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1.  KIF20A Regulates Porcine Oocyte Maturation and Early Embryo Development 
PLoS ONE  2014;9(7):e102898.
KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development.
doi:10.1371/journal.pone.0102898
PMCID: PMC4103861  PMID: 25036038
2.  Non-destructively shattered mesoporous silica for protein drug delivery 
Mesoporous silicas have been extensively used for entrapping small chemical molecules and biomacromolecules for drug delivery. We hypothesize that the loading density of biomacromlecules such as proteins in mesoporous silicas could be limited due to disordering in the pore structure and long diffusion time in the pore channels. We shattered mesoporous silicas non-destructively resulting in improved intramesoporous structures and reduced particle sizes in aqueous solutions by a powerful sonication, where the mesoporous structures were still well maintained. The sonication-shattered mesoporous silica can increase the protein loading density to nearly 2.7 times as high as that of the non-shattered one, demonstrating that significantly more mesopore space of the silica could be accessible by the protein molecules, which may result in more sustained protein drug delivery.
doi:10.1016/j.micromeso.2013.03.022
PMCID: PMC3653322  PMID: 23687455
Mesoporous silica; Non-destructive shattering; Intramesoporous structure; Protein drug delivery
3.  Anti-Epileptic Effect of Ganoderma Lucidum Polysaccharides by Inhibition of Intracellular Calcium Accumulation and Stimulation of Expression of CaMKII α in Epileptic Hippocampal Neurons 
PLoS ONE  2014;9(7):e102161.
Purpose
To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated.
Method
Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg2+ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg2+ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca2+ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca2+ turnover was observed under a laser scanning confocal microscope.
Results
The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca2+ fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes.
Conclusion
GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.
doi:10.1371/journal.pone.0102161
PMCID: PMC4092074  PMID: 25010576
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  The Diagnostic Value of Cervical Lymph Node Metastasis in Head and Neck Squamous Carcinoma by Using Diffusion-Weighted Magnetic Resonance Imaging and Computed Tomography Perfusion 
BioMed Research International  2014;2014:260859.
Purpose. The aim of this study was to compare diffusion-weighted magnetic resonance imaging (DWI) with computed tomography perfusion (CTP) for preoperative detection of metastases to lymph nodes (LNs) in head and neck squamous cell carcinoma (SCC). Methods. Between May 2010 and April 2012, 30 patients with head and neck SCC underwent preoperative DWI and CTP. Two radiologists measured apparent diffusion coefficient (ADC) values and CTP parameters independently. Surgery and histopathologic examinations were performed on all patients. Results. On DWI, 65 LNs were detected in 30 patients. The mean ADC value of metastatic nodes was lower than benign nodes and the difference was statistically significant (P < 0.05). On CTP images, the mean value in metastatic nodes of blood flow (BF) and blood volume (BV) was higher than that in benign nodes, and mean transit time (MTT) in metastatic nodes was lower than that in benign nodes. There were significant differences in BF and MTT values between metastatic and benign LNs (P < 0.05). There were significant differences between the AUCs of DWI and CTP (Z=4.612, P < 0.001). Conclusion. DWI with ADC value measurements may be more accurate than CTP for the preoperative diagnosis of cervical LN metastases.
doi:10.1155/2014/260859
PMCID: PMC4094719  PMID: 25050333
11.  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
12.  microRNA-99a acts as a tumor suppressor and is down-regulated in bladder cancer 
BMC Urology  2014;14:50.
Background
Increasing evidences have documented that microRNAs (miRNAs) act as oncogenes or tumor suppressors in a variety types of cancer. The discovery of tumor associated miRNAs in serum of patients gives rise to extensive investigation of circulating miRNAs in many human cancers which support the use of plasma/serum miRNAs as noninvasive means of cancer detection. However, the aberrant expression of miRNAs and the circulating miRNAs in bladder cancer are less reported.
Methods
We used Taqman probe stem-loop real-time PCR to accurately measure the levels of miR-99a in bladder cancer cell lines, 100 pairs of bladder cancer tissues, the adjacent non-neoplastic tissues and plasma collected from bladder cancer patients or control patients. miR-99a mimics were re-introduced into bladder cancer cells to investigate its role on regulating cell proliferation which was measured by CCK-8 assay and cell cycle analysis.
Results
miR-99a was significantly down-regulated in bladder cancer tissues, and even the lower expression of miR-99a was correlative with the more aggressive phenotypes of bladder cancer. Meanwhile, enforced expression of miR-99a can inhibit the cell proliferation of bladder cancer cells. Furthermore, investigation of the expression of miR-99a in plasma of bladder cancer patients showed that miR-99a was also decreased in plasma of bladder cancer patients. The results strongly supported miR-99a as the potential diagnostic marker of bladder cancer.
Conclusions
Our data indicated that miR-99a might act as a tumor suppressor in bladder cancer and was significantly down-regulated in development of bladder cancer.
doi:10.1186/1471-2490-14-50
PMCID: PMC4083872  PMID: 24957100
Bladder cancer; miR-99a; Circulation miRNA
13.  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
14.  Number of Polyploid Giant Cancer Cells and Expression of EZH2 Are Associated with VM Formation and Tumor Grade in Human Ovarian Tumor 
BioMed Research International  2014;2014:903542.
To investigate the associations among the number of polyploid giant cancer cells (PGCCs) and vasculogenic mimicry (VM), EZH2 expression, and serous ovarian tumor grade, a total of 80 paraffin-embedded serous ovarian tumor samples including 21 cases of primary carcinoma and their metastatic tumors, 26 cases of primary carcinoma without metastasis, and 12 cases of serous borderline cystadenoma were analyzed. PGCCs and VM were detected in human serous ovarian tumor. The metastatic foci of ovarian carcinoma had the highest number of PGCCs and VM. The number of PGCCs and VM increased with the grade of ovarian carcinomas. PGCCs generated erythrocytes via budding and together they formed VM. Tumor cells and cancer-associated fibroblasts were positive for EZH2 immunohistochemical staining. The tumor cells and cancer associated fibroblasts in the metastatic foci had the highest staining index of EZH2 staining. Both tumor cells and cancer-associated fibroblasts express EZH2 which then contributes to the malignant grade of serous ovarian tumor.
doi:10.1155/2014/903542
PMCID: PMC4082869  PMID: 25025074
15.  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
16.  Hydrogen sulfide reduces kidney injury due to urinary-derived sepsis by inhibiting NF-κB expression, decreasing TNF-α levels and increasing IL-10 levels 
The present study aimed to investigate the effect of hydrogen sulfide (H2S) on kidney injury induced by urinary-derived sepsis. Rabbits were randomly divided into control, sham, sepsis, NaHS 2.8 μmol/kg and NaHS 8.4 μmol/kg groups, with six rabbits in each group. Upper urinary tract obstruction and acute infection was induced to establish the sepsis model. Blood was collected to carry out a white blood cell (WBC) count, and creatinine (Cr) and blood urea nitrogen (BUN) analysis. Morphological changes were observed by hematoxylin and eosin (H&E) staining and transmission electron microscopy. Immunohistochemical staining was used to detect the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-10 and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Cystathionine-γ-lyase (CSE) activity was measured by the spectrophotometric methylene blue method and the blood H2S concentration was measured by deproteinization. WBC, Cr and BUN levels were significantly elevated in the sepsis group compared with those in the control group (P<0.05). Following treatment with NaHS, the WBC, Cr and BUN levels were significantly decreased in the NaHS groups compared with those in the sepsis group (P<0.05). The pathological features of kidney injury were also alleviated by NaHS. In the sepsis group, the levels of TNF-α, IL-10 and NF-κB were significantly increased compared with those in the control group (P<0.05). In the NaHS groups, the TNF-α and NF-κB levels were significantly reduced whereas the IL-10 level was significantly increased compared with the respective levels in the sepsis group (P<0.05). The H2S concentration was significantly decreased in the sepsis group and this reduction was attenuated in the NaHS groups (P<0.05). Furthermore, the NaHS 8.4 μmol/kg dose revealed a more potent effect than the NaHS 2.8 μmol/kg dose. Thus, exogenous H2S reduced kidney injury from urinary-derived sepsis by decreasing the levels of NF-κB and TNF-α, and increasing the level of IL-10.
doi:10.3892/etm.2014.1781
PMCID: PMC4079422  PMID: 25009602
hydrogen sulfide; kidney injury; urinary-derived sepsis; NF-κB; TNF-α; IL-10
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  Qualitative Analysis of the Helical Electronic Energy of Inherently Chiral Calix[4]arenes: An Approach to Effectively Assign Their Absolute Configuration 
For all microhelices on aromatic rings of inherently chiral calix[4]arene, an expression was derived from one approximation and one hypothesis on the basis of the electron-on-a-helix model of Tinoco and Woody as follows: , where µ = 1 for the right-handed microhelix and µ = −1 for the left-handed microhelix; and H and K are constant and greater than zero. The expression correlates microhelical electronic energy (E) with the atom polarizability difference (Δα) on both microhelix ends, which intuitively and clearly shows the impact of helical substituent polarizability on helical electronic energy. The case analysis almost entirely proves that the qualitative analysis of the helical electronic energy of inherently chiral Calix[4]arenes with the expression is scientific and can be used to effectively assign their absolute configuration.
doi:10.3390/ijms15069844
PMCID: PMC4100125  PMID: 24897021
inherently chiral; Calix[4]arene; helix theory; absolute configuration
24.  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
25.  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

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