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1.  Hyperspectral Imaging for Mapping of Total Nitrogen Spatial Distribution in Pepper Plant 
PLoS ONE  2014;9(12):e116205.
Visible/near-infrared (Vis/NIR) hyperspectral imaging was employed to determine the spatial distribution of total nitrogen in pepper plant. Hyperspectral images of samples (leaves, stems, and roots of pepper plants) were acquired and their total nitrogen contents (TNCs) were measured using Dumas combustion method. Mean spectra of all samples were extracted from regions of interest (ROIs) in hyperspectral images. Random frog (RF) algorithm was implemented to select important wavelengths which carried effective information for predicting the TNCs in leaf, stem, root, and whole-plant (leaf-stem-root), respectively. Based on full spectra and the selected important wavelengths, the quantitative relationships between spectral data and the corresponding TNCs in organs (leaf, stem, and root) and whole-plant (leaf-stem-root) were separately developed using partial least-squares regression (PLSR). As a result, the PLSR model built by the important wavelengths for predicting TNCs in whole-plant (leaf-stem-root) offered a promising result of correlation coefficient (R) for prediction (RP = 0.876) and root mean square error (RMSE) for prediction (RMSEP = 0.426%). Finally, the TNC of each pixel within ROI of the sample was estimated to generate the spatial distribution map of TNC in pepper plant. The achievements of the research indicated that hyperspectral imaging is promising and presents a powerful potential to determine nitrogen contents spatial distribution in pepper plant.
doi:10.1371/journal.pone.0116205
PMCID: PMC4280196  PMID: 25549353
2.  Allokutzneria multivorans sp. nov., an actinomycete isolated from soil 
An actinomycete with well-branched mycelia, designated strain YIM 120521T, was isolated from soil collected from the banks of the Nujiang River, Yunnan Province, south-west China. Both aerial and substrate mycelia were white and non-pigmented. Growth was observed at 4–40 °C (optimum 28 °C), pH 6.0–9.0 (optimum 7.0) and in 0–4 % (w/v) NaCl (optimum 0 %). Analysis of the 16S rRNA gene sequence revealed that strain YIM 120521T belongs to the genus Allokutzneria with the highest sequence similarity to Allokutzneria albata DSM 44149T (98.4 %). However, the mean DNA–DNA relatedness value between the two strains was below 70 %. Chemotaxonomic characteristics supported the inclusion of strain YIM 120521T in the genus Allokutzneria, with rhamnose, arabinose, glucose, galactose and mannose as the whole-cell sugars, meso-diaminopimelic acid as the cell-wall diamino acid and MK-9(H4) as the predominant menaquinone. On the basis of physiological, biochemical and chemotaxonomic characteristics, strain YIM 120521T is considered to represent a novel species of the genus Allokutzneria, for which the name Allokutzneria multivorans sp. nov. is proposed. The type strain is YIM 120521T ( = JCM 17342T = DSM 45532T).
doi:10.1099/ijs.0.054411-0
PMCID: PMC3836494  PMID: 23811139
3.  Development and Characterisation of Ursolic Acid Nanocrystals Without Stabiliser Having Improved Dissolution Rate and In Vitro Anticancer Activity 
AAPS PharmSciTech  2013;15(1):11-19.
Ursolic acid (UA), which is a natural pentacyclic triterpenoid, has the potential to be developed as an anticancer drug, whereas its poor aqueous solubility and dissolution rate limit its clinical application. The aim of the present study was to develop UA nanocrystals to enhance its aqueous dispersibility, dissolution rate and anticancer activity. Following the investigation on the effects of stabiliser, the ratio of organic phase to aqueous solution and drug concentration, the UA nanocrystals without stabiliser were successfully prepared by anti-solvent precipitation approach. The nanocrystals maintained similar crystallinity with particle size, polydispersion index and zeta potential values of 188.0 ± 4.4 nm, 0.154 ± 0.022, and −25.0 ± 5.9 mV, respectively. Compared with the raw material, the UA nanocrystals showed good aqueous dispensability and a higher dissolution rate, and they could be completely dissolved in 0.5% SDS solution within 120 min. Moreover, the suspension of UA nanocrystals was physically stable after storage at 4°C for 7 weeks. By inducing G2/M phase cell cycle arrest, the UA nanocrystals significantly induced stronger cell growth inhibition activity against MCF-7 cells compared with free drug in vitro, although the uptake of free UA was approximately twice higher than that of the UA nanocrystals. The UA nanocrystals may be used as a potential delivery formulation for intravenous injection with enhanced dissolution velocity and anticancer activity.
doi:10.1208/s12249-013-0028-0
PMCID: PMC3909170  PMID: 24022345
anticancer; dissolution; MCF-7; nanocrystals; ursolic acid
4.  Augmenter of liver regeneration ameliorates renal fibrosis in rats with obstructive nephropathy 
Bioscience Reports  2014;34(5):e00135.
Renal fibrosis is a hallmark in CKD (chronic kidney disease) and is strongly correlated to the deterioration of renal function that is characterized by tubulointerstitial fibrosis, tubular atrophy, glomerulosclerosis and disruption of the normal architecture of the kidney. ALR (augmenter of liver regeneration) is a growth factor with biological functions similar to those of HGF (hepatocyte growth factor). In this study, our results indicate that endogenous ALR is involved in the pathological progression of renal fibrosis in UUO (unilateral ureteral obstruction) rat model. Moreover, we find that administration of rhALR (recombinant human ALR) significantly alleviates renal interstitial fibrosis and reduces renal-fibrosis-related proteins in UUO rats. Further investigation reveals that rhALR suppresses the up-regulated expression of TGF-β1 (transforming growth factor β1) induced by UUO operation in the obstructed kidney, and inhibits Smad2 and Smad3 phosphorylation activated by the UUO-induced injury in the animal model. Therefore we suggest that ALR is involved in the progression of renal fibrosis and administration of rhALR protects the kidney against renal fibrosis by inhibition of TGF-β/Smad activity.
doi:10.1042/BSR20140038
PMCID: PMC4155836  PMID: 24844766
augmenter of liver regeneration; renal fibrosis; Smads protein; transforming growth factor-β1; tubular epithelial–mesenchymal transition; ALR, augmenter of liver regeneration; CKD, chronic kidney disease; ECM, extracellular matrix; EMT, epithelial–mesenchymal transition; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HGF, hepatocyte growth factor; IgG, immunoglobulin G; rhALR, recombinant human ALR; RT, reverse transcription; TGF-β, transforming growth factor β; UUO, unilateral ureteral obstruction
5.  Transplacental transfer and metabolism of 17α-hydroxyprogesterone caproate 
Objective
Determine transplacental transfer and metabolism of 17α-hydroxyprogesterone caproate (17-HPC) and its distribution between the tissue, maternal, and fetal circuits of the dually perfused placental lobule.
Study design
17-HPC (21ng/ml) and its dual labeled isotope, 17α-hydroxy-[3H]progesterone [14C] caproate were added to the maternal circuit. The concentrations of the drug and its metabolite in trophoblast tissue and both circuits were determined by High Performance Liquid Chromatography (HPLC) and liquid scintillation spectrometry.
Results
The data obtained revealed that 17-HPC is transferred from the maternal to fetal circuit. At the end of a 4 hour perfusion period, a metabolite of 17-HPC that retained both progesterone and caproate moiety was identified in the tissue, maternal and fetal circuits. Neither 17-HPC nor its metabolite, at the concentrations tested, had any adverse effect on the determined viability and functional parameters of placental tissue.
Conclusions
17α-hydroxyprogesterone caproate is metabolized by term placental lobule during its perfusion and both parent compound and its metabolite transferred to the fetal circuit.
doi:10.1016/j.ajog.2007.11.065
PMCID: PMC4046337  PMID: 18674659
preterm delivery; 17α-hydroxyprogesterone caproate; dual perfusion; metabolism
6.  Laser-induced Greenish-Blue Photoluminescence of Mesoporous Silicon Nanowires 
Scientific Reports  2014;4:4940.
Solid silicon nanowires and their luminescent properties have been widely studied, but lesser is known about the optical properties of mesoporous silicon nanowires (mp-SiNWs). In this work, we present a facile method to generate greenish-blue photoluminescence (GB-PL) by fast scanning a focused green laser beam (wavelength of 532 nm) on a close-packed array of mp-SiNWs to carry out photo-induced chemical modification. The threshold of laser power is 5 mW to excite the GB-PL, whose intensity increases with laser power in the range of 5–105 mW. The quenching of GB-PL comes to occur beyond 105 mW. The in-vacuum annealing effectively excites the GB-PL in the pristine mp-SiNWs and enhances the GB-PL of the laser-modified mp-SiNWs. A complex model of the laser-induced surface modification is proposed to account for the laser-power and post-annealing effect. Moreover, the fast scanning of focused laser beam enables us to locally tailor mp-SiNWs en route to a wide variety of micropatterns with different optical functionality, and we demonstrate the feasibility in the application of creating hidden images.
doi:10.1038/srep04940
PMCID: PMC4018655  PMID: 24820533
7.  A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge 
Journal of Virology  2014;88(9):4624-4631.
ABSTRACT
Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use.
IMPORTANCE A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.
doi:10.1128/JVI.00005-14
PMCID: PMC3993805  PMID: 24522928
8.  Hendra Virus Vaccine, a One Health Approach to Protecting Horse, Human, and Environmental Health 
Emerging Infectious Diseases  2014;20(3):372-379.
In recent years, the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health. For example, Hendra virus (HeV), a zoonotic paramyxovirus, was discovered in 1994, and since then, infections have occurred in 7 humans, each of whom had a strong epidemiologic link to similarly affected horses. As a consequence of these outbreaks, eradication of bat populations was discussed, despite their crucial environmental roles in pollination and reduction of the insect population. We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse, human, and environmental health. The HeV vaccine for horses is a key example of a One Health approach to the control of human disease.
doi:10.3201/eid2003.131159
PMCID: PMC3944873  PMID: 24572697
Hendra virus; HeV; vaccine; HeV vaccine; One Health; G glycoprotein; zoonoses; viruses; zoonotic paramyxovirus; flying foxes; horses; humans; Pteropus bats; Australia; environment; vaccination
9.  Effects of resveratrol on apoptosis in a rat model of vascular dementia 
Resveratrol is a natural polyphenol widely present in plants, particularly in the skin of red grapes and in wine. It possesses a wide range of biological effects and exhibits neuroprotective effects in numerous diseases. However, data evaluating the effects of resveratrol in vascular dementia (VaD) are lacking. In the present study, the permanent, bilateral common carotid artery occlusion rat model was used to study the effects of resveratrol on VaD. The Morris water maze was used to test the spatial learning and memory performance of the rats. The expression levels of Bax, Bcl-2, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) in the hippocampus were measured. The results showed that resveratrol inhibited memory impairment in the VaD rat model, and attenuated the increases in the expression levels of Bax, cleaved caspase-3 and cleaved PARP and the reductions in the expression levels of Bcl-2 that were induced by VaD. These results provide a novel insight into the neuroprotective effects of resveratrol and its possible therapeutic role in VaD.
doi:10.3892/etm.2014.1542
PMCID: PMC3961111  PMID: 24660032
vascular dementia; resveratrol; apoptosis; caspase-3; poly(ADP-ribose) polymerase
10.  Cadherin-11 regulates cell-cell tension necessary for calcific nodule formation by valvular myofibroblasts 
Objective
Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MEK1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of TGF-β1-induced cadherin-11 expression in calcific nodule formation.
Methods and Results
As shown previously, porcine AVICs treated with TGF-β1 prior to cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, TGF-β1 treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in TGF-β1 treated AVICs possessing cadherin-11. Furthermore, when siRNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell-cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets.
Conclusions
These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2 dependent pathway.
doi:10.1161/ATVBAHA.112.300278
PMCID: PMC3536033  PMID: 23162011
11.  Biotransformation of ginsenoside Rb1 via the gypenoside pathway by human gut bacteria 
Chinese Medicine  2013;8:22.
Background
Bacterial conversion of ginsenosides is crucial for the health-promoting effects of ginsenosides. Previous studies on the biotransformation of ginsenoside Rb1 (Rb1) by gut bacteria have focused on the ginsenoside Rd (Rd) pathway (Rb1 → Rd → ginsenoside F2 (F2) → compound K (Cpd K)). This study aims to examine the gypenoside pathway in human gut bacteria in vitro.
Methods
The metabolic pathways of ginsenoside Rb1 and its metabolites ginsenoside Rd and gypenoside XVII in human gut bacteria were investigated by incubating the compounds anaerobically with pooled or individual gut bacteria samples from healthy volunteers. Ginsenoside Rb1, the metabolites generated by human gut bacteria, and degraded products in simulated gastric fluid (SGF) were qualitatively analyzed using an LC/MSD Trap system in the negative ion mode and quantitatively determined by HPLC-UV analysis.
Results
When incubated anaerobically with pooled gut bacteria, Rb1 generated five metabolites, namely Rd, F2, Cpd K, and the rare gypenosides XVII (G-XVII) and LXXV (G-LXXV). The gypenoside pathway (Rb1 → G-XVII → G-LXXV → Cpd K) was rapid, intermediate, and minor, and finally converted Rb1 to Cpd K via G-XVII → F2 (major)/G-LXXV (minor). Both the Rd and gypenoside pathways exhibited great inter-individual variations in age-and sex-independent manners (P > 0.05). Rb1 was highly acid-labile and degraded rapidly to form F2, ginsenoside Rg3, ginsenoside Rh2, and Cpd K, but did not generate the gypenosides in SGF. The formation of the gypenosides might be explained by the involvement of a gut bacteria-mediated enzymatic process.
Conclusions
Rb1 was metabolized to G-XVII, F2 (major) or G-LXXL (minor), and finally Cpd K by human gut bacteria in vitro.
doi:10.1186/1749-8546-8-22
PMCID: PMC4175505  PMID: 24267405
12.  Correction: Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites 
PLoS ONE  2013;8(11):10.1371/annotation/9cb2000b-a962-453c-ad8b-088f91095f6d.
doi:10.1371/annotation/9cb2000b-a962-453c-ad8b-088f91095f6d
PMCID: PMC3838210
13.  The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Three-Dimensional Model to Culture Human Pluripotent Stem Cells 
Stem Cells and Development  2013;23(4):380-392.
Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.
doi:10.1089/scd.2013.0314
PMCID: PMC3920753  PMID: 24188453
14.  Enteractinococcus coprophilus gen. nov., sp. nov., of the family Micrococcaceae, isolated from Panthera tigris amoyensis faeces, and transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as Enteractinococcus fodinae comb. nov. 
A novel actinobacterium, designated strain YIM 100590T, was isolated from Panthera tigris amoyensis faeces collected from Yunnan Wild Animal Park in Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 100590T is a member of the family Micrococcaceae. Cells were coccoid to oval (0.7–1.5 µm in diameter) occurring singly or in clusters. Growth was observed at 10–37 °C (optimum 28 °C) and at pH 7.0–11.0 (optimum pH 8.0). The major fatty acids were iso-C15 : 0 (32.22 %), anteiso-C15 : 0 (31.64 %) and iso-C16 : 0 (17.38 %). The peptidoglycan was of A4α type (l-Lys–Gly–l-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, dimannosyl diacylglycerol, an unknown glycolipid and two unknown phospholipids. The quinone system comprised menaquinones MK-7 (91.9 %) and MK-8 (8.3 %). The DNA G+C content of strain YIM 100590T was 56.2 mol%. Chemotaxonomic data indicated that the strain belongs to the family Micrococcaceae. On the basis of morphological and chemotaxonomic data and phylogenetic analysis, strain YIM 100590T is considered to represent a novel species of a new genus within the family Micrococcaceae, for which the name Enteractinococcus coprophilus gen. nov., sp. nov. is proposed. The type strain of Enteractinococcus coprophilus is YIM 100590T ( = DSM 24083T = JCM 17352T). Yaniella fodinae DSM 22966T was transferred to the new genus as Enteractinococcus fodinae comb. nov. (type strain G5T = DSM 22966T = JCM 17931T = MTCC 9846T).
doi:10.1099/ijs.0.034249-0
PMCID: PMC4080730  PMID: 22228667
15.  Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites 
PLoS ONE  2013;8(10):e75338.
1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1−/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1−/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.
doi:10.1371/journal.pone.0075338
PMCID: PMC3792969  PMID: 24116037
16.  Cathepsin L Plays a Role in Quinolinic Acid-Induced NF-Κb Activation and Excitotoxicity in Rat Striatal Neurons 
PLoS ONE  2013;8(9):e75702.
The present study seeks to investigate the role of cathepsin L in glutamate receptor-induced transcription factor nuclear factor-kappa B (NF-κB) activation and excitotoxicity in rats striatal neurons. Stereotaxic administration of the N-methyl-d-aspartate (NMDA) receptor agonist Quinolinic acid (QA) into the unilateral striatum was used to produce the in vivo excitotoxic model. Co-administration of QA and the cathepsin L inhibitor Z-FF-FMK or 1-Naphthalenesulfonyl-IW-CHO (NaphthaCHO) was used to assess the contribution of cathepsin L to QA-induced striatal neuron death. Western blot analysis and cathepsin L activity assay were used to assess the changes in the levels of cathepsin L after QA treatment. Western blot analysis was used to assess the changes in the protein levels of inhibitor of NF-κB alpha isoform (IκB-α) and phospho-IκB alpha (p-IκBα) after QA treatment. Immunohistochemical analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced NF-κB. Western blot analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced IκB-α phosphorylation and degradation, changes in the levels of IKKα, p-IKKα, TP53, caspase-3, beclin1, p62, and LC3II/LC3I. The results show that QA-induced loss of striatal neurons were strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced degradation of IκB-α, NF-κB nuclear translocation, up-regulation of NF-κB responsive gene TP53, and activation of caspase-3 was strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced increases in beclin 1, LC3II/LC3I, and down-regulation of p62 were reduced by Z-FF-FMK or NaphthaCHO. These results suggest that cathepsin L is involved in glutamate receptor-induced NF-κB activation. Cathepsin L inhibitors have neuroprotective effects by inhibiting glutamate receptor-induced IκB-α degradation and NF-κB activation.
doi:10.1371/journal.pone.0075702
PMCID: PMC3779166  PMID: 24073275
17.  Estimation of glomerular filtration rate by a radial basis function neural network in patients with type-2 diabetes mellitus 
BMC Nephrology  2013;14:181.
Background
Accurate and precise estimates of glomerular filtration rate (GFR) are essential for clinical assessments, and many methods of estimation are available. We developed a radial basis function (RBF) network and assessed the performance of this method in the estimation of the GFRs of 207 patients with type-2 diabetes and CKD.
Methods
Standard GFR (sGFR) was determined by 99mTc-DTPA renal dynamic imaging and GFR was also estimated by the 6-variable MDRD equation and the 4-variable MDRD equation.
Results
Bland-Altman analysis indicated that estimates from the RBF network were more precise than those from the other two methods for some groups of patients. However, the median difference of RBF network estimates from sGFR was greater than those from the other two estimates, indicating greater bias. For patients with stage I/II CKD, the median absolute difference of the RBF network estimate from sGFR was significantly lower, and the P50 of the RBF network estimate (n = 56, 87.5%) was significantly higher than that of the MDRD-4 estimate (n = 49, 76.6%) (p < 0.0167), indicating that the RBF network estimate provided greater accuracy for these patients.
Conclusions
In patients with type-2 diabetes mellitus, estimation of GFR by our RBF network provided better precision and accuracy for some groups of patients than the estimation by the traditional MDRD equations. However, the RBF network estimates of GFR tended to have greater bias and higher than those indicated by sGFR determined by 99mTc-DTPA renal dynamic imaging.
doi:10.1186/1471-2369-14-181
PMCID: PMC3766235  PMID: 23988079
Type 2 diabetes; Chronic kidney disease; Glomerular filtration rate; Artificial neural network
18.  Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months 
Virology Journal  2013;10:237.
Background
Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family Paramyxoviridae. Since NiV was first identified in 1999, outbreaks have continued to occur in humans in Bangladesh and India on an almost annual basis with case fatality rates reported between 40% and 100%.
Methods
Ferrets were vaccinated with 4, 20 or 100 μg HeVsG formulated with the human use approved adjuvant, CpG, in a prime-boost regime. One half of the ferrets were exposed to NiV at 20 days post boost vaccination and the other at 434 days post vaccination. The presence of virus or viral genome was assessed in ferret fluids and tissues using real-time PCR, virus isolation, histopathology, and immunohistochemistry; serology was also carried out. Non-immunised ferrets were also exposed to virus to confirm the pathogenicity of the inoculum.
Results
Ferrets exposed to Nipah virus 20 days post vaccination remained clinically healthy. Virus or viral genome was not detected in any tissues or fluids of the vaccinated ferrets; lesions and antigen were not identified on immunohistological examination of tissues; and there was no increase in antibody titre during the observation period, consistent with failure of virus replication. Of the ferrets challenged 434 days post vaccination, all five remained well throughout the study period; viral genome – but not virus - was recovered from nasal secretions of one ferret given 20 μg HeVsG and bronchial lymph nodes of the other. There was no increase in antibody titre during the observation period, consistent with lack of stimulation of a humoral memory response.
Conclusions
We have previously shown that ferrets vaccinated with 4, 20 or 100 μg HeVsG formulated with CpG adjuvant, which is currently in several human clinical trials, were protected from HeV disease. Here we show, under similar conditions of use, that the vaccine also provides protection against NiV-induced disease. Such protection persists for at least 12 months post-vaccination, with data supporting only localised and self-limiting virus replication in 2 of 5 animals. These results augur well for acceptability of the vaccine to industry.
doi:10.1186/1743-422X-10-237
PMCID: PMC3718761  PMID: 23867060
Nipah virus; Hendra virus; Henipavirus; Paramyxovirus; Ferret; Immunity; Vaccination; Glycoprotein; Subunit vaccine; Longevity
19.  Nonomuraea soli sp. nov., an actinomycete isolated from soil 
A straight-chain, spore-forming actinobacterium, strain YIM 120770T, was isolated from soil. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons revealed that the isolate represents a distinct cluster within the clade comprising the genus Nonomuraea and is related most closely to Nonomuraea rhizophila YIM 67092T (96.5 % similarity). Cells of strain YIM 120770T grew in the presence of 0–3 % (w/v) NaCl, at 15–37 °C and at pH 7.0–8.0. The diagnostic amino acid was meso-diaminopimelic acid, cell hydrolysates contained madurose, glucose, mannose, ribose and galactose, the predominant cellular fatty acids were 10-methyl C17 : 0 and iso-C16 : 0, and the DNA G+C content was 66.4 mol%, data consistent with affiliation of strain YIM 120770T to the genus Nonomuraea. Strain YIM 120770T shared low levels of 16S rRNA gene sequence similarity (<97 %) with the type strains of recognized species of the genus Nonomuraea and could be differentiated from its closest phylogenetic relative based on phenotypic characteristics. These results suggested that strain YIM 120770T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea soli sp. nov. is proposed. The type strain is YIM 120770T ( = DSM 45533T = JCM 17347T).
doi:10.1099/ijs.0.034751-0
PMCID: PMC3541754  PMID: 21890732
20.  An Integrative Proteomics and Interaction Network-Based Classifier for Prostate Cancer Diagnosis 
PLoS ONE  2013;8(5):e63941.
Aim
Early diagnosis of prostate cancer (PCa), which is a clinically heterogeneous-multifocal disease, is essential to improve the prognosis of patients. However, published PCa diagnostic markers share little overlap and are poorly validated using independent data. Therefore, we here developed an integrative proteomics and interaction network-based classifier by combining the differential protein expression with topological features of human protein interaction networks to enhance the ability of PCa diagnosis.
Methods and Results
By two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MS using PCa and adjacent benign tissues of prostate, a total of 60 proteins with the differential expression in PCa tissues were identified as the candidate markers. Then, their networks were analyzed by GeneGO Meta-Core software and three hub proteins (PTEN, SFPQ and HDAC1) were chosen. After that, a PCa diagnostic classifier was constructed by support vector machine (SVM) modeling based on the microarray gene expression data of the genes which encode the hub proteins mentioned above. Validations of diagnostic performance showed that this classifier had high predictive accuracy (85.96∼90.18%) and area under ROC curve (approximating 1.0). Furthermore, the clinical significance of PTEN, SFPQ and HDAC1 proteins in PCa was validated by both ELISA and immunohistochemistry analyses. More interestingly, PTEN protein was identified as an independent prognostic marker for biochemical recurrence-free survival in PCa patients according to the multivariate analysis by Cox Regression.
Conclusions
Our data indicated that the integrative proteomics and interaction network-based classifier which combines the differential protein expression and topological features of human protein interaction network may be a powerful tool for the diagnosis of PCa. We also identified PTEN protein as a novel prognostic marker for biochemical recurrence-free survival in PCa patients.
doi:10.1371/journal.pone.0063941
PMCID: PMC3667836  PMID: 23737958
21.  The effect of esterases on 17α-hydroxyprogesteronecaproate 
Objectives
The aim of this investigation is to determine whether 17α-hydroxyprogesterone caproate is hydrolyzed, in vitro, to 17α-hydroxyprogesterone and caproate.
Study design
The in vitro hydrolysis of dual radioactively labeled 17α-hydroxy-[3H] progesterone [14C] caproate by human plasma, hepatic and placental S9 fractions as well as recombinant esterases was investigated. The formation of [3H]-17α-hydroxyprogesterone and [14C]-caproate were determined using HPLC equipped with an online radioactivity detector. The presence and activity of carboxylesterase and butyrylcholinesterase in the human derived preparations was confirmed by the hydrolysis of their prototypic substrates p-nitrophenyl acetate, p-nitrophenyl butyrate and butyrylthiocholine, respectively.
Results
The aforementioned human derived preparations hydrolyzed p-nitrophenyl acetate, p-nitrophenyl butyrate and butyrylthiocholine. However, when 17α-hydroxyprogesterone caproate was incubated with the human derived preparations under identical experimental conditions neither [3H]-17α-hydroxyprogesterone nor [14C]-caproate was detected.
Conclusion
17α-Hydroxyprogesterone caproate is not hydrolyzed in vitro by the esterase enzymes present in human plasma, liver, preterm or term placenta.
doi:10.1016/j.ajog.2007.07.038
PMCID: PMC3622550  PMID: 17936237
Spontaneous preterm labor; 17α-hydroxyprogesterone caproate; hydrolysis
22.  Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus 
Glycobiology  2011;22(4):572-584.
Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.
doi:10.1093/glycob/cwr180
PMCID: PMC3287018  PMID: 22171062
glycopeptides; Hendra virus (HeV); mass spectrometry; N- and O-linked glycosylation
23.  Reproducibility of intracardiac and transpulmonary biomarkers in the evaluation of pulmonary hypertension 
Pulmonary Circulation  2013;3(2):345-349.
Intracardiac and transpulmonary levels of natriuretic peptides (NPs) and cyclic guanosine monophosphate (cGMP) provide insight into the pathophysiology of pulmonary hypertension (PH) secondary to left-heart failure but have not been evaluated in established or suspected pulmonary arterial hypertension (PAH). Demonstrating adequate reproducibility of these markers is an important precursor to further study. We hypothesized that the reproducibility of intracardiac and transpulmonary NPs and cGMP is similar to the reproducibility of these markers sampled from the peripheral venous circulation. In outpatients undergoing right-heart catheterization for PH, blood samples were obtained from a peripheral venous site, superior vena cava, inferior vena cava, coronary sinus, pulmonary artery, and pulmonary capillary wedge position. At each site, a repeat sample was collected approximately 60 seconds after the initial measurement. Reproducibility was assessed using the slope of the regression line between initial and follow-up levels. We enrolled 10 patients: Six had PAH, two had pulmonary venous hypertension, and two had normal pulmonary pressure. At all sites, the slopes of the regression lines for BNP were close to identity. BNP was generally more reproducible than NT-pro-BNP. For the NPs and cGMP, reproducibility at intracardiac and transpulmonary sites was similar to the peripheral venous site. Reproducibility of NPs was not influenced by PH severity, access site, or time between measurements. The two patients with the highest transpulmonary pressure gradients had high transpulmonary BNP uptake, but their transpulmonary cGMP gradients were negative. In patients evaluated for PH, reproducibility of NPs and cGMP at intracardiac and transpulmonary sites is high and is comparable to that of peripheral venous measurements.
doi:10.4103/2045-8932.114762
PMCID: PMC3757828  PMID: 24015334
cyclic guanosine monophosphate; natriuretic peptides; right-heart catheterization
24.  Neutralizing Antibody Responses in Macaques Induced by Human Immunodeficiency Virus Type 1 Monovalent or Trivalent Envelope Glycoproteins 
PLoS ONE  2013;8(3):e59803.
A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb). In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs) would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q), was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B) and 14/00/4 (subtype F), both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q). The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.
doi:10.1371/journal.pone.0059803
PMCID: PMC3606129  PMID: 23533650
25.  A Hendra Virus G Glycoprotein Subunit Vaccine Protects African Green Monkeys from Nipah Virus Challenge 
Science translational medicine  2012;4(146):146ra107.
In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use.
doi:10.1126/scitranslmed.3004241
PMCID: PMC3516289  PMID: 22875827

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