive NR8383 cells. Results. In LPS-induced NR8383 cells, TNF-α, IL-1β, and PAI-1 expression levels increased remarkably. Upon PAI-1 knockdown, TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1 levels were decreased, while mTOR increased. Conversely, overexpression of PAI-1 resulted in increased amounts of TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1. However, no significant change was observed in mTOR expression. Conclusions. In NR8383 cells, PAI-1 contributes in the regulation of LPS-induced inflammation, likely by promoting autophagy.
doi:10.1155/2014/189168
PMCID: PMC4122156  PMID: 25133205
15.  Primary intrathoracic liposarcoma: a clinicopathologic study and prognostic analysis of 23 cases 
Background
Primary intrathoracic liposarcoma is an extremely rare malignancy as well as a rare histologic subtype of intrathoracic sarcoma. Relatively few reports appear in the world literatures. We explored the clinicopathologic features and prognostic factors of this tumor in this study.
Methods
We retrospectively analyzed the clinicopathological data of 23 patients with primary intrathoracic liposarcoma who were treated in Shanghai chest Hospital affiliated to Jiao Tong University, from January 2003 to March 2013. These patients were classified into three groups according to the distinct tumor locations, including mediastinum, pleura and lung liposarcoma. Also, these patients could be divided into four types, including well-differentiated, myxoid, dedifferentiated and pleomorphic liposarcoma. The influences of age, sex, tumor size, tumor location, tumor histologic type and therapy on the prognosis of the patients were analyzed.
Results
There were no significant difference for survival among distinct liposarcoma locations. However, significant difference for survival among distinct liposarcoma types were observed. Poor disease-free survival (DFS) was observed in the myxoid, pleomorphic and dedifferentiated types as compared to well-differentiated type (P = 0.038). Inferior overall-survival (OS) was observed in dedifferentiated, pleomorphic and myxoid types relative to well-differentiated type (P = 0.027). The radical surgery was a favorable prognostic factor for OS, as demonstrated by the better OS of the radical surgery group as compared to that of the non-radical surgery group ( P = 0.029). Notably, there were no significant differences for DFS and OS in other clinical parameters including tumor size, gender and age. In addition, radiotherapy and/or chemotherapy could not improve the prognosis of the patients receiving non-radical surgery or suffering from relapse.
Conclusions
The histological type and the radical surgery are the factors that influence the behavior and prognosis of liposarcoma. In general, radiotherapy and chemotherapy are believed to be ineffective therapeutic modalities for survival. So it is essential to completely resect the primary intrathoracic liposarcoma as radical cure of the disease.
doi:10.1186/1749-8090-9-119
PMCID: PMC4088306  PMID: 24993036
Intrathoracic liposarcoma; Histological type; Radical surgery; Overall survival; Disease-free survival
16.  Inefficient Translation Renders the Enterococcus faecalis fabK Enoyl-Acyl Carrier Protein Reductase Phenotypically Cryptic 
Journal of Bacteriology  2014;196(1):170-179.
Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the bacterial fatty acid elongation cycle. Enterococcus faecalis is unusual in that it encodes two unrelated enoyl-ACP reductases, FabI and FabK. We recently reported that deletion of the gene encoding FabI results in an unsaturated fatty acid (UFA) auxotroph despite the presence of fabK, a gene encoding a second fully functional enoyl-ACP reductase. By process of elimination, our prior report argued that poor expression was the reason that fabK failed to functionally replace FabI. We now report that FabK is indeed poorly expressed and that the expression defect is at the level of translation rather than transcription. We isolated four spontaneous mutants that allowed growth of the E. faecalis ΔfabI strain on fatty acid-free medium. Each mutational lesion (single base substitution or deletion) extended the fabK ribosome binding site. Inactivation of fabK blocked growth, indicating that the mutations acted only on fabK rather than a downstream gene. The mutations activated fabK translation to levels that supported fatty acid synthesis and hence cell growth. Furthermore, site-directed and random mutagenesis experiments showed that point mutations that resulted in increased complementarity to the 3′ end of the 16S rRNA increased FabK translation to levels sufficient to support growth, whereas mutations that decreased complementarity blocked fabK translation.
doi:10.1128/JB.01148-13
PMCID: PMC3911128  PMID: 24163335
17.  Downregulation of ROS-FIG inhibits cell proliferation, colony-formation, cell cycle progression, migration and invasion, while inducing apoptosis in intrahepatic cholangiocarcinoma cells 
Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with poor responsiveness to existing drug therapies. Therefore, novel treatment strategies against ICC are required to improve survival. The aim of this study was to demonstrate the role of fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS) fusion gene in ICC. ROS was positively expressed in ICC tissues and HUCCT1 cells. Plasmids expressing ROS- and FIG-specific shRNAs were constructed and transfected into HUCCT1 cells. The results showed that single transfection of ROS- or FIG-specific shRNA inhibited HUCCT1 cell proliferation, colony formation, cell cycle progression, migration and invasion, while inducing apoptosis. Moreover, the co-inhibition of ROS- and FIG-specific shRNA exhibited stronger effects on HUCCT1 cell proliferation, apoptosis, colony formation, cell cycle progression, migration and invasion, when compared to single inhibition of ROS and FIG. Furthermore, findings of this study suggested that the AKT signaling pathway was involved in the ROS-FIG-mediated biological processes of HUCCT1 cells. In summary, the results suggest that FIG-ROS plays an oncogenic role in ICC. Additionally, ROS1-6290 and FIG-363 segments may become effective therapeutic targets for ICC harboring ROS-FIG fusion protein.
doi:10.3892/ijmm.2014.1823
PMCID: PMC4121345  PMID: 24968753
intrahepatic cholangiocarcinoma; fused-in-glioblastoma-c-ros-oncogene1; oncogene; HUCCT1 cell
18.  Gold Nanoparticle Based Activatable Probe for Sensing Ultra-Low Levels of Prostate Specific Antigen 
ACS nano  2013;7(6):5568-5576.
It is still in high demand to develop extremely sensitive and accurate clinical tools for biomarkers of interest for early diagnosis and monitoring of diseases. In this report, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based fluorescence activatable probe for sensing ultra-low levels of prostate-specific antigen (PSA) in patient serum samples. The limit of detection of the newly-developed probe for PSA was pushed down to 0.032 pg/mL, which is more than two orders of magnitude lower than that of the conventional fluorescence probe. The ultrahigh sensitivity of this probe was attributed to the high loading efficiency of the dyes on AuNP surfaces and high fluorescence quenching unquenching abilities of the dye-AuNP pairs. The efficiency and robustness of this probe was investigated in patient serum samples, demonstrating the great potential of this probe in real-world applications.
doi:10.1021/nn401837q
PMCID: PMC3696512  PMID: 23683064
fluorescence activatable probe; gold nanoparticle; prostate specific antigen (PSA); Rhodamine B isothiocyanate (RBITC)
19.  Toll-Like Receptors Expressed by Synovial Fibroblasts Perpetuate Th1 and Th17 Cell Responses in Rheumatoid Arthritis 
PLoS ONE  2014;9(6):e100266.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone and cartilage erosion. Synovial fibroblast- and T cell-mediated inflammation plays crucial roles in the pathogenesis of RA. However how this inflammation is initiated, propagated, and maintained remains controversial. Here, we systemically examined the contribution of toll-like receptors (TLRs) to the inflammatory mediator production as well as Th1 and Th17 cell hyperactivity in RA. Our results show that rheumatoid arthritis synovial fibroblasts (RASF) express a series of TLRs, including TLR2, TLR3, TLR4, and TLR9, with the predominant expression of TLR3. Moreover, the expression levels of these TLRs were higher than those in osteoarthritis synovial fibroblasts (OASF). Ligation of TLR3, as well as TLR2 and TLR4, resulted in vigorous production of inflammatory cytokines, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) in RASF, with activation of the NF-κB, MAPK, and IRF3 pathways. More important, activation of these TLRs expressed by RASF exacerbated inflammatory Th1 and Th17 cell expansion both in cell-cell contact-dependent and inflammatory cytokine-dependent manners, which induced more IFN-γ and IL-17 accumulation. Targeting TLRs may modulate the inflammation in RA and provide new therapeutic strategies for overcoming this persistent disease.
doi:10.1371/journal.pone.0100266
PMCID: PMC4061069  PMID: 24936783
20.  Cryopreservation and replantation of amputated rat hind limbs 
Background
In spite of the relatively high success rate of limb replantation, many patients cannot undergo replantation surgery because the preservation time of an amputated limb is only about six hours. In addition, although allotransplantation of composite tissues is being performed more commonly with increasingly greater success rates, the shortage of donors limits the number of patients that can be treated. So the purpose of this study is to examine the feasibility of cryopreservation and replantation of limbs in a rat model.
Methods
Twelve five-month-old Sprague-Dawley rats were divided evenly into group A (above-knee amputation) and group B (Syme’s amputation). One hind limb was amputated from each rat. The limbs were irrigated with cryoprotectant, cooled in a controlled manner to -140°C, and placed in liquid nitrogen. Thawing and replantation were performed 14 days later.
Results
In group A, the limbs became swollen after restoration of blood flow resulting in blood vessel compression and all replantations failed. In group B, restoration of blood flow was noted in all limbs after replantation. In one case, the rat chewed the replanted limb and replantation failed. The other five rats were followed for three months with no abnormalities noted in the replanted limbs.
Conclusions
Limbs with a minimal amount of muscle tissue can be successfully cryopreserved and replanted.
doi:10.1186/2047-783X-19-28
PMCID: PMC4048362  PMID: 24886622
Amputation; Cryopreservation; Injury; Rat model; Replantation
21.  A Coding Method for Efficient Subgraph Querying on Vertex- and Edge-Labeled Graphs 
PLoS ONE  2014;9(5):e97178.
Labeled graphs are widely used to model complex data in many domains, so subgraph querying has been attracting more and more attention from researchers around the world. Unfortunately, subgraph querying is very time consuming since it involves subgraph isomorphism testing that is known to be an NP-complete problem. In this paper, we propose a novel coding method for subgraph querying that is based on Laplacian spectrum and the number of walks. Our method follows the filtering-and-verification framework and works well on graph databases with frequent updates. We also propose novel two-step filtering conditions that can filter out most false positives and prove that the two-step filtering conditions satisfy the no-false-negative requirement (no dismissal in answers). Extensive experiments on both real and synthetic graphs show that, compared with six existing counterpart methods, our method can effectively improve the efficiency of subgraph querying.
doi:10.1371/journal.pone.0097178
PMCID: PMC4031119  PMID: 24853266
22.  Integrated and Passive 1,2,3-Triazolyl Groups in Fluorescent Indicators for Zinc(II) Ions – Thermodynamic and Kinetic Evaluations 
Inorganic chemistry  2013;52(10):5838-5850.
In addition to being a covalent linker in molecular conjugation chemistry, the function of a 1,2,3-triazolyl moiety resulting from the copper(I)-catalyzed azide-alkyne cycloaddition reaction as a ligand for metal ions is receiving considerable attention. In this work, we characterize the thermodynamic and kinetic effects of incorporating a 1,2,3-triazolyl group in a multidentate ligand scaffold on metal coordination in the context of fluorescent zinc(II) indicator development. Ligands L14, BrL14, and FL14 (1,4-isomers) contain the 1,4- disubstituted-1,2,3-triazolyl group that is capable of binding with zinc(II) in conjunction with a di(2-picolylamino) (DPA) moiety within a multidentate ligand scaffold. The 1,2,3-triazolyl in the 1,4-isomers is therefore “integrated” in chelation. The 1,5-isomers L15, BrL15, and FL15 contain 1,2,3-triazolyls that are excluded from participating in zinc(II) coordination. These 1,2,3- triazolyls are “passive linkers”. Zinc(II) complexes of 2:1 (ligand/metal) stoichiometry are identified in solution using 1H NMR spectroscopy and isothermal titration calorimetry (ITC), and in one case, characterized in the solid state. The 1:1 ligand/zinc(II) affinity ratio of L14 over L15, which is attributed to the affinity enhancement of a 1,2,3-triazolyl group to zinc(II) over that of the solvent acetonitrile, is quantified at 18 (−1.7 kcal/mol at 298 K) using an ITC experiment. Fluorescent ligands FL14 and FL15 are evaluated for their potential in zinc(II) sensing applications under pH neutral aqueous conditions. The 1,4-isomer FL14 binds zinc(II) both stronger and faster than the 1,5-isomer FL15. Visualization of free zinc(II) ion distribution in live HeLa cells is achieved using both FL14 and FL15. The superiority of FL14 in staining endogenous zinc(II) ions in live rat hippocampal slices is evident. In summation, this work is a fundamental study of 1,2,3-triazole coordination chemistry, with a demonstration of its utility in developing fluorescent indicators.
doi:10.1021/ic302798u
PMCID: PMC3687791  PMID: 23621758
1,2,3-triazole; zinc sensor; fluorescence; isothermal titration calorimetry; stoppedflow; hippocampus; photoinduced electron transfer
23.  Specific Changes of Serum Proteins in Parkinson's Disease Patients 
PLoS ONE  2014;9(4):e95684.
The aim of this study is to identify and validate protein change in the serum from PD patients. We used serum samples from 21 PD patients and 20 age-matched normal people as control to conduct a comparative proteomic study. We performed 2-DE and analyzed the differentially expressed protein spots by LC-MS/MS. In PD group 13 spots were shown to be differentially expressed compared to control group. They were identified as 6 proteins. Among these, 3 proteins were confirmed by Western blot analysis. It showed that the frequency of fibrinogen γ-chain (FGG) appeared 70% in PD, which could not be detected in control group. The protein of inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4) was found to exist two forms in serum. The full size (120 kDa) of the protein was increased and the fragmented ITI-H4 (35 kDa) was decreased in PD group. The ratio of full size ITI-H4 to fragmented ITI-H4 in PD patients was 3.85±0.29-fold higher than in control group. Furthermore, fragmented Apo A-IV (∼26 kDa) was mainly detected in control group, while it was rare to be found in PD group. Above findings might be useful for diagnosis of PD. When the expressions of FGG and 120 kDa ITI-H4 are increase, as well as ∼26 kDa Apo A-IV disappear would provide strong evidence for PD.
doi:10.1371/journal.pone.0095684
PMCID: PMC4000217  PMID: 24769800
24.  The Expression and Clinical Significance of Different Forms of Mer Receptor Tyrosine Kinase in Systemic Lupus Erythematosus 
Journal of Immunology Research  2014;2014:431896.
Objective. To investigate the expression and clinical significance of trans-membrane MerTK (mMer) on circulating CD14+ monocytes/macrophages and soluble MerTK (sMer) levels in plasma in systemic lupus erythematosus (SLE). Method. 108 SLE patients and 42 healthy controls were recruited in this study. The expression of mMer on the surfaces of CD14+ monocytes/macrophages was evaluated by flow cytometry (FCM). The sMer levels were measured by ELISA. Real-time quantitative PCR was applied to evaluate the mRNA levels of MerTK and ADAM17. Results. Both mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma significantly increased in SLE patients compared to healthy subjects. The frequency of anti-inflammatory MerTK expressing CD14+CD16+ monocytes decreased in SLE. mMer expression was positively correlated with CD163 expression on CD14+ cells. Both the mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma were positively correlated with SLEDAI. Furthermore, more elevated mMer and sMer levels were found in patients with higher SLEDAI, presence of anti-SSA, anti-Sm autoantibodies, and lupus nephritis. Conclusion. Both mMer and sMer levels significantly increased in SLE and positively correlated with disease activity and severity. The upregulation of MerTK expression may serve as a biomarker of the disease activity and severity of SLE.
doi:10.1155/2014/431896
PMCID: PMC3987794  PMID: 24741600
25.  Accelerating Content-Based Image Retrieval via GPU-Adaptive Index Structure 
The Scientific World Journal  2014;2014:829059.
A tremendous amount of work has been conducted in content-based image retrieval (CBIR) on designing effective index structure to accelerate the retrieval process. Most of them improve the retrieval efficiency via complex index structures, and few take into account the parallel implementation of them on underlying hardware, making the existing index structures suffer from low-degree of parallelism. In this paper, a novel graphics processing unit (GPU) adaptive index structure, termed as plane semantic ball (PSB), is proposed to simultaneously reduce the work of retrieval process and exploit the parallel acceleration of underlying hardware. In PSB, semantics are embedded into the generation of representative pivots and multiple balls are selected to cover more informative reference features. With PSB, the online retrieval of CBIR is factorized into independent components that are implemented on GPU efficiently. Comparative experiments with GPU-based brute force approach demonstrate that the proposed approach can achieve high speedup with little information loss. Furthermore, PSB is compared with the state-of-the-art approach, random ball cover (RBC), on two standard image datasets, Corel 10 K and GIST 1 M. Experimental results show that our approach achieves higher speedup than RBC on the same accuracy level.
doi:10.1155/2014/829059
PMCID: PMC3980781  PMID: 24782668

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