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1.  Hydrogen Sulfide Treatment Promotes Glucose Uptake by Increasing Insulin Receptor Sensitivity and Ameliorates Kidney Lesions in Type 2 Diabetes 
Abstract
Aims: To examine if hydrogen sulfide (H2S) can promote glucose uptake and provide amelioration in type 2 diabetes. Results: Treatment with sodium hydrosulfide (NaHS, an H2S donor) increased glucose uptake in both myotubes and adipocytes. The H2S gas solution showed similar effects. The NaHS effects were blocked by an siRNA-mediated knockdown of the insulin receptor (IR). NaHS also increased phosphorylation of the IR, PI3K, and Akt. In Goto-Kakizaki (GK) diabetic rats, chronic NaHS treatment (30 μmol·kg−1·day−1) decreased fasting blood glucose, increased insulin sensitivity, and increased glucose tolerance with increased phosphorylation of PI3K and Akt in muscles. Similar insulin-sensitizing effects of NaHS treatment were also observed in Wistar rats. Moreover, glucose uptake was reduced in the cells with siRNA-mediated knockdown of the H2S-generating enzyme cystathionine γ-lyase in the presence or absence of exogenous H2S. Moreover, chronic NaHS treatment reduced oxygen species and the number of crescentic glomeruli in the kidney of GK rats. Innovation and Conclusion: This study provides the first piece of evidence for the insulin-sensitizing effect of NaHS/H2S in the both in vitro and in vivo models of insulin resistance. Rebound Track: This work was rejected during a standard peer review and rescued by the Rebound Peer Review (Antoxid Redox Signal 16: 293–296, 2012) with the following serving as open reviewers: Jin-Song Bian, Samuel Dudley, Hideo Kimura, and Xian Wang. Antioxid. Redox Signal. 19, 5–23.
doi:10.1089/ars.2012.5024
PMCID: PMC3676664  PMID: 23293908
2.  Erythropoietin Protects against Murine Cerebral Malaria through Actions on Host Cellular Immunity 
Infection and Immunity  2014;82(1):165-173.
Cerebral malaria (CM) is associated with excessive host proinflammatory responses and endothelial activation. The hematopoietic hormone erythropoietin (EPO) possesses neuroprotective functions in animal models of ischemic-hypoxic, traumatic, and inflammatory injuries. In the Plasmodium berghei ANKA model of experimental CM (ECM), recombinant human EPO (rhEPO) has shown evident protection against ECM. To elucidate the mechanism of EPO in this ECM model, we investigated the effect of rhEPO on host cellular immune responses. We demonstrated that improved survival of mice with ECM after rhEPO treatment was associated with reduced endothelial activation and improved integrity of the blood-brain barrier. Our results revealed that rhEPO downregulated the inflammatory responses by directly inhibiting the levels and functions of splenic dendritic cells. Conversely, rhEPO treatment led to significant expansion of regulatory T cells and increased expression of the receptor cytotoxic T lymphocyte antigen 4 (CTLA-4). The data presented here provide evidence of the direct effect of rhEPO on host cellular immunity during ECM.
doi:10.1128/IAI.00929-13
PMCID: PMC3911832  PMID: 24126529
3.  Visualization of Distinct Substrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy 
The eukaryotic exosome is a multi-subunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3’ to 5’ exo- and endo-ribonuclease activities. RNA substrates have been shown to be recruited through the core to reach Rrp44's exoribonuclease (EXO) site. Using single particle electron microscopy and biochemical analysis, we provide visual evidence that two distinct substrate recruitment pathways exist. In the through-core route, channeling of the single stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place and the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate specific modes of degradation by this complex.
doi:10.1038/nsmb.2736
PMCID: PMC3976988  PMID: 24336220
4.  Deep Bottleneck Features for Spoken Language Identification 
PLoS ONE  2014;9(7):e100795.
A key problem in spoken language identification (LID) is to design effective representations which are specific to language information. For example, in recent years, representations based on both phonotactic and acoustic features have proven their effectiveness for LID. Although advances in machine learning have led to significant improvements, LID performance is still lacking, especially for short duration speech utterances. With the hypothesis that language information is weak and represented only latently in speech, and is largely dependent on the statistical properties of the speech content, existing representations may be insufficient. Furthermore they may be susceptible to the variations caused by different speakers, specific content of the speech segments, and background noise. To address this, we propose using Deep Bottleneck Features (DBF) for spoken LID, motivated by the success of Deep Neural Networks (DNN) in speech recognition. We show that DBFs can form a low-dimensional compact representation of the original inputs with a powerful descriptive and discriminative capability. To evaluate the effectiveness of this, we design two acoustic models, termed DBF-TV and parallel DBF-TV (PDBF-TV), using a DBF based i-vector representation for each speech utterance. Results on NIST language recognition evaluation 2009 (LRE09) show significant improvements over state-of-the-art systems. By fusing the output of phonotactic and acoustic approaches, we achieve an EER of 1.08%, 1.89% and 7.01% for 30 s, 10 s and 3 s test utterances respectively. Furthermore, various DBF configurations have been extensively evaluated, and an optimal system proposed.
doi:10.1371/journal.pone.0100795
PMCID: PMC4077656  PMID: 24983963
5.  Analysis of Altered MicroRNA Expression Profiles in Proximal Renal Tubular Cells in Response to Calcium Oxalate Monohydrate Crystal Adhesion: Implications for Kidney Stone Disease 
PLoS ONE  2014;9(7):e101306.
Background
Calcium oxalate monohydrate (COM) is the major crystalline component in kidney stones and its adhesion to renal tubular cells leads to tubular injury. However, COM-induced toxic effects in renal tubular cells remain ambiguous. MicroRNAs (miRNAs) play an important role in gene regulation at the posttranscriptional levels.
Objective
The present study aimed to assess the potential changes in microRNAs of proximal renal tubular cells in response to the adhesion of calcium oxalate monohydrate (COM) crystals.
Methodology
Lactate dehydrogenase (LDH) activity and DAPI staining were used to measure the toxic effects of HK-2 cells exposed to COM crystals. MicroRNA microarray and mRNA microarray were applied to evaluate the expression of HK-2 cells exposed to COM crystals. Quantitative real-time PCR (qRT-PCR) technology was used to validate the microarray results. Target prediction, Gene Ontology (GO) analysis and pathway analysis were applied to predict the potential roles of microRNAs in biological processes.
Principal Findings
Our study showed that COM crystals significantly altered the global expression profile of miRNAs in vitro. After 24 h treatment with a dose (1 mmol/L), 25 miRNAs were differentially expressed with a more than 1.5-fold change, of these miRNAs, 16 were up-regulated and 9 were down-regulated. A majority of these differentially expressed miRNAs were associated with cell death, mitochondrion and metabolic process. Target prediction and GO analysis suggested that these differentially expressed miRNAs potentially targeted many genes which were related to apoptosis, regulation of metabolic process, intracellular signaling cascade, insulin signaling pathway and type 2 diabetes.
Conclusion
Our study provides new insights into the role of miRNAs in the pathogenesis associated with nephrolithiasis.
doi:10.1371/journal.pone.0101306
PMCID: PMC4077747  PMID: 24983625
6.  Ginsenoside Rg1 Prevents Cognitive Impairment and Hippocampus Senescence in a Rat Model of D-Galactose-Induced Aging 
PLoS ONE  2014;9(6):e101291.
Neurogenesis continues throughout the lifetime in the hippocampus, while the rate declines with brain aging. It has been hypothesized that reduced neurogenesis may contribute to age-related cognitive impairment. Ginsenoside Rg1 is an active ingredient of Panax ginseng in traditional Chinese medicine, which exerts anti-oxidative and anti-aging effects. This study explores the neuroprotective effect of ginsenoside Rg1 on the hippocampus of the D-gal (D-galactose) induced aging rat model. Sub-acute aging was induced in male SD rats by subcutaneous injection of D-gal (120 mg/kg·d) for 42 days, and the rats were treated with ginsenoside Rg1 (20 mg/kg·d, intraperitoneally) or normal saline for 28 days after 14 days of D-gal injection. In another group, normal male SD rats were treated with ginsenoside Rg1 alone (20 mg/kg·d, intraperitoneally) for 28 days. It showed that administration of ginsenoside Rg1 significantly attenuated all the D-gal-induced changes in the hippocampus, including cognitive capacity, senescence-related markers and hippocampal neurogenesis, compared with the D-gal-treated rats. Further investigation showed that ginsenoside Rg1 protected NSCs/NPCs (neural stem cells/progenitor cells) shown by increased level of SOX-2 expression; reduced astrocytes activation shown by decrease level of Aeg-1 expression; increased the hippocampal cell proliferation; enhanced the activity of the antioxidant enzymes GSH-Px (glutathione peroxidase) and SOD (Superoxide Dismutase); decreased the levels of IL-1β, IL-6 and TNF-α, which are the proinflammatory cytokines; increased the telomere lengths and telomerase activity; and down-regulated the mRNA expression of cellular senescence associated genes p53, p21Cip1/Waf1 and p19Arf in the hippocampus of aged rats. Our data provides evidence that ginsenoside Rg1 can improve cognitive ability, protect NSCs/NPCs and promote neurogenesis by enhancing the antioxidant and anti-inflammatory capacity in the hippocampus.
doi:10.1371/journal.pone.0101291
PMCID: PMC4076296  PMID: 24979747
7.  Continuous intra-articular infusion anesthesia for pain control after total knee arthroplasty: study protocol for a randomized controlled trial 
Trials  2014;15:245.
Background
Postoperative pain control after total knee arthroplasty (TKA) remains a great challenge. The management of pain in the immediate postoperative period is one of the most critical aspects to allow speedier rehabilitation and reduce the risk of postoperative complications. Recently, periarticular infiltration anesthesia has become popular, but the outcome is controversial. Some studies have shown transient effects, “rebound pain”, or no effectiveness in pain control. Continuous intra-articular infusion technique has been introduced to improve these transient effects, but more clinical studies are needed. Furthermore, the potential risk of early periprosthetic joint infection is causing concerning. We plan to compare continuous intra-articular infusion anesthesia with epidural infusion anesthesia after TKA to assess the effectiveness of this technique in reducing pain, in improving postoperative function, and to look at the evidence for risk of early infection.
Methods/design
This trial is a randomized, controlled study. Patients (n = 214) will be randomized into two groups: to receive continuous intra-articular infusion anesthesia (group C); and epidural infusion anesthesia (group E). For the first 3 postoperative days, pain at rest, active range of motion (A-ROM), rescue analgesia and side effects will be recorded. At 3-month and 6-month follow-up, A-ROM, C-reactive protein, erythrocyte sedimentation rate, and synovial fluid cell count and culture will be analyzed.
Discussion
The results from this study will provide clinical evidence on the efficacy of a continuous intra-articular infusion technique in reducing pain, postoperative functional improvement and safety. It will be the first randomized controlled trial to investigate infection risk with local anesthesia after TKA.
Trial registration
ClinicalTrials.gov identifier: ChiCTR-TRC-13003999
doi:10.1186/1745-6215-15-245
PMCID: PMC4074607  PMID: 24958315
Total knee arthroplasty; Postoperative pain control; Continuous intra-articular infusion anesthesia; Randomized controlled trial
8.  Rapid Birth-and-Death Evolution of Imprinted snoRNAs in the Prader-Willi Syndrome Locus: Implications for Neural Development in Euarchontoglires 
PLoS ONE  2014;9(6):e100329.
Imprinted small nucleolar RNAs (snoRNAs) are only found in eutherian genomes and closely related to brain functions. A complex human neurological disease, Prader-Willi syndrome (PWS), is primarily attributed to the deletion of imprinted snoRNAs in chromosome 15q11-q13. Here we investigated the snoRNA repertoires in the PWS locus of 12 mammalian genomes and their evolution processes. A total of 613 imprinted snoRNAs were identified in the PWS homologous loci and the gene number was highly variable across lineages, with a peak in Euarchontoglires. Lineage-specific gene gain and loss events account for most extant genes of the HBII-52 (SNORD115) and the HBII-85 (SNORD116) gene family, and remarkable high gene-birth rates were observed in the primates and the rodents. Meanwhile, rapid sequence substitution occurred only in imprinted snoRNA genes, rather than their flanking sequences or the protein-coding genes located in the same imprinted locus. Strong selective constraints on the functional elements of these imprinted snoRNAs further suggest that they are subjected to birth-and-death evolution. Our data suggest that the regulatory role of HBII-52 on 5-HT2CR pre-mRNA might originate in the Euarchontoglires through adaptive process. We propose that the rapid evolution of PWS-related imprinted snoRNAs has contributed to the neural development of Euarchontoglires.
doi:10.1371/journal.pone.0100329
PMCID: PMC4063771  PMID: 24945811
9.  Intestinal obstruction due to dual gastrointestinal atresia in infants: diagnosis and management of 3 cases 
BMC Gastroenterology  2014;14:108.
Background
Several types of congenital lesions can cause complete or incomplete obstruction of the intestine. Our purpose is to present 3 neonates with dual intestinal type I atresia, i.e., simultaneous obstructive lesions at 2 locations in which the atresia manifested as diaphragm-like tissue.
Case presentation
All 3 cases were female infants ranging in age from 2 to 14 months. The common symptom in all cases was intermittent persistent vomiting. In some cases the vomitus was bilious, and other symptoms included abdominal distention and delayed meconium passage. Prior surgeries at another hospital were unsuccessful at relieving the symptoms in one case. One case had dual lesions in the colon, one dual lesions in the duodenum, and one atresia at both the distal portion of the ileum and the descending colon. Surgical exploration and removal of the lesions at our hospital was successful in all cases, and the infants were discharged in good condition.
Conclusions
Type I atresia can manifest as a diaphragm-like tissue obstructing the continuity of gastrointestinal tract, and in rare cases multiple areas may be present. Base on the intermittent nature of the associated symptoms, diagnosis can be difficult and is often delayed. Physicians should be aware of this condition during the work-up of an infant with persistent intermittent vomiting.
doi:10.1186/1471-230X-14-108
PMCID: PMC4064280  PMID: 24928109
Type I Atresia; Gastrointestinal diaphragm; Congenital; Intestinal obstruction; Infant
10.  New clade of enigmatic early archosaurs yields insights into early pseudosuchian phylogeny and the biogeography of the archosaur radiation 
Background
The origin and early radiation of archosaurs and closely related taxa (Archosauriformes) during the Triassic was a critical event in the evolutionary history of tetrapods. This radiation led to the dinosaur-dominated ecosystems of the Jurassic and Cretaceous, and the high present-day archosaur diversity that includes around 10,000 bird and crocodylian species. The timing and dynamics of this evolutionary radiation are currently obscured by the poorly constrained phylogenetic positions of several key early archosauriform taxa, including several species from the Middle Triassic of Argentina (Gracilisuchus stipanicicorum) and China (Turfanosuchus dabanensis, Yonghesuchus sangbiensis). These species act as unstable ‘wildcards’ in morphological phylogenetic analyses, reducing phylogenetic resolution.
Results
We present new anatomical data for the type specimens of G. stipanicicorum, T. dabanensis, and Y. sangbiensis, and carry out a new morphological phylogenetic analysis of early archosaur relationships. Our results indicate that these three previously enigmatic taxa form a well-supported clade of Middle Triassic archosaurs that we refer to as Gracilisuchidae. Gracilisuchidae is placed basally within Suchia, among the pseudosuchian (crocodile-line) archosaurs. The approximately contemporaneous and morphologically similar G. stipanicicorum and Y. sangbiensis may be sister taxa within Gracilisuchidae.
Conclusions
Our results provide increased resolution of the previously poorly constrained relationships of early archosaurs, with increased levels of phylogenetic support for several key early pseudosuchian clades. Moreover, they falsify previous hypotheses suggesting that T. dabanensis and Y. sangbiensis are not members of the archosaur crown group. The recognition of Gracilisuchidae provides further support for a rapid phylogenetic diversification of crown archosaurs by the Middle Triassic. The disjunct distribution of the gracilisuchid clade in China and Argentina demonstrates that early archosaurs were distributed over much or all of Pangaea although they may have initially been relatively rare members of faunal assemblages.
doi:10.1186/1471-2148-14-128
PMCID: PMC4061117  PMID: 24916124
Archosauria; Argentina; Biogeography; China; Gracilisuchidae; Pangaea; Phylogenetics; Suchia; Triassic
11.  Treatment of Unstable Thoracolumbar Fractures through Short Segment Pedicle Screw Fixation Techniques Using Pedicle Fixation at the Level of the Fracture: A Finite Element Analysis 
PLoS ONE  2014;9(6):e99156.
Objective
To compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques to treat thoracic 12 vertebral fractures, especially the mono-segment pedicle screw fixation and intermediate unilateral pedicle screw fixation techniques.
Methods
Finite element methods were utilised to investigate the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques (S4+2: traditional short-segment 4 pedicle screw fixation [SPSF]; M4+2: mono-segment pedicle screw fixation; I6+2: intermediate bilateral pedicle screw fixation; and I5+2: intermediate unilateral pedicle screw fixation).
Results
The range of motion (ROM) in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but lateral bending was the largest in the M4+2 fixation model. The maximal stress of the upper pedicle screw is larger than the lower pedicle screw in S4+2 and M4+2. The largest maximal von Mises stress was observed in the upper pedicle screw in the S4+2 and M4+2 fixation models and in the lower pedicle screw in the I6+2 and I5+2 fixation models. The values of the largest maximal von Mises stress of the pedicle screws and rods during all states of motion were 263.1 MPa and 304.5 MPa in the S4+2 fixation model, 291.6 MPa and 340.5 MPa in the M4+2 fixation model, 182.9 MPa and 263.2 MPa in the I6+2 fixation model, and 269.3 MPa and 383.7 MPa in the I5+2 fixation model, respectively. Comparing the stress between different spinal loadings, the maximal von Mises stress of the implants were observed in flexion in all implanted models.
Conclusion
Additional bilateral pedicle screws at the level of the fracture to SPSF may result in a stiffer construct and less von Mises stress for pedicle screws and rods. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique.
doi:10.1371/journal.pone.0099156
PMCID: PMC4051693  PMID: 24914815
12.  Investigation of cholecystokinin receptors in the human lower esophageal sphincter 
AIM: To compare the binding of cholecystokinin (CCK)-8 to CCK receptors in sling and clasp fibers of the human lower esophageal sphincter.
METHODS: Esophageal sling and clasp fibers were isolated from eight esophagectomy specimens, resected for squamous cell carcinoma in the upper two thirds of the esophagus, which had been maintained in oxygenated Kreb’s solution. Western blot was used to measure CCK-A and CCK-B receptor subtypes in the two muscles. A radioligand binding assay was used to determine the binding parameters of 3H-CCK-8S to the CCK receptor subtypes. The specificity of binding was determined by the addition of proglumide, which blocks the binding of CCK to both receptor subtypes.
RESULTS: There was no significant difference between the sling and clasp fibers of the human lower esophageal sphincter in the amount of CCK-A [integrated optical density (IOD) value: 22.65 ± 0.642 vs 22.328 ± 1.042, P = 0.806] or CCK-B receptor protein (IOD value: 13.20 ± 0.423 vs 12.45 ± 0.294, P = 0.224) as measured by Western blot. The maximum binding of radio-labeled CCK-8S was higher in the sling fibers than in the clasp fibers (595.75 ± 3.231 cpm vs 500.000 ± 10.087 cpm, P < 0.001) and dissociation constant was lower (Kd: 1.437 ± 0.024 nmol/L vs 1.671 ± 0.024 nmol/L, P < 0.001). The IC50 of the receptor specific antagonists were lower for the CCK-A receptors than for the CCK-B (P < 0.01).
CONCLUSION: CCK binding modulates the contractile function of the lower esophageal sphincter through differential binding to the CCK-A receptor on the sling and clasp fibers.
doi:10.3748/wjg.v20.i21.6554
PMCID: PMC4047341  PMID: 24914377
Cholecystokinins; Cholecystokinins-A receptor; Cholecystokinins-B receptor; Radioligand binding; Lower esophageal sphincter; Sling fibers; Clasp fibers
13.  EFFICIENT DRUG SCREENING AND GENE CORRECTION FOR TREATING LIVER DISEASE USING PATIENT-SPECIFIC STEM CELLS 
Hepatology (Baltimore, Md.)  2013;57(6):2458-2468.
Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drugand cell- therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug- or gene- therapy available, we established a platform to discover new drug candidates and to correct disease-causing mutation with a high efficiency. A high-throughput format screening assay based on our hepatic differentiation protocol was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds, the Johns Hopkins Drug Library, with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease (TALEN) technology, we achieved high gene targeting efficiency in AAT-deficiency patient iPSCs with 25–33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications. Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs; both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases.
doi:10.1002/hep.26237
PMCID: PMC3633649  PMID: 23325555
14.  Bayesian hierarchical model of protein-binding microarray k-mer data reduces noise and identifies transcription factor subclasses and preferred k-mers 
Bioinformatics  2013;29(11):1390-1398.
Motivation: Sequence-specific transcription factors (TFs) regulate the expression of their target genes through interactions with specific DNA-binding sites in the genome. Data on TF-DNA binding specificities are essential for understanding how regulatory specificity is achieved.
Results: Numerous studies have used universal protein-binding microarray (PBM) technology to determine the in vitro binding specificities of hundreds of TFs for all possible 8 bp sequences (8mers). We have developed a Bayesian analysis of variance (ANOVA) model that decomposes these 8mer data into background noise, TF familywise effects and effects due to the particular TF. Adjusting for background noise improves PBM data quality and concordance with in vivo TF binding data. Moreover, our model provides simultaneous identification of TF subclasses and their shared sequence preferences, and also of 8mers bound preferentially by individual members of TF subclasses. Such results may aid in deciphering cis-regulatory codes and determinants of protein–DNA binding specificity.
Availability and implementation: Source code, compiled code and R and Python scripts are available from http://thebrain.bwh.harvard.edu/hierarchicalANOVA.
Contact: bojiang83@gmail.com or mlbulyk@receptor.med.harvard.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt152
PMCID: PMC3661050  PMID: 23559638
15.  Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii 
Eukaryotic Cell  2013;12(12):1588-1599.
Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii.
doi:10.1128/EC.00082-13
PMCID: PMC3889574  PMID: 23873863
16.  Itraconazole, a commonly used anti-fungal that inhibits Hedgehog pathway activity and cancer growth 
Cancer cell  2010;17(4):388-399.
SUMMARY
In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (Smo) by a mechanism distinct from that of cyclopamine and other known Smo antagonists, and prevents the ciliary accumulation of Smo normally caused by Hh stimulation.
doi:10.1016/j.ccr.2010.02.027
PMCID: PMC4039177  PMID: 20385363
17.  Free posterior tibial flap reconstruction for hypopharyngeal squamous cell carcinoma 
Objectives
The aim of this article was to determine outcomes in patients with squamous cell carcinoma of the hypopharynx (SCCHP) in whom the free posterior tibial flap was used for primary reconstruction of hypopharynx defects after cancer resection.
Subjects and methods
Between August 2009 and February 2012, 10 patients with SCCHP underwent posterior tibial flap reconstruction for hypopharynx defects. The corresponding clinical data were retrospectively collected and analyzed.
Results
Despite the multistep and time-consuming procedure, the posterior tibial flap survival rate was 100%. Operation-induced complications did not occur in four patients. Six patients developed postoperative hypoproteinemia, four patients developed postoperative pulmonary infections, and four patients developed pharyngeal fistula. The pharyngeal and laryngeal functions of all patients were preserved.
Conclusion
Our experience demonstrates that the posterior tibial flap is a safe and reliable choice for the reconstruction of hypopharynx defects.
doi:10.1186/1477-7819-12-163
PMCID: PMC4038592  PMID: 24884631
Hypopharyngeal cancer; Posterior tibial flap; Reconstruction
18.  Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidase-1 in Cells 
Journal of medicinal chemistry  2013;56(10):3996-4016.
Methionine aminopeptidases (MetAPs) which remove the initiator methionine from nascent peptides are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1–4). But all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1–4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.
doi:10.1021/jm400227z
PMCID: PMC3714011  PMID: 23634668
19.  Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi 
PLoS ONE  2014;9(5):e98338.
The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.
doi:10.1371/journal.pone.0098338
PMCID: PMC4032328  PMID: 24859001
20.  Powerful inner/outer controlled multi-target magnetic nanoparticle drug carrier prepared by liquid photo-immobilization 
Scientific Reports  2014;4:4990.
Nanomagnetic materials offer exciting avenues for advancing cancer therapies. Most researches have focused on efficient delivery of drugs in the body by incorporating various drug molecules onto the surface of nanomagnetic particles. The challenge is how to synthesize low toxic nanocarriers with multi-target drug loading. The cancer cell death mechanisms associated with those nanocarriers remain unclear either. Following the cell biology mechanisms, we develop a liquid photo-immobilization approach to attach doxorubicin, folic acid, tumor necrosis factor-α, and interferon-γ onto the oleic acid molecules coated Fe3O4 magnetic nanoparticles to prepare a kind of novel inner/outer controlled multi-target magnetic nanoparticle drug carrier. In this work, this approach is demonstrated by a variety of structural and biomedical characterizations, addressing the anti-cancer effects in vivo and in vitro on the HeLa, and it is highly efficient and powerful in treating cancer cells in a valuable programmed cell death mechanism for overcoming drug resistance.
doi:10.1038/srep04990
PMCID: PMC4028896  PMID: 24845203
21.  Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii 
The intracellular parasite Toxoplasma gondii is a leading cause of congenital neurological defects. To cause disease, it must reiterate its lytic cycle through host cell invasion, replication,and parasite egress. This requires the parasite to sense changes in its environment and switch between the non-motile (for replication) and motile (for invasion and egress) states appropriately. Recently, we discovered a previously unknown mechanism of motility regulation in T. gondii, mediated by a lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). When AKMT is absent, activation of motility is inhibited, which compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Although the methyltransferase activity of AKMT has been established, the phylogenetic relationship of AKMT with other better studied lysine methyltransferases (KMTs) was not known. Also unknown was the functional relationships between different domains of AKMT. In this work we carried out phylogenetic analyses, which show that AKMT orthologs form a new subfamily of KMTs. We systematically generated truncation mutants of AKMT, and discovered that the predicted enzymatic domain alone is a very poor enzyme and cannot complement the function of AKMT in vivo. Interestingly, the N- and C-terminal domains of the AKMT have drastically different impacts on its enzyme activity, localization as well as in vivo function. Our results thus reveal that AKMT is an unusual, parasite-specific enzyme and identified regions and interactions within this novel lysine methyltransferase that can be used as drug targets.
doi:10.1016/j.molbiopara.2013.05.004
PMCID: PMC3740173  PMID: 23685344
lysine methyltransferase; AKMT; motility; Toxoplasma gondii; Plasmodium; KMT phylogeny
22.  Induced pluripotent stem cell-derived mesenchymal stem cell seeding on biofunctionalized calcium phosphate cements 
Bone research  2013;4:371-384.
Induced pluripotent stem cells (iPSCs) have great potential due to their proliferation and differentiation capability. The objectives of this study were to generate iPSC-derived mesenchymal stem cells (iPSC-MSCs), and investigate iPSC-MSC proliferati on and osteogenic differentiation on calcium phosphate cement (CPC) containing biofunctional agents for the first time. Human iPSCs were derived from marrow CD34+ cells which were reprogrammed by a single episomal vector. iPSCs were cultured to form embryoid bodies (EBs), and MSCs migrated out of EBs. Five biofunctional agents were incorporated into CPC: RGD (Arg-Gly-Asp) peptides, fibronectin (Fn), fibronectin-like engineered polymer protein (FEPP), extracellular matrix Geltrex, and platelet concentrate. iPSC-MSCs were seeded on five biofunctionalized CPCs: CPC-RGD, CPC-Fn, CPC-FEPP, CPC-Geltrex, and CPC-Platelets. iPSC-MSCs on biofunctional CPCs had enhanced proliferation, actin fiber expression, osteogenic differentiation and mineralization, compared to control. Cell proliferation was greatly increased on biofunctional CPCs. iPSC-MSCs underwent osteogenic differentiation with increased alkaline phosphatase, Runx2 and collagen-I expressions. Mineral synthesis by iPSC-MSCs on CPC-Platelets was 3-fold that of CPC control. In conclusion, iPSCs showed high potential for bone engineering. iPSC-MSCs on biofunctionalized CPCs had cell proliferation and bone mineralization that were much better than traditional CPC. iPSC-MSC-CPC constructs are promising to promote bone regeneration in craniofacial/orthopedic repairs.
doi:10.4248/BR201304008
PMCID: PMC4023512  PMID: 24839581
induced pluripotent stem cells; RGD; fibronectin; platelet concentrate; biofunctionalized calcium phosphate cement; bone tissue engineering
23.  Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression 
Oncology Reports  2014;32(1):419-424.
Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.
doi:10.3892/or.2014.3174
PMCID: PMC4067428  PMID: 24841903
tilorone; synthetic lethality; cyclin-dependent kinase 5; prostate cancer
24.  High magnetic field phase diagram in electron-doped manganites La0.4Ca0.6Mn1−yCryO3 
Scientific Reports  2014;4:4902.
We report the charge-order to ferromagnetic phase transition induced by pulsed high magnetic field and impurity doping effects in manganites La0.4Ca0.6(Mn1−yCry)O3 (0 ≤ y ≤ 0.2). Significant charge-order suppression and ferromagnetic tendency upon the Cr3+-doping are evidenced, and three different ground states are identified, namely the charge-order state, the phase separated state, and the spin-glass like state. Phase diagram in the H-y plane at 4.2 K is determined by the high magnetic field study, in which the charge-order and ferromagnetic phase boundary is clearly figured out. The critical magnetic field for melting the charge-order phase of La0.4Ca0.6MnO3 is revealed to reach up to 46 T at 4.2 K. Interestingly, distinct responses of the three states to the high magnetic field are observed, indicating the special physics regarding the charge order melting process in each state. The mechanism of the doping induced charge-order suppression and ferromagnetism promotion can be understood by the competition between the antiferromagnetic interaction of Cr-Mn and local enhancement of electron hopping by Cr3+.
doi:10.1038/srep04902
PMCID: PMC4014982  PMID: 24811441
25.  The intrinsically disordered membrane protein selenoprotein S is a reductase in vitro 
Biochemistry  2013;52(18):3051-3061.
Selenoprotein S (SelS, VIMP) is an intrinsically disordered membrane enzyme that provides protection against reactive oxidative species. SelS is a member of the endoplasmic reticulum associated protein degradation pathway but its precise enzymatic function is unknown. Since it contains the rare amino acid selenocysteine, it belongs to the family of selenoproteins, which are typically oxidoreductases. Its exact enzymatic function is key to understanding how the cell regulates the response to oxidative stress and thus influences human health and aging. In order to identify its enzymatic function, we have isolated the selenocysteine-containing enzyme by relying on the aggregation of forms that do not have this reactive residue. That allows us to establish that SelS is primarily a thioredoxin-dependent reductase. It is capable of reducing hydrogen peroxide but is not an efficient or broad-spectrum peroxidase. Only the selenocysteine-containing enzyme is active. In addition, the reduction potential of SelS was determined to be −234 mV using electrospray ionization mass spectrometry. This value agrees with SelS being a partner of thioredoxin. Based on this information, SelS can directly combat reactive oxygen species but is also likely to participate in a signaling pathway, via a yet unidentified substrate.
doi:10.1021/bi4001358
PMCID: PMC3675161  PMID: 23566202

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