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1.  Deep sequencing reveals microbiota dysbiosis of tongue coat in patients with liver carcinoma 
Scientific Reports  2016;6:33142.
Liver carcinoma (LC) is a common malignancy worldwide, associated with high morbidity and mortality. Characterizing microbiome profiles of tongue coat may provide useful insights and potential diagnostic marker for LC patients. Herein, we are the first time to investigate tongue coat microbiome of LC patients with cirrhosis based on 16S ribosomal RNA (rRNA) gene sequencing. After strict inclusion and exclusion criteria, 35 early LC patients with cirrhosis and 25 matched healthy subjects were enrolled. Microbiome diversity of tongue coat in LC patients was significantly increased shown by Shannon, Simpson and Chao 1 indexes. Microbiome on tongue coat was significantly distinguished LC patients from healthy subjects by principal component analysis. Tongue coat microbial profiles represented 38 operational taxonomic units assigned to 23 different genera, distinguishing LC patients. Linear discriminant analysis (LDA) effect size (LEfSe) reveals significant microbial dysbiosis of tongue coats in LC patients. Strikingly, Oribacterium and Fusobacterium could distinguish LC patients from healthy subjects. LEfSe outputs show microbial gene functions related to categories of nickel/iron_transport, amino_acid_transport, energy produced system and metabolism between LC patients and healthy subjects. These findings firstly identify microbiota dysbiosis of tongue coat in LC patients, may providing novel and non-invasive potential diagnostic biomarker of LC.
PMCID: PMC5015078  PMID: 27605161
2.  Origin of Structural Transformation in Mono- and Bi-Layered Molybdenum Disulfide 
Scientific Reports  2016;6:26666.
Mono- and multi-layered molybdenum disulfide (MoS2) is considered to be one of the next generation anode materials for rechargeable ion batteries. Structural transformation from trigonal prismatic (2H) to octahedral (1T) upon lithium or sodium intercalation has been in-situ observed experimentally using transmission electron microscope during studies of their electrochemical dynamics processes. In this work, we explored the fundamental mechanisms of this structural transformation in both mono- and bi-layered MoS2 using density functional theory. For the intercalated MoS2, the Li and Na donate their electrons to the MoS2. Based on the theoretical analysis, we confirmed that, for the first time, electron transfer is dominant in initiating this structural transformation, and the results provide an in-depth understanding of the transformation mechanism induced by the electron doping. The critical values of electron concentrations for this structural transformation are decreased with increasing the layer thickness.
PMCID: PMC4880921  PMID: 27225416
3.  Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays 
Molecular Medicine Reports  2016;14(1):175-183.
Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53-mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53-independent NS pathway. NS expression was silenced using lentivirus-mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse-transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c-Jun N-terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54-independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.
PMCID: PMC4918620  PMID: 27374947
nucleostemin; microarray; gene expression profiling; NB4
4.  A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis 
PLoS ONE  2015;10(12):e0146163.
It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis.
PMCID: PMC4696740  PMID: 26717241
5.  Functional Redundancy Instead of Species Redundancy Determines Community Stability in a Typical Steppe of Inner Mongolia 
PLoS ONE  2015;10(12):e0145605.
The redundancy hypothesis predicts that the species redundancy in a plant community enhances community stability. However, numerous studies in recent years questioned the positive correlation between redundancy and stability.
We explored the relationship between the species redundancy, functional redundancy and community stability in typical steppe grassland in Northern China by sampling grassland vegetation along a gradient of resource availability caused by micro-topography. We aimed to test whether community redundancy enhanced community stability, and to quantify the relative importance of species redundancy and functional redundancy in maintaining community stability.
Our results showed that the spatial stability of plant community production increases with increased supply of soil resources, and the functional redundancy instead of species diversity or species redundancy is correlated with the community stability. Our results supported the redundancy hypothesis and have implications for sustainable grassland management.
PMCID: PMC4689422  PMID: 26699133
6.  Defect-Mediated Lithium Adsorption and Diffusion on Monolayer Molybdenum Disulfide 
Scientific Reports  2015;5:18712.
Monolayer Molybdenum Disulfide (MoS2) is a promising anode material for lithium ion batteries because of its high capacities. In this work, first principle calculations based on spin density functional theory were performed to investigate adsorption and diffusion of lithium on monolayer MoS2 with defects, such as single- and few-atom vacancies, antisite, and grain boundary. The values of adsorption energies on the monolayer MoS2 with the defects were increased compared to those on the pristine MoS2. The presence of defects causes that the Li is strongly bound to the monolayer MoS2 with adsorption energies in the range between 2.81 and 3.80 eV. The donation of Li 2s electron to the defects causes an enhancement of adsorption of Li on the monolayer MoS2. At the same time, the presence of defects does not apparently affect the diffusion of Li, and the energy barriers are in the range of 0.25–0.42 eV. The presence of the defects can enhance the energy storage capacity, suggesting that the monolayer MoS2 with defects is a suitable anode material for the Li-ion batteries.
PMCID: PMC4686938  PMID: 26692345
7.  The association between fine particulate matter exposure during pregnancy and preterm birth: a meta-analysis 
Although several previous studies have assessed the association of fine particulate matter (PM2.5) exposure during pregnancy with preterm birth, the results have been inconsistent and remain controversial. This meta-analysis aims to quantitatively summarize the association between maternal PM2.5 exposure and preterm birth and to further explore the sources of heterogeneity in findings on this association.
We searched for all studies published before December 2014 on the association between PM2.5 exposure during pregnancy and preterm birth in the MEDLINE, PUBMED and Embase databases as well as the China Biological Medicine and Wanfang databases. A pooled OR for preterm birth in association with each 10 μg/m3 increase in PM2.5 exposure was calculated by a random-effects model (for studies with significant heterogeneity) or a fixed-effects model (for studies without significant heterogeneity).
A total of 18 studies were included in this analysis. The pooled OR for PM2.5 exposure (per 10 μg/m3 increment) during the entire pregnancy on preterm birth was 1.13 (95 % CI = 1.03–1.24) in 13 studies with a significant heterogeneity (Q = 80.51, p < 0.001). The pooled ORs of PM2.5 exposure in the first, second and third trimester were 1.08 (95 % CI = 0.92–1.26), 1.09 (95 % CI = 0.82–1.44) and 1.08 (95 % CI = 0.99–1.17), respectively. The corresponding meta-estimates of PM2.5 effects in studies assessing PM2.5 exposure at individual, semi-individual and regional level were 1.11 (95 % CI = 0.89–1.37), 1.14 (95 % CI = 0.97–1.35) and 1.07 (95 % CI = 0.94–1.23). In addition, significant meta-estimates of PM2.5 exposures were found in retrospective studies (OR = 1.10, 95 % CI = 1.01–1.21), prospective studies (OR = 1.42, 95 % CI = 1.08–1.85), and studies conducted in the USA (OR = 1.16, 95 % CI = 1.05–1.29).
Maternal PM2.5 exposure during pregnancy may increase the risk of preterm birth,but significant heterogeneity was found between studies. Exposure assessment methods, study designs and study settings might be important sources of heterogeneity, and should be taken into account in future meta-analyses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12884-015-0738-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4650291  PMID: 26581753
Fine particulate matter; Preterm birth; Meta-analysis; Adverse pregnancy outcome
8.  GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, Plays Differential Roles in Plant Responses to Bicarbonate, Salt and Osmotic Stresses 
PLoS ONE  2015;10(11):e0141888.
Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca2+ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na+, K+) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca2+-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.
PMCID: PMC4638360  PMID: 26550992
9.  Modified glasgow prognostic score as a prognostic factor in gastriccancer patients: a systematic review and meta-analysis 
Objective: Modified Glasgow prognostic score (mGPS) had been reported to associate with the prognosis ofgastric cancer (GC), butits significance in gastric cancer patients has not been studied fully. Methods: PubMed; EMBASE; Web of Science and CNKI data base were searched to identify studies using the mGPS in gastric cancer patients. Outcome measures that were evaluated included overall survival (OS), lymphatic invasion and venous invasion inpatients with gastric cancer. Results: A total of seven studies comprising 3206 patients were included in the meta-analysisof which all used OS as an outcome measure, three studies reported lymphatic invasionand three evaluated venous invasion. The results show that OS was worse in patients with an mGPS=1 and 2 (odds ratio [OR]=2.54, 95% [CI]: 1.62-3.98 and OR=12.02, 95% [CI]: 6.79-21.28, respectively) compared with those with a score of 0 (both P<0.01). Furthermore, gastric cancer patients with mGPS≥1 have higher rates of lymphatic and venous invasion with ORs of 2.51 (95% CI: 1.80-3.51) and 2.63 (95% CI: 1.35-5.11) respectively (both P<0.01). Conclusions: Them GPS could be used as a prognosis predictorfor gastric cancer patients and associated lymphatic and venous invasion.
PMCID: PMC4658896  PMID: 26629007
mGPS; gastric cancer; prognostic factor; meta-analysis
10.  Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress 
PLoS ONE  2015;10(6):e0129998.
Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants.
PMCID: PMC4474918  PMID: 26091094
11.  A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity 
eLife  null;4:e05449.
Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.
eLife digest
Huntington's disease is an inherited disorder of the central nervous system. Symptoms typically begin between the ages of 30 and 50, and initially include clumsiness and uncontrollable movements, as well as personality changes and mood swings. Symptoms worsen over time and life expectancy is usually around 10 to 25 years following diagnosis.
The disease is caused by a mutation in the ‘huntingtin’ gene, which leads to the production of an abnormal form of ‘huntingtin’ protein. This accumulates inside neurons in a region of the brain called the striatum, which is involved in the control of movement, and destroys them. However, it is not clear why other regions of the brain that also produce the mutant huntingtin protein are not affected.
Yao, Cui, Al-Ramahi, Sun et al. have now identified a protein that could explain this phenomenon and open up new therapeutic possibilities for Huntington's disease. The protein, which is called Gpr52, is a receptor located within the outer membrane of neurons, particularly those in the striatum. Reducing the levels of this protein reduced the amount of mutant huntingtin protein that was able to accumulate inside cells grown in culture. Moreover, mice that were genetically engineered to possess a mutant huntingtin gene, but only a single copy of the gene for Gpr52, accumulated less mutant huntingtin in the striatum than mice with two copies of the Gpr52 gene.
Further experiments revealed that Gpr52 protects mutant huntingtin from being broken down inside cells: it does this by activating a signaling pathway involving the cellular messenger cAMP. Encouragingly, when genetic techniques were used to reduce Gpr52 synthesis in a fruit fly model of Huntington's disease, the treated flies showed fewer movement impairments than flies that had not been treated. In addition, reduced levels of Gpr52 were observed to lead to dramatic protective effects in neurons derived from the stem cells of a patient with Huntington's disease.
The fact that Gpr52 is located on the surface of neurons means that it might be possible to design drugs that can block its activity and thus reduce accumulation of mutant huntingtin. Such a treatment would be the first to target the causal mechanism behind Huntington's disease, rather than simply addressing the symptoms. The strategy could also be relevant to Alzheimer's disease, Parkinson's disease and other neurodegenerative disorders in which death of neurons is triggered by abnormal accumulation or aggregation of proteins.
PMCID: PMC4372774  PMID: 25738228
polyQ; neurodegeneration; GPCR; huntington's disease; cyclic AMP; intron; D. melanogaster; human; mouse
12.  Partner of Sld five 3: a potential prognostic biomarker for colorectal cancer 
Diagnostic Pathology  2014;9:217.
Partner of Sld five 3 (PSF3) is a member of the evolutionarily conserved heterotetrameric complex “Go-Ichi-Ni-San” (GINS), which consists of SLD5, PSF1, PSF2, and PSF3. Previous studies have suggested that some GINS complex members are upregulated in cancer, but the status of PSF3 expression in colorectal cancer has not been investigated.
We investigated the status of PSF3 expression in 137 consecutive resected colorectal caners by quantitative reverse-transcription polymerase chain reaction. Univariable and multivariable Cox regression analyses were performed to assess independent prognostic factors for overall survival in colorectal cancer.
In 137 restected colorectal cancer samples, median messenger RNA (mRNA) expression levels of PSF3 were significantly higher in tumor tissues (1.35 × 10−3, range 2.88 × 10−4 to 3.16 × 10−2) than in adjacent normal tissues (2.94 × 10−4, range 5.48 × 10−5 to 1.27 × 10−3) (P < 0.05). Moreover, high expression of PSF3 in tumor tissues was associated with shorter disease-free survival and overall survival. When analyzed with a Cox regression model, the PSF3 expression was an independent prognostic factor for overall survival. In addition, in patients with early stage (stage I and II) colorectal cancer, the overall survival rate of the high PSF3 expression group was significantly lower than that of the low PSF3 expression group (P < 0.001).
The PSF3 expression plays an important role in the progression of colorectal cancer and acts as a factor significantly affecting the prognosis of patients.
Virtual Slides
The virtual slide(s) for this article can be found here:
PMCID: PMC4244056  PMID: 25403684
Partner of Sld five 3; Colorectal cancer; Overall survival
13.  Association between miR-27a genetic variants and susceptibility to colorectal cancer 
Diagnostic Pathology  2014;9:146.
MicroRNAs (miRNAs) are short, non-coding RNAs that negatively regulate target genes. A single nucleotide polymorphism (SNP) in a miRNA sequence may alter miRNA expression and/or maturation, which was proposed to associate with the development and progression of cancer. The rs895819 polymorphism, located in the terminal loop of pre-miR-27a, has been reported to have relevance to several cancers. In this study, we investigated the possibility of association between polymorphism in rs895819 and susceptibility to colorectal cancer (CRC).
We identified a single SNP, rs895819 in pre-miR-27a, for further investigation, were determined in 205 CRC patients and 455 healthy controls.
When taking the AA genotype as a reference, we found that AG genotype was not statistically significantly associated with the risk of CRC (AG vs. AA, OR 1.245, 95% CI: 0.806 – 1.923). However, the GG genotype was significantly associated with risk of CRC (GG vs. AA, OR 1.599, 95% CI: 1.052 – 2.430). In the AG + GG vs GG group, no significant difference was detected (OR 1.424, 95% CI, 0.974 – 1.801). GG genotype and G allele was associated with an increased risk of metastasis in this study (P < 0.001 and P = 0.003, respectively).
This study found significant association between rs895819 polymorphism in pre-miR-27a and CRC risk. Population-based studies with large number of subjects and long-term follow-up are needed to verify the association of miR-27a polymorphism with CRC susceptibility and severity.
Virtual Slides
The virtual slide(s) for this article can be found here:
PMCID: PMC4261532  PMID: 25078482
Single nucleotide polymorphism; Colorectal cancer; miR-27a; Risk factor
14.  Ectopic Expression of GsPPCK3 and SCMRP in Medicago sativa Enhances Plant Alkaline Stress Tolerance and Methionine Content 
PLoS ONE  2014;9(2):e89578.
So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H+-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops.
PMCID: PMC3934933  PMID: 24586886
15.  Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis 
PLoS ONE  2013;8(8):e73295.
Flowering is a critical event in the life cycle of plants; the WRKY-type transcription factors are reported to be involved in many developmental processes sunch as trichome development and epicuticular wax loading, but whether they are involved in flowering time regulation is still unknown. Within this study, we provide clear evidence that GsWRKY20, a member of WRKY gene family from wild soybean, is involved in controlling plant flowering time. Expression of GsWRKY20 was abundant in the shoot tips and inflorescence meristems of wild soybean. Phenotypic analysis showed that GsWRKY20 over-expression lines flowered earlier than the wild-type plants under all conditions: long-day and short-day photoperiods, vernalization, or exogenous GA3 application, indicating that GsWRKY20 may mainly be involved in an autonomous flowering pathway. Further analyses by qRT-PCR and microarray suggests that GsWRKY20 accelerating plant flowering might primarily be through the regulation of flowering-related genes (i.e., FLC, FT, SOC1 and CO) and floral meristem identity genes (i.e., AP1, SEP3, AP3, PI and AG). Our results provide the evidence demonstrating the effectiveness of manipulating GsWRKY20 for altering plant flowering time.
PMCID: PMC3753250  PMID: 23991184
16.  S100P Expression in response to sex steroids during the implantation window in human endometrium 
S100P, a protein originally detected in the human placenta, has been found to play an important role in the development and invasion of tumors. Interestingly, we have recently discovered using data mining that S100P was considerably up-regulated during the window of implantation in the human endometrium, but little further information has been available.
Real-time PCR and immunofluorescence were performed to examine the expression and location of S100P in the human endometrium and endometrial cells. Estrogen and progesterone were added to the cultured cells to test the response of S100P to sex steroids.
A dramatic peak, approximately a 100-fold increase in comparison with the proliferative and early- and late-secretory phases, was observed in the endometrium during the mid-secretory phase, which corresponds to the time of embryo implantation. Progesterone regulated the expression of S100P in both primary endometrial epithelial and stromal cells, but estrogen had no significant effect.
The results indicate that S100P participates in the periodic change of the endometrium under the regulation of progesterone, may be used as a unique biomarker of the receptive endometrium and play an important role in embryo implantation.
PMCID: PMC3551790  PMID: 23216986
Calcium-binding protein S100P; Endometrial receptivity; Hormonal regulation
17.  Liuwei Dihuang (LWDH), a Traditional Chinese Medicinal Formula, Protects against β-Amyloid Toxicity in Transgenic Caenorhabditis elegans 
PLoS ONE  2012;7(8):e43990.
Liuwei Dihuang (LWDH), a classic Chinese medicinal formula, has been used to improve or restore declined functions related to aging and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory. Here, we report on the effect and possible mechanisms of LWDH mediated protection of β-amyloid (Aβ) induced paralysis in Caenorhabditis elegans using ethanol extract (LWDH-EE) and water extract (LWDH-WE). Chemical profiling and quantitative analysis revealed the presence of different levels of bioactive components in these extracts. LWDH-WE was rich in polar components such as monosaccharide dimers and trimers, whereas LWDH-EE was enriched in terms of phenolic compounds such as gallic acid and paeonol. In vitro studies revealed higher DPPH radical scavenging activity for LWDH-EE as compared to that found for LWDH-WE. Neither LWDH-EE nor LWDH-WE were effective in inhibiting aggregation of Aβ in vitro. By contrast, LWDH-EE effectively delayed Aβ induced paralysis in the transgenic C. elegans (CL4176) model which expresses human Aβ1–42. Western blot revealed no treatment induced reduction in Aβ accumulation in CL4176 although a significant reduction was observed at an early stage with respect to β-amyloid deposition in C. elegans strain CL2006 which constitutively expresses human Aβ1–42. In addition, LWDH-EE reduced in vivo reactive oxygen species (ROS) in C. elegans (CL4176) that correlated with increased survival of LWDH-EE treated N2 worms under juglone-induced oxidative stress. Analysis with GFP reporter strain TJ375 revealed increased expression of hsp16.2::GFP after thermal stress whereas a minute induction was observed for sod3::GFP. Quantitative gene expression analysis revealed that LWDH-EE repressed the expression of amy1 in CL4176 while up-regulating hsp16.2 induced by elevating temperature. Taken together, these results suggest that LWDH extracts, particularly LWDH-EE, alleviated β-amyloid induced toxicity, in part, through up-regulation of heat shock protein, antioxidant activity and reduced ROS in C. elegans.
PMCID: PMC3431378  PMID: 22952840
18.  A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery 
The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol.
Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency.
CMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS- CLDPD from serum interaction but also enhance gene transfection efficiency.
These results demonstrated that multifunctional CMCS-CLDPD had pH- sensitivity, which may provide a new approach for the antitumor gene delivery.
PMCID: PMC3289447  PMID: 22393290
layer-by-layer; multifunctional nanovector; pH-sensitivity; gene delivery
19.  Two-color STED microscopy in living cells 
Biomedical Optics Express  2011;2(8):2364-2371.
Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAPf and CLIPf technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.
PMCID: PMC3149534  PMID: 21833373
(170.3880) Medical and biological imaging; (180.2520) Fluorescence microscopy; (350.5730) Resolution
20.  Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth 
Molecular Biology of the Cell  2011;22(5):624-633.
The small GTPase Cdc42p is a master regulator of cell polarity. We analyzed Cdc42p localization using yeast mutants and found that endo-exocytic trafficking and septin-based diffusion barrier synergistically control Cdc42p polarization during asymmetric cell growth.
Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.
PMCID: PMC3046059  PMID: 21209323
21.  Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection 
Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.
PMCID: PMC3083711  PMID: 21541064
PLA-PEG-PLL; nanoparticles; non-viral gene vector; gene transfection; tri-block copolymer
22.  Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth 
Molecular Biology of the Cell  2005;16(3):1500-1512.
Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.
PMCID: PMC551511  PMID: 15647373
23.  Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging 
Chembiochem  2011;12(14):2217-2226.
The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
PMCID: PMC3213346  PMID: 21793150
cell imaging; covalent labeling; fluorescent probes; fluorogenic substrates; protein modifications

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