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1.  Material Properties of Aged Human Mitral Valve Leaflets 
Objective
To characterize the mechanical properties of aged human anterior (AML) and posterior (PML) mitral leaflets.
Materials and Methods
The AML and PML samples from explanted human hearts (n = 21, mean age of 82.62 ± 8.77 years old) were subjected to planar biaxial mechanical tests. The material stiffness, extensibility and degree of anisotropy of the leaflet samples were quantified. The microstructure of the samples was assessed through histology.
Results
Both the AML and PML samples exhibited a nonlinear and anisotropic behavior with the circumferential direction being stiffer than the radial direction. The AML samples were significantly stiffer than the PML samples in both directions, suggesting that they should be modeled with separate sets of material properties in computational studies. Histological analysis indicated the changes in the tissue elastic constituents, including the fragmented and disorganized elastin network, the presence of fibrosis and proteoglycan/glycosaminoglycan infiltration and calcification, suggesting possible valvular degenerative characteristics in the aged human leaflet samples. Overall, stiffness increased and areal strain decreased with calcification severity. In addition, leaflet tissues from hypertensive individuals also exhibited a higher stiffness and low areal strain than normotensive individuals.
Conclusion
There are significant differences in the mechanical properties of the two human mitral valve leaflets from this advanced age group. The morphologic changes in the tissue composition and structure also infer the structural and functional difference between aged human valves and those of animals.
doi:10.1002/jbm.a.34939
PMCID: PMC4033712  PMID: 24039052
human mitral valve; anterior mitral leaflet; posterior mitral leaflet; mechanical properties; planar biaxial test
2.  Simulation of long-term fatigue damage in bioprosthetic heart valves: effects of leaflet and stent elastic properties 
One of the major failure modes of bioprosthetic heart valves (BHVs) is noncalcific structural deterioration due to fatigue of the tissue leaflets; yet, the mechanisms of fatigue are not well understood. BHV durability is primarily assessed based on visual inspection of the leaflets following accelerated wear testing. In this study, we developed a computational framework to simulate BHV leaflet fatigue, which is both efficient and quantitative, making it an attractive alternative to traditional accelerated wear testing. We utilize a phenomenological soft tissue fatigue damage model developed previously to describe the stress softening and permanent set of the glutaraldehyde-treated bovine pericardium leaflets in BHVs subjected to cyclic loading. A parametric study was conducted to determine the effects of altered leaflet and stent elastic properties on the fatigue of the leaflets. The simulation results show that heterogeneity of the leaflet elastic properties, poor leaflet coaptation, and little stent-tip deflection may accelerate leaflet fatigue, which agrees with clinical findings. Therefore, the developed framework may be an invaluable tool for evaluating leaflet durability in new tissue valve designs, including traditional BHVs as well as new transcatheter valves.
doi:10.1007/s10237-013-0532-x
PMCID: PMC3976462  PMID: 24092257
Bioprosthetic heart valve durability; Soft tissue fatigue; Finite element analysis
3.  Partial Correlation Matrix Estimation using Ridge Penalty Followed by Thresholding and Reestimation 
Biometrics  2014;70(3):762-770.
Summary
Motivated by the problem of construction gene co-expression network, we propose a statistical framework for estimating high-dimensional partial correlation matrix by a three-step approach. We first obtain a penalized estimate of a partial correlation matrix using ridge penalty. Next we select the non-zero entries of the partial correlation matrix by hypothesis testing. Finally we reestimate the partial correlation coefficients at these non-zero entries. In the second step, the null distribution of the test statistics derived from penalized partial correlation estimates has not been established. We address this challenge by estimating the null distribution from the empirical distribution of the test statistics of all the penalized partial correlation estimates. Extensive simulation studies demonstrate the good performance of our method. Application on a yeast cell cycle gene expression data shows that our method delivers better predictions of the protein-protein interactions than the Graphic Lasso.
doi:10.1111/biom.12186
PMCID: PMC4239206  PMID: 24845967
Co-expression network; Empirical null distribution; Graphical model; Partial correlation matrix; Ridge regression
4.  A Proteomic Analysis of Individual and Gender Variations in Normal Human Urine and Cerebrospinal Fluid Using iTRAQ Quantification 
PLoS ONE  2015;10(7):e0133270.
Urine and cerebrospinal fluid (CSF) are two important biofluids used for disease biomarker discovery. For differential proteomic analysis, it is essential to evaluate individual and gender variations. In this study, we characterized urinary and CSF proteomes of 14 healthy volunteers with regard to individual and gender variations using 2DLC-MS/MS analysis and 8-plex iTRAQ quantification. A total of 968/512 urinary/CSF proteins were identified, with 406/280 quantified in all individuals. The median inter-individual coefficients of variation (CVs) were 0.262 and 0.183 for urinary and CSF proteomes, respectively. Cluster analysis showed that male and female urinary proteomes exhibited different patterns, though CSF proteome showed no remarkable gender differences. In comparison with CSF proteome, urinary proteome showed higher individual variation. Further analysis revealed that individual variation was not correlated with protein abundance. The minimum sample size for proteomic analysis with a 2-fold change was 10 (4/5 for males/females using iTRAQ quantification) for urinary or 8 for CSF proteome. Intracellular proteins leaked from exfoliative cells tended to have higher CVs, and extracellular proteins secreted from urinary tract or originating from plasma tended to have lower CVs. The above results might be beneficial for differential proteomic analysis and biomarker discovery.
doi:10.1371/journal.pone.0133270
PMCID: PMC4519152  PMID: 26222143
5.  A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo 
BMC Cancer  2015;15:527.
Background
Tumor lymphangiogenesis plays an important role in promoting growth and metastasis of tumors, but no antilymphangiogenic agent is used clinically. Based on the effect of norcantharidin (NCTD) on lymphangiogenesis of human lymphatic endothelial cells (LECs), we firstly investigated the antilymphangiogenic activity of NCTD as a tumor lymphangiogenic inhibitor for human colonic adenocarcinomas (HCACs).
Methods
In vivo and in vitro experiments to determine the effects of NCTD on tumor growth and lymphangiogenesis of the in-situ colonic xenografts in nude mice, and lymphatic tube formation of the three-dimensional (3-D) of the co-culture system of HCAC HT-29 cells and LECs were done. Proliferation, apoptosis, migration, invasion, Ki-67, Bcl-2 and cell cycle of LECs and the co-culture system in vitro were respectively determined. Streparidin-peroxidase staining, SABC, western blotting and RT-PCR were respectively used to examine the expression of LYVE-1, D2-40, CK20 (including their LMVD), and VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in vitro and in vivo.
Results
NCTD inhibited tumor growth and lymphangiogenesis of the in-situ colonic xenografts in vivo, and these observations were confirmed by facts that lymphatic tube formation, proliferation, apoptosis, migration, invasion, S-phase cell cycle, and Ki-67 and Bcl-2 expression in vitro, and LYVE-1, D2-40, CK20 expression and their LMVD in vitro and in vivo were inhibited and affected. Furthermore, the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 at protein/mRNA levels in the process of lymphatic tube formation in vitro and tumor lymphangiogenesis in vivo was downregulated; NCTD in combination with mF4-31C1 or Sorafenib enhanced these effects.
Conclusions
NCTD inhibits tumor growth and lymphangiogenesis of HCACs through “multi-points priming” mechanisms i.e. affecting related malignant phenotypes, inhibiting Ki-67 and Bcl-2 expression, inducing S-phase cell cycle arrest, and directly or indirectly downregulating VEGF-A,-C,-D/VEGFR-2,-3 signaling pathways. The present finding strongly suggests that NCTD could serve as a potential antilymphangiogenic agent for tumor lymphangiogenesis and is of importance to explore NCTD is used for antitumor metastatic comprehensive therapy for HCACs.
doi:10.1186/s12885-015-1521-5
PMCID: PMC4506614  PMID: 26187792
Colonic neoplasm; Norcantharidin; Tumor growth; Lymphangiogenesis; Antilymphangiogenic therapy
6.  Quantification of Structural Compliance of Aged Human and Porcine Aortic Root Tissues 
The structural compliance of the aortic root has a significant implication for valve procedures such as transcatheter aortic valve implantation and valve-sparing aortic root replacement. However, a detailed quantification of human aortic roots structural compliance, particularly in different regions has been incomplete. In this study, the structural properties of human aortic roots (81 ± 8.74 years, n=10) were characterized and compared with those of porcine ones (6–9 months, n=10) using a vessel pressure-inflation test. The test involves tracking three-dimensional deformation of the markers affixed on the different surface regions of the aortic roots, including the three sinuses: the non-coronary sinus (NCS), the left coronary sinus (LCS) and the right coronary sinus (RCS), and at three regions along the longitudinal direction of each sinus: the upper sinus (US), the middle sinus (MS) and the lower sinus (LS), and the ascending aorta (AA) region above the NCS. We found that tissue stiffness at a physiological pressure range was similar among the three human sinuses. The regional structural stiffness of human aorta was observed. In the circumferential direction, the LS regions were the stiffest in the LCS and RCS sinuses, while NCS had relatively uniform stiffness. In the longitudinal direction, the human AA regions were more compliant than all sinuses. There was a significant difference in tissue stiffness between human and porcine aortic tissues, suggesting that the mechanical properties of porcine may not be analogous to aged human ones.
doi:10.1002/jbm.a.34884
PMCID: PMC3903671  PMID: 23894117
Heart valve mechanics; Aortic root compliance; Inflation test; Surface deformation; Direct linear transformation
7.  Possible Loss of GABAergic Inhibition in Mice With Induced Adenomyosis and Treatment With Epigallocatechin-3-Gallate Attenuates the Loss With Improved Hyperalgesia 
Reproductive Sciences  2014;21(7):869-882.
We have previously reported that induction of adenomyosis in mice results in progressive hyperalgesia, uterine hyperactivity, and elevated plasma corticosterone levels and that epigallocatechin-3-gallate (EGCG) treatment dose dependently suppressed myometrial infiltration and improved generalized hyperalgesia. In this study, we examined whether adenomyosis induced in mice results in the loss of GABAergic inhibition as manifested by the diminished glutamate decarboxylase (GAD) 65-expressing neurons in the brainstem nucleus raphe magnus (NRM) that could correlate with heightened hyperalgesia. We also evaluated whether EGCG treatment would reverse these changes and also improve the expression of some proteins known to be involved in adenomyosis. Adenomyosis was induced in 28 female ICR mice and additional 12 were used as blank controls, as reported previously. At the 16th week, all mice with induced adenomyosis received low- or high-dose EGCG treatment or untreated. Mice without adenomyosis received no treatment. After 3 weeks of treatment, their uterine horns and brains were harvested. The right uterine horn was used for immunohistochemistry analysis and for counting the number of macrophages infiltrating into the ectopic endometrium. The brainstem NRM sections were subjected to immunofluorescence staining for GAD65. We found that mice with induced adenomyosis had significantly diminished GAD65-expressing neurons, concomitant with heightened hyperalgesia. Treatment with EGCG increased these neurons in conjunction with improved hyperalgesia, reduced the expression of p-p65, cycloxygenase 2, oxytocin receptor, collagen I and IV, and transient receptor potential vanilloid type 1 in ectopic endometrium or myometrium, reduced the number of macrophages infiltrating into the ectopic endometrium while elevated the expression of progesterone receptor isoform B. Thus, adenomyosis-induced pain resembles neuropathic pain in that there is a remarkable central plasticity.
doi:10.1177/1933719113518984
PMCID: PMC4107564  PMID: 24492488
adenomyosis; epigallocatechin-3-gallate; GABAergic inhibition; hyperalgesia; mouse; pain; treatment
8.  Intra-ventral tegmental area HIV-1 Tat1–86 attenuates nicotine-mediated locomotor sensitization and alters mesocorticolimbic ERK and CREB signaling in rats 
Cigarette smoking prevalence in the HIV-positive individuals is profoundly higher than that in the HIV-negative individuals. We have demonstrated that HIV-1 transgenic rats exhibit attenuated nicotine-mediated locomotor activity, altered cAMP response element binding protein (CREB) and extracellular regulated kinase (ERK1/2) signaling in the mesocorticolimbic regions. This study investigated the role of HIV-1 transactivator of transcription (Tat) protein in the alterations of nicotine-mediated behavior and the signaling pathway observed in the HIV-1 transgenic rats. Rats received bilateral microinjection of recombinant Tat1–86 (25 μg/side) or vehicle directed at ventral tegmental area (VTA) followed by locomotor testing in response to 13 daily intravenous injections of nicotine (0.05 mg/kg, freebase, once/day) or saline. Further, we examined the phosphorylated levels of CREB (pCREB) and ERK1/2 (pERK1/2) in the prefrontal cortex (PFC), nucleus accumbens (NAc) and VTA. Tat diminished baseline activity in saline control rats, and attenuated nicotine-induced behavioral sensitization. Following repeated saline injection, the basal levels of pERK1 in the NAc and VTA and pERK2 in VTA were lower in the vehicle control group, relative to the Tat group. After repeated nicotine injection, pERK1 in NAc and VTA and pERK2 in VTA were increased in the vehicle group, but not in the Tat group. Moreover, repeated nicotine injections decreased pCREB in the PFC and VTA in the Tat group but not in the vehicle group. Thus, these findings indicate that the direct injection of Tat at the VTA may mediate CREB and ERK activity in response to nicotine-induced locomotor activity.
doi:10.3389/fmicb.2015.00540
PMCID: PMC4473058  PMID: 26150803
HIV-1 Tat; nicotine; locomotor activity; behavioral sensitization; CREB; ERK; rat
9.  Analysis on the psychological characteristics of patients with acute iridocyclitis 
doi:10.3980/j.issn.2222-3959.2015.03.36
PMCID: PMC4458677  PMID: 26086022
10.  Natural hybridization and asymmetric introgression at the distribution margin of two Buddleja species with a large overlap 
BMC Plant Biology  2015;15:146.
Background
Natural hybridization in plants is universal and plays an important role in evolution. Based on morphology it has been presumed that hybridization occurred in the genus Buddleja, though genetic studies confirming this assumption have not been conducted to date. The two species B. crispa and B. officinalis overlap in their distributions over a wide range in South-West China, and we aimed to provide genetic evidence for ongoing hybridization in this study.
Results
We investigated the occurrence of hybrids between the two species at the southern-most edge of the distribution of B. crispa using five nuclear loci and pollination experiments. The genetic data suggest substantial differentiation between the two species as species-specific alleles are separated by at least 7–28 mutations. The natural hybrids found were nearly all F1s (21 of 23), but backcrosses were detected, and some individuals, morphologically indistinguishable from the parental species, showed introgression. Pollen viability test shows that the percentage of viable pollen grains was 50 ± 4 % for B. crispa, and 81 ± 2 % for B. officinalis. This difference is highly significant (t = 7.382, p < 0.0001). Hand cross-pollination experiments showed that B. crispa is not successful as pollen-parent, but B. officinalis is able to pollinate B. crispa to produce viable hybrid seed. Inter-specific seed-set is low (8 seeds per fruit, as opposed to about 65 for intra-specific pollinations), suggesting post-zygotic reproductive barriers. In addition, one of the reference populations also suggests a history of introgression at other localities.
Conclusions
The occurrence of morphologically intermediate individuals between B. crispa and B. officinalis at Xishan Mountain is unequivocally linked to hybridization and almost all examined individuals of the putative hybrids were likely F1s. Despite pollination experiments indicating higher chances for introgression into B. officinalis (hybrids only produced viable seed when crossed with B. officinalis), observed introgression was asymmetrical into B. crispa. This could be due to seeds produced by hybrids not contributing to seedlings, or other factors favoring the establishment of backcrosses towards B. crispa. However, further research will be needed to confirm these observations, as the small number of plants used for the pollination experiments could have introduced an artifact, for example if used individuals were more or less compatible than the species average, and also the small number of loci used could convey a picture of introgression that is not representative for the whole genome.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0539-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-015-0539-9
PMCID: PMC4470074  PMID: 26081944
Asymmetric introgression; Buddleja; Hybridization; Nuclear genes; Reproductive isolation
11.  The Effects of Acupuncture on Cerebral and Muscular Microcirculation: A Systematic Review of Near-Infrared Spectroscopy Studies 
Acupuncture produces physiological effects via stimulating acupoints, proximal or distal to the region of effect. Near-infrared spectroscopy (NIRS) noninvasively measures tissue-level hemodynamics in real time. We review the literature investigating the effect of acupuncture on muscular and/or cerebral microcirculation. As the basis, we queried PubMed in June 2014 for articles mentioning both acupuncture and NIRS in title/abstract. The reviewed papers investigated either cerebral (n = 11) or muscular hemodynamics (n = 5) and, based on STRICTA for reporting acupuncture methodology, were overall poor in quality. Acupuncture was found to influence regional oxygen saturation in cerebral and muscular tissue. The cortical response in healthy subjects varied across studies. For subjects with stroke or cerebrovascular dementia, findings suggest that acupuncture may modulate dysfunction in cerebral autoregulation. The muscular response to pressure techniques was more intense than that to needling or laser. Probe proximity could impact measurement sensitivity. No one study simultaneously investigated the direct and remote responses. Research utilizing NIRS to investigate the hemodynamics of acupuncture presently lacks in scope and quality. Improved designs, for example, placebo-controlled, randomized trials, and standardized intervention reporting will raise study quality. Exploiting NIRS in clinical settings, such as stroke, migraine, or other pain conditions, is worthwhile.
doi:10.1155/2015/839470
PMCID: PMC4480930  PMID: 26221180
12.  Neuronal Transgene Expression in Dominant-Negative SNARE Mice 
The Journal of Neuroscience  2014;34(50):16594-16604.
Experimental advances in the study of neuroglia signaling have been greatly accelerated by the generation of transgenic mouse models. In particular, an elegant manipulation that interferes with astrocyte vesicular release of gliotransmitters via overexpression of a dominant-negative domain of vesicular SNARE (dnSNARE) has led to documented astrocytic involvement in processes that were traditionally considered strictly neuronal, including the sleep–wake cycle, LTP, cognition, cortical slow waves, depression, and pain. A key premise leading to these conclusions was that expression of the dnSNARE was specific to astrocytes. Inconsistent with this premise, we report here widespread expression of the dnSNARE transgene in cortical neurons. We further demonstrate that the activity of cortical neurons is reversibly suppressed in dnSNARE mice. These findings highlight the need for independent validation of astrocytic functions identified in dnSNARE mice and thus question critical evidence that astrocytes contribute to neurotransmission through SNARE-dependent vesicular release of gliotransmitters.
doi:10.1523/JNEUROSCI.2585-14.2014
PMCID: PMC4261088  PMID: 25505312
adenosine; EEG; GFAP; sleep; SNARE
13.  Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense 
Scientific Reports  2015;5:11087.
Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.
doi:10.1038/srep11087
PMCID: PMC4457147  PMID: 26046933
14.  Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants 
Annals of Botany  2014;113(7):1219-1233.
Background and Aims
The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized.
Methods
Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato.
Key Results
Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family.
Conclusions
This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants.
doi:10.1093/aob/mcu061
PMCID: PMC4030818  PMID: 24812252
OVATE family proteins; OFP; land plants; angiosperm; phylogenetic analyses; Arabidopsis thaliana; Oryza sativa; Solanum lycopersicum; segmental duplication; tandem duplication
15.  Disruption of chaperone-mediated autophagy-dependent degradation of MEF2A by oxidative stress-induced lysosome destabilization 
Autophagy  2014;10(6):1015-1035.
Oxidative stress has been implicated in both normal aging and various neurodegenerative disorders and it may be a major cause of neuronal death. Chaperone-mediated autophagy (CMA) targets selective cytoplasmic proteins for degradation by lysosomes and protects neurons against various extracellular stimuli including oxidative stress. MEF2A (myocyte enhancer factor 2A), a key transcription factor, protects primary neurons from oxidative stress-induced cell damage. However, the precise mechanisms of how the protein stability and the transcriptional activity of MEF2A are regulated under oxidative stress remain unknown. In this study, we report that MEF2A is physiologically degraded through the CMA pathway. In pathological conditions, mild oxidative stress (200 μM H2O2) enhances the degradation of MEF2A as well as its activity, whereas excessive oxidative stress (> 400 μM H2O2) disrupts its degradation process and leads to the accumulation of nonfunctional MEF2A. Under excessive oxidative stress, an N-terminal HDAC4 (histone deacetylase 4) cleavage product (HDAC4-NT), is significantly induced by lysosomal serine proteases released from ruptured lysosomes in a PRKACA (protein kinase, cAMP-dependent, catalytic, α)-independent manner. The production of HDAC4-NT, as a MEF2 repressor, may account for the reduced DNA-binding and transcriptional activity of MEF2A. Our work provides reliable evidence for the first time that MEF2A is targeted to lysosomes for CMA degradation; oxidative stress-induced lysosome destabilization leads to the disruption of MEF2A degradation as well as the dysregulation of its function. These findings may shed light on the underlying mechanisms of pathogenic processes of neuronal damage in various neurodegenerative-related diseases.
doi:10.4161/auto.28477
PMCID: PMC4091166  PMID: 24879151
chaperone-mediated autophagy; histone deacetylase 4; myocyte enhancer factor 2A; neurodegenerative diseases; oxidative stress
16.  Microbiota prevents cholesterol loss from the body by regulating host gene expression in mice 
Scientific Reports  2015;5:10512.
We have previously observed that knockout of Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol transporter essential for intestinal cholesterol absorption, reduces the output of dry stool in mice. As the food intake remains unaltered in NPC1L1-knockout (L1-KO) mice, we hypothesized that NPC1L1 deficiency may alter the gut microbiome to reduce stool output. Consistently, here we demonstrate that the phyla of fecal microbiota differ substantially between L1-KO mice and their wild-type controls. Germ-free (GF) mice have reduced stool output. Inhibition of NPC1L1 by its inhibitor ezetimibe reduces stool output in specific pathogen-free (SPF), but not GF mice. In addition, we show that GF versus SPF mice have reduced intestinal absorption and increased fecal excretion of cholesterol, particularly after treatment with ezetimibe. This negative balance of cholesterol in GF mice is associated with reduced plasma and hepatic cholesterol, and likely caused by reduced expression of NPC1L1 and increased expression of ABCG5 and ABCG8 in small intestine. Expression levels of other genes in intestine and liver largely reflect a state of cholesterol depletion and a decrease in intestinal sensing of bile acids. Altogether, our findings reveal a broad role of microbiota in regulating whole-body cholesterol homeostasis and its response to a cholesterol-lowering drug, ezetimibe.
doi:10.1038/srep10512
PMCID: PMC4444975  PMID: 26015368
17.  Effects of Water and Nitrogen Addition on Ecosystem Carbon Exchange in a Meadow Steppe 
PLoS ONE  2015;10(5):e0127695.
A changing precipitation regime and increasing nitrogen deposition are likely to have profound impacts on arid and semiarid ecosystem C cycling, which is often constrained by the timing and availability of water and nitrogen. However, little is known about the effects of altered precipitation and nitrogen addition on grassland ecosystem C exchange. We conducted a 3-year field experiment to assess the responses of vegetation composition, ecosystem productivity, and ecosystem C exchange to manipulative water and nitrogen addition in a meadow steppe. Nitrogen addition significantly stimulated aboveground biomass and net ecosystem CO2 exchange (NEE), which suggests that nitrogen availability is a primary limiting factor for ecosystem C cycling in the meadow steppe. Water addition had no significant impacts on either ecosystem C exchange or plant biomass, but ecosystem C fluxes showed a strong correlation with early growing season precipitation, rather than whole growing season precipitation, across the 3 experimental years. After we incorporated water addition into the calculation of precipitation regimes, we found that monthly average ecosystem C fluxes correlated more strongly with precipitation frequency than with precipitation amount. These results highlight the importance of precipitation distribution in regulating ecosystem C cycling. Overall, ecosystem C fluxes in the studied ecosystem are highly sensitive to nitrogen deposition, but less sensitive to increased precipitation.
doi:10.1371/journal.pone.0127695
PMCID: PMC4444226  PMID: 26010888
18.  Triptolide Attenuates Podocyte Injury by Regulating Expression of miRNA-344b-3p and miRNA-30b-3p in Rats with Adriamycin-Induced Nephropathy 
Objectives. We investigated the action of triptolide in rats with adriamycin-induced nephropathy and evaluated the possible mechanisms underlying its protective effect against podocyte injury. Methods. In total, 30 healthy male Sprague-Dawley rats were randomized into three groups (normal group, model group, and triptolide group). On days 7, 28, 42, and 56, 24 h urine samples were collected. All rats were sacrificed on day 56, and their blood and renal tissues were collected for determination of biochemical and molecular biological parameters. Expression of miRNAs in the renal cortex was analyzed by a biochip assay and RT-PCR was used to confirm observed differences in miRNA levels. Results. Triptolide decreased proteinuria, improved renal function without apparent adverse effects on the liver, and alleviated renal pathological lesions. Triptolide also elevated the nephrin protein level. Furthermore, levels of miR-344b-3p and miR-30b-3p were elevated in rats with adriamycin-induced nephropathy, while triptolide treatment reversed the increase in the expression of these two miRNAs. Conclusions. These results suggest that triptolide may attenuate podocyte injury in rats with adriamycin-induced nephropathy by regulating expression of miRNA-344b-3p and miRNA-30b-3p.
doi:10.1155/2015/107814
PMCID: PMC4452866  PMID: 26078766
19.  Cordycepin Decreases Compound Action Potential Conduction of Frog Sciatic Nerve In Vitro Involving Ca2+-Dependent Mechanisms 
Neural Plasticity  2015;2015:927817.
Cordycepin has been widely used in oriental countries to maintain health and improve physical performance. Compound nerve action potential (CNAP), which is critical in signal conduction in the peripheral nervous system, is necessary to regulate physical performance, including motor system physiological and pathological processes. Therefore, regulatory effects of cordycepin on CNAP conduction should be elucidated. In this study, the conduction ability of CNAP in isolated frog sciatic nerves was investigated. Results revealed that cordycepin significantly decreased CNAP amplitude and conductive velocity in a reversible and concentration-dependent manner. At 50 mg/L cordycepin, CNAP amplitude and conductive velocity decreased by 62.18 ± 8.06% and 57.34% ± 6.14% compared with the control amplitude and conductive velocity, respectively. However, the depressive action of cordycepin on amplitude and conductive velocity was not observed in Ca2+-free medium or in the presence of Ca2+ channel blockers (CdCl2/LaCl3). Pretreatment with L-type Ca2+ channel antagonist (nifedipine/deltiazem) also blocked cordycepin-induced responses; by contrast, T-type and P-type Ca2+ channel antagonists (Ni2+) failed to block such responses. Therefore, cordycepin decreased the conduction ability of CNAP in isolated frog sciatic nerves via L-type Ca2+ channel-dependent mechanism.
doi:10.1155/2015/927817
PMCID: PMC4452462  PMID: 26078886
20.  The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis 
PLoS ONE  2015;10(5):e0127180.
Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples.
doi:10.1371/journal.pone.0127180
PMCID: PMC4436214  PMID: 25984759
21.  Rapid Identification and Verification of Indirubin-Containing Medicinal Plants 
Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.
doi:10.1155/2015/484670
PMCID: PMC4451998  PMID: 26089942
22.  Tinnitus and hyperacusis involve hyperactivity and enhanced connectivity in auditory-limbic-arousal-cerebellar network 
eLife  null;4:e06576.
Hearing loss often triggers an inescapable buzz (tinnitus) and causes everyday sounds to become intolerably loud (hyperacusis), but exactly where and how this occurs in the brain is unknown. To identify the neural substrate for these debilitating disorders, we induced both tinnitus and hyperacusis with an ototoxic drug (salicylate) and used behavioral, electrophysiological, and functional magnetic resonance imaging (fMRI) techniques to identify the tinnitus–hyperacusis network. Salicylate depressed the neural output of the cochlea, but vigorously amplified sound-evoked neural responses in the amygdala, medial geniculate, and auditory cortex. Resting-state fMRI revealed hyperactivity in an auditory network composed of inferior colliculus, medial geniculate, and auditory cortex with side branches to cerebellum, amygdala, and reticular formation. Functional connectivity revealed enhanced coupling within the auditory network and segments of the auditory network and cerebellum, reticular formation, amygdala, and hippocampus. A testable model accounting for distress, arousal, and gating of tinnitus and hyperacusis is proposed.
DOI: http://dx.doi.org/10.7554/eLife.06576.001
eLife digest
One in three adults over the age of 65 will experience a significant loss of hearing. This is often worsened by related conditions, such as: tinnitus, an unexplained constant buzzing or ringing sound; and hyperacusis, whereby everyday sounds are perceived as too loud or painful.
Most hearing loss is caused by damage to the sound-sensitive cells within a structure in the inner ear called the cochlea. Some studies have also identified regions of the brain that show abnormal activity in people with tinnitus and hyperacusis. However, the results from different patients have often been inconsistent and sometimes contradictory, and so it remains unclear what exactly causes these conditions.
To overcome this problem, Chen et al. made use of the fact that tinnitus and hyperacusis are common short-term side effects of certain drugs and measured the brain activity in rats before and after they were given one such drug. Before receiving the drug, the rats had first been trained to expect to receive a food pellet from the left side of their cage when they heard a steady buzzing sound. The rats were also trained to expect a food pellet from their right if they heard nothing at all. Shortly after receiving the drug, the rats often failed to respond correctly in the ‘quiet tests’ and behaved like they were already experiencing a constant buzzing sound, as would be expected if they had tinnitus. Further tests confirmed that the drug also triggered behavior in the rats that is typical of people with hyperacusis.
Chen et al. then discovered that the drug treatment reduced the nerve signals that are sent from a rat's cochlea. Moreover, the drug treatment greatly increased the activity in response to sound within parts of the rat's brain; these and other parts of the brain also became overactive in drug-treated rats in the absence of sound. Finally, further experiments revealed that drug-treated rats had stronger connections between these brain regions than in normal rats.
Chen et al. used these results to propose a model to explain the underlying causes of tinnitus and hyperacusis. However, because the drug treatment only induces short-term hearing impairment, further studies are needed to see if this model also applies when these conditions are long-term.
DOI: http://dx.doi.org/10.7554/eLife.06576.002
doi:10.7554/eLife.06576
PMCID: PMC4426664  PMID: 25962854
salicylate; tinnitus; hyperacusis; functional MRI; ALFF; functional connectivity; rat
23.  Casting inorganic structures with DNA molds 
Science (New York, N.Y.)  2014;346(6210):1258361.
We report a general strategy for designing and synthesizing inorganic nanostructures with arbitrarily prescribed three-dimensional shapes. Computationally designed DNA strands self-assemble into a stiff “nano-mold” that contains a user-specified three-dimensional cavity and encloses a nucleating gold “seed”. Under mild conditions, this seed grows into a larger cast structure that fills and thus replicates the cavity. We synthesized a variety of nanoparticles with three nanometer resolution: three distinct silver cuboids with three independently tunable dimensions, silver and gold nanoparticles with diverse cross sections, and composite structures with homo-/heterogeneous components. The designer equilateral silver triangular and spherical nanoparticles exhibited plasmonic properties consistent with electromagnetism-based simulations. Our framework is generalizable to more complex geometries and diverse inorganic materials, offering a range of applications in biosensing, photonics, and nanoelectronics.
doi:10.1126/science.1258361
PMCID: PMC4260265  PMID: 25301973
24.  Calcium- and integrin-binding protein-1 is down-regulated in the sperm of patients with oligoasthenozoospermia 
Purpose
The aim of this study was to determine whether altered expression and distribution of calcium- and integrin-binding protein-1 (CIB1) is involved in the pathogenesis of patients with oligoasthenozoospermia.
Methods
Sperm samples were obtained from 25 infertile Chinese men who had failed to achieve conception after a period of 1–2 y and had been referred to the Reproductive Laboratory of the second hospital affiliated to the Shandong University of Traditional Chinese Medicine. Participants were divided into two groups: oligoasthenozoospermia (n = 13) and asthenozoospermia (n = 12); as a third group, fertile men (n = 19) were included as controls. The expression levels of mRNA and protein levels of CIB1 and cyclin-dependent kinase 1 (CDK1) were measured using qRT-PCR and western blotting.
Results
mRNA and protein expression levels of CIB1 were decreased in the oligoasthenozoospermia patients. Interestingly mRNA and protein expression levels of CDK1 were increased in the oligoasthenozoospermia patients.
Conclusion
The results of the present study indicate that that CIB1 may be involved in the pathogenesis of oligoasthenozoospermia by the CDK1 signaling pathway.
doi:10.1007/s10815-014-0177-4
PMCID: PMC4016369  PMID: 24464679
CDK1; CIB1; Male infertility; Oligoasthenozoospermia; Sperm

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