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1.  The Human Gastrointestinal Tract, a Potential Autologous Neural Stem Cell Source 
PLoS ONE  2013;8(9):e72948.
Stem cell therapies seem to be an appropriate tool for the treatment of a variety of diseases, especially when a substantial cell loss leads to a severe clinical impact. This is the case in most neuronal cell losses. Unfortunately, adequate neural stem cell sources are hard to find and current alternatives, such as induced programmed stem cells, still have incalculable risks. Evidence of neurogenesis in the adult human enteric nervous system brought up a new perspective. In humans the appendix harbors enteric neuronal tissue and is an ideal location where the presence of neural stem cells is combined with a minimal invasive accessibility. In this study appendices from adults and children were investigated concerning their neural stem cell potential. From each appendix tissue samples were collected, and processed for immunohistochemistry or enteric neural progenitor cell generation. Free-floating enteric neurospheres (EnNS’s) could be generated after 6 days in vitro. EnNS’s were either used for transplantation into rat brain slices or differentiation experiments. Both transplanted spheres and control cultures developed an intricate network with glia, neurons and interconnecting fibers, as seen in primary enteric cultures before. Neuronal, glial and neural stem cell markers could be identified both in vitro and in vivo by immunostaining. The study underlines the potential of the enteric nervous system as an autologous neural stem cell source. Using the appendix as a potential target opens up a new perspective that might lead to a relatively unproblematic harvest of neural stem cells.
doi:10.1371/journal.pone.0072948
PMCID: PMC3762931  PMID: 24023797
2.  Comparison of Journal Self-Citation Rates between Some Chinese and Non-Chinese International Journals 
PLoS ONE  2012;7(11):e49001.
Background
The past 3 decades have witnessed a boost in science development in China; in parallel, more and more Chinese scientific journals are indexed by the Journal Citation Reports issued by Thomson Reuters (SCI). Evaluation of the performance of these Chinese SCI journals is necessary and helpful to improve their quality. This study aimed to evaluate these journals by calculating various journal self-citation rates, which are important parameters influencing a journal impact factor.
Methodology/Principal Findings
We defined three journal self-citation rates, and studied these rates for 99 Chinese scientific journals, almost exhausting all Chinese SCI journals currently available. Likewise, we selected 99 non-Chinese international (abbreviated as ‘world’) journals, with each being in the same JCR subject category and having similar impact factors as their Chinese counterparts. Generally, Chinese journals tended to be higher in all the three self-citation rates than world journal counterparts. Particularly, a few Chinese scientific journals had much higher self-citation rates.
Conclusions/Significance
Our results show that generally Chinese scientific journals have higher self-citation rates than those of world journals. Consequently, Chinese scientific journals tend to have lower visibility and are more isolated in the relevant fields. Considering the fact that sciences are rapidly developing in China and so are Chinese scientific journals, we expect that the differences of journal self-citation rates between Chinese and world scientific journals will gradually disappear in the future. Some suggestions to solve the problems are presented.
doi:10.1371/journal.pone.0049001
PMCID: PMC3500263  PMID: 23173041
3.  Three-Dimensional Microscopy Characterization of Death Receptor 5 Expression by Over-Activated Human Primary CD4+ T Cells and Apoptosis 
PLoS ONE  2012;7(3):e32874.
Activation-induced cell death is a natural process that prevents tissue damages from over-activated immune cells. TNF-Related apoptosis ligand (TRAIL), a TNF family member, induces apoptosis of infected and tumor cells by binding to one of its two death receptors, DR4 or DR5. TRAIL was reported to be secreted by phytohemagglutinin (PHA)-stimulated CD4+ T cells in microvesicles.
We investigate here TRAIL and DR5 regulation by activated primary CD4+ T cells and its consequence on cell death. We observed that PHA induced CD4+ T cell apoptosis in a dose-dependent manner. Thus, we investigated molecules involved in PHA-mediated cell death and demonstrated that TRAIL and DR5 were over-expressed on the plasma membrane of PHA-stimulated CD4+ T cells. Surprisingly, DR5 was constitutively expressed in naive CD4+ T cells at messenger RNA (mRNA) and protein levels. Thus, using 3 dimensional microscopy and intracellular staining assays, we show that DR5 is constitutively expressed in CD4+ T cells and is pre-stocked in the cytoplasm. When cells are stimulated by PHA, DR5 is relocalized from cytoplasm to plasma membrane. Small interference RNA (siRNA) and blocking antibody assays demonstrate that TRAIL/DR5 interaction is mainly responsible for PHA-mediated CD4+ T cell apoptosis. Thus, membrane DR5 expression leading to TRAIL-mediated apoptosis may represent one of the pathways responsible for eradication of over-activated CD4+ T cells during immune responses.
doi:10.1371/journal.pone.0032874
PMCID: PMC3295789  PMID: 22412938
4.  De Novo Analysis of Transcriptome Dynamics in the Migratory Locust during the Development of Phase Traits 
PLoS ONE  2010;5(12):e15633.
Locusts exhibit remarkable density-dependent phenotype (phase) changes from the solitary to the gregarious, making them one of the most destructive agricultural pests. This phenotype polyphenism arises from a single genome and diverse transcriptomes in different conditions. Here we report a de novo transcriptome for the migratory locust and a comprehensive, representative core gene set. We carried out assembly of 21.5 Gb Illumina reads, generated 72,977 transcripts with N50 2,275 bp and identified 11,490 locust protein-coding genes. Comparative genomics analysis with eight other sequenced insects was carried out to indentify the genomic divergence between hemimetabolous and holometabolous insects for the first time and 18 genes relevant to development was found. We further utilized the quantitative feature of RNA-seq to measure and compare gene expression among libraries. We first discovered how divergence in gene expression between two phases progresses as locusts develop and identified 242 transcripts as candidates for phase marker genes. Together with the detailed analysis of deep sequencing data of the 4th instar, we discovered a phase-dependent divergence of biological investment in the molecular level. Solitary locusts have higher activity in biosynthetic pathways while gregarious locusts show higher activity in environmental interaction, in which genes and pathways associated with regulation of neurotransmitter activities, such as neurotransmitter receptors, synthetase, transporters, and GPCR signaling pathways, are strongly involved. Our study, as the largest de novo transcriptome to date, with optimization of sequencing and assembly strategy, can further facilitate the application of de novo transcriptome. The locust transcriptome enriches genetic resources for hemimetabolous insects and our understanding of the origin of insect metamorphosis. Most importantly, we identified genes and pathways that might be involved in locust development and phase change, and may thus benefit pest management.
doi:10.1371/journal.pone.0015633
PMCID: PMC3012706  PMID: 21209894
5.  Synapse Clusters Are Preferentially Formed by Synapses with Large Recycling Pool Sizes 
PLoS ONE  2010;5(10):e13514.
Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1–43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1–43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.
doi:10.1371/journal.pone.0013514
PMCID: PMC2958124  PMID: 20976002
6.  Abnormal Behaviors and Developmental Disorder of Hippocampus in Zinc Finger Protein 521 (ZFP521) Mutant Mice 
PLoS ONE  2014;9(3):e92848.
Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.
doi:10.1371/journal.pone.0092848
PMCID: PMC3968043  PMID: 24676388
7.  Excess Burden of Depression among HIV-Infected Persons Receiving Medical Care in the United States: Data from the Medical Monitoring Project and the Behavioral Risk Factor Surveillance System 
PLoS ONE  2014;9(3):e92842.
Background
With increased life expectancy for HIV-infected persons, there is concern regarding comorbid depression because of its common occurrence and association with behaviors that may facilitate HIV transmission. Our objectives were to estimate the prevalence of current depression among HIV-infected persons receiving care and assess the burden of major depression, relative to that in the general population.
Methods and Findings
We used data from the Medical Monitoring Project (MMP) and the Behavioral Risk Factors Surveillance System (BRFSS). The eight-item Patient Health Questionnaire was used to identify depression. To assess the burden of major depression among HIV-infected persons receiving care, we compared the prevalence of current major depression between the MMP and BRFSS populations using stratified analyses that simultaneously controlled for gender and, in turn, each of the potentially confounding demographic factors of age, race/ethnicity, education, and income. Each unadjusted comparison was summarized as a prevalence ratio (PR), and each of the adjusted comparisons was summarized as a standardized prevalence ratio (SPR). Among HIV-infected persons receiving care, the prevalence of a current episode of major depression and other depression, respectively, was 12.4% (95% CI: 11.2, 13.7) and 13.2% (95% CI: 12.0%, 14.4%). Overall, the PR comparing the prevalence of current major depression between HIV-infected persons receiving care and the general population was 3.1. When controlling for gender and each of the factors age, race/ethnicity, and education, the SPR (3.3, 3.0, and 2.9, respectively) was similar to the PR. However, when controlling for gender and annual household income, the SPR decreased to 1.5.
Conclusions
Depression remains a common comorbidity among HIV-infected persons. The overall excess burden among HIV-infected persons receiving care is about three-times that among the general population and is associated with differences in annual household income between the two populations. Relevant efforts are needed to reduce this burden.
doi:10.1371/journal.pone.0092842
PMCID: PMC3963963  PMID: 24663122
8.  Therapeutic Benefit of Bone Marrow–Derived Endothelial Progenitor Cell Transplantation after Experimental Aneurysm Embolization with Coil in Rats 
PLoS ONE  2014;9(2):e90069.
Aneurysm embolization with coil is now widely used clinically. However, the recurrence of aneurysms after embolization has always plagued neurosurgeons because the endothelial layer of the aneurysm neck loses its integrity after being embolized by coil. Bone marrow–derived endothelial progenitor cells (BM-EPCs) could be incorporated into injured endothelium and differentiate into mature endothelial cells during vascular repairing processes. The aim of our study is to explore the effects of BM-EPCs on aneurysm repairing and remodeling in a rat embolization model of abdominal aortic aneurysm. BM-EPC proliferation, migration and tube formation were not affected by super-paramagnetic iron oxide nanoparticle (SPIO) labeling compared to the controls (p>0.05). The number of SPIO-labeled cells greatly increased in EPC transplanted rats compared to that of phosphate buffered saline treated rats. SPIO-labeled EPC (SPIO-EPC) are mainly located in the aneurysm neck and surrounded by fibrous tissue. A histology study showed that the aneurysm orifice was closed with neointima and the aneurysm was filled with newly formed fibrous tissue. The SPIO-EPC accumulated in the aneurysm neck, which accelerated focal fibrous tissue remodeling, suggesting that BM-EPCs play a crucial role in repairing and remodeling the aneurysm neck orifice.
doi:10.1371/journal.pone.0090069
PMCID: PMC3938595  PMID: 24587209
9.  Monocytes-Derived Macrophages Mediated Stable Expression of Human Brain-Derived Neurotrophic Factor, a Novel Therapeutic Strategy for NeuroAIDS 
PLoS ONE  2014;9(2):e82030.
HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF) may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS). It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (h)BDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM) and murine myeloid monocyte-derived macrophages (mMDM). These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.
doi:10.1371/journal.pone.0082030
PMCID: PMC3914783  PMID: 24505242
10.  Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro 
PLoS ONE  2014;9(1):e87228.
We recently reported that neural stem cells (NSCs) become senescent and commit to astrocytic differentiation upon X-ray irradiation. Surprisingly, under self-renewing culture conditions, some of these senescent cells undergo p53-independent apoptosis, which can be suppressed by caspase inhibition and BCL2 overexpression. Inhibition of apoptosis proved beneficial for astroglial differentiation efficiency; hence the toxicity of DNA damage on NSCs was specifically tested in context of the culture conditions. In this regard, self-renewal-promoting culture conditions proved incompatible with terminal astrocyte differentiation and impacted negatively on the viability of NSCs following DNA damage-induced cell cycle exit. On the contrary, a switch to differentiation-supporting conditions ablated apoptosis and conveyed tolerance to DNA damage. Thus, stem cell death has likely not originated from DNA break toxicity, while the potentially confounding effect of stem cell niche should always be taken in consideration in stem cell irradiation experiments.
doi:10.1371/journal.pone.0087228
PMCID: PMC3903639  PMID: 24475256
11.  A Self-Report Risk Index to Predict Occurrence of Dementia in Three Independent Cohorts of Older Adults: The ANU-ADRI 
PLoS ONE  2014;9(1):e86141.
Background and Aims
The Australian National University AD Risk Index (ANU-ADRI, http://anuadri.anu.edu.au) is a self-report risk index developed using an evidence-based medicine approach to measure risk of Alzheimer's disease (AD). We aimed to evaluate the extent to which the ANU-ADRI can predict the risk of AD in older adults and to compare the ANU-ADRI to the dementia risk index developed from the Cardiovascular Risk Factors, Aging and Dementia (CAIDE) study for middle-aged cohorts.
Methods
This study included three validation cohorts, i.e., the Rush Memory and Aging Study (MAP) (n = 903, age ≥53 years), the Kungsholmen Project (KP) (n = 905, age ≥75 years), and the Cardiovascular Health Cognition Study (CVHS) (n = 2496, age ≥65 years) that were each followed for dementia. Baseline data were collected on exposure to the 15 risk factors included in the ANU-ADRI of which MAP had 10, KP had 8 and CVHS had 9. Risk scores and C-statistics were computed for individual participants for the ANU-ADRI and the CAIDE index.
Results
For the ANU-ADRI using available data, the MAP study c-statistic was 0·637 (95% CI 0·596–0·678), for the KP study it was 0·740 (0·712–0·768) and for the CVHS it was 0·733 (0·691–0·776) for predicting AD. When a common set of risk and protective factors were used c-statistics were 0.689 (95% CI 0.650–0.727), 0.666 (0.628–0.704) and 0.734 (0.707–0.761) for MAP, KP and CVHS respectively. Results for CAIDE ranged from c-statistics of 0.488 (0.427–0.554) to 0.595 (0.565–0.625).
Conclusion
A composite risk score derived from the ANU-ADRI weights including 8–10 risk or protective factors is a valid, self-report tool to identify those at risk of AD and dementia. The accuracy can be further improved in studies including more risk factors and younger cohorts with long-term follow-up.
doi:10.1371/journal.pone.0086141
PMCID: PMC3900468  PMID: 24465922
12.  Development of a Screening Algorithm for Alzheimer's Disease Using Categorical Verbal Fluency 
PLoS ONE  2014;9(1):e84111.
We developed a weighted composite score of the categorical verbal fluency test (CVFT) that can more easily and widely screen Alzheimer's disease (AD) than the mini-mental status examination (MMSE). We administered the CVFT using animal category and MMSE to 423 community-dwelling mild probable AD patients and their age- and gender-matched cognitively normal controls. To enhance the diagnostic accuracy for AD of the CVFT, we obtained a weighted composite score from subindex scores of the CVFT using a logistic regression model: logit (case)  = 1.160+0.474× gender +0.003× age +0.226× education level – 0.089× first-half score – 0.516× switching score -0.303× clustering score +0.534× perseveration score. The area under the receiver operating curve (AUC) for AD of this composite score AD was 0.903 (95% CI = 0.883 – 0.923), and was larger than that of the age-, gender- and education-adjusted total score of the CVFT (p<0.001). In 100 bootstrapped re-samples, the composite score consistently showed better diagnostic accuracy, sensitivity and specificity for AD than the total score. Although AUC for AD of the CVFT composite score was slightly smaller than that of the MMSE (0.930, p = 0.006), the CVFT composite score may be a good alternative to the MMSE for screening AD since it is much briefer, cheaper, and more easily applicable over phone or internet than the MMSE.
doi:10.1371/journal.pone.0084111
PMCID: PMC3879263  PMID: 24392109
13.  Bone Marrow-Derived Mesenchymal Stem Cells Maintain the Resting Phenotype of Microglia and Inhibit Microglial Activation 
PLoS ONE  2013;8(12):e84116.
Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS), and then culturing these microglia with BMSC-conditioned medium (BMSC-CM). We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO) were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.
doi:10.1371/journal.pone.0084116
PMCID: PMC3877190  PMID: 24391898
14.  Surgery-Related Thrombosis Critically Affects the Brain Infarct Volume in Mice Following Transient Middle Cerebral Artery Occlusion 
PLoS ONE  2013;8(9):e75561.
Transient middle cerebral artery occlusion (tMCAO) model is widely used to mimic human focal ischemic stroke in order to study ischemia/reperfusion brain injury in rodents. In tMCAO model, intraluminal suture technique is widely used to achieve ischemia and reperfusion. However, variation of infarct volume in this model often requires large sample size, which hinders the progress of preclinical research. Our previous study demonstrated that infarct volume was related to the success of reperfusion although the reason remained unclear. The aim of present study is to explore the relationship between focal thrombus formation and model reproducibility with respect to infarct volume. We hypothesize that suture-induced thrombosis causes infarct volume variability due to insufficient reperfusion after suture withdrawal. Seventy-two adult male CD-1 mice underwent 90 minutes of tMCAO with or without intraperitoneal administration of heparin. Dynamic synchrotron radiation microangiography (SRA) and laser speckle contrast imaging (LSCI) were performed before and after tMCAO to observe the cerebral vascular morphology and to measure the cerebral blood flow in vivo. Infarct volume and neurological score were examined to evaluate severity of ischemic brain injury. We found that the rate of successful reperfusion was much higher in heparin-treated mice compared to that in heparin-free mice according to the result of SRA and LSCI at 1 and 3 hours after suture withdrawal (p<0.05). Pathological features and SRA revealed that thrombus formed in the internal carotid artery, middle cerebral artery or anterior cerebral artery, which blocked reperfusion following tMCAO. LSCI showed that cortical collateral circulation could be disturbed by thrombi. Our results demonstrated that suture-induced thrombosis was a critical element, which affects the success of reperfusion. Appropriate heparin management provides a useful approach for improving reproducibility of reperfusion model in mice.
doi:10.1371/journal.pone.0075561
PMCID: PMC3782513  PMID: 24086572
15.  Expansion of Multipotent Stem Cells from the Adult Human Brain 
PLoS ONE  2013;8(8):e71334.
The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells.
doi:10.1371/journal.pone.0071334
PMCID: PMC3743777  PMID: 23967194
16.  Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN 
PLoS ONE  2013;8(8):e71225.
Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.
doi:10.1371/journal.pone.0071225
PMCID: PMC3731303  PMID: 23936495
17.  Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP 
PLoS ONE  2013;8(6):e67701.
It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.
doi:10.1371/journal.pone.0067701
PMCID: PMC3688615  PMID: 23818986
18.  Prevalence of TB/HIV Co-Infection in Countries Except China: A Systematic Review and Meta-Analysis 
PLoS ONE  2013;8(5):e64915.
Background
TB and HIV co-epidemic is a major public health problem in many parts of the world. But the prevalence of TB/HIV co-infection was diversified among countries. Exploring the reasons of the diversity of TB/HIV co-infection is important for public policy, planning and development of collaborative TB/HIV activities. We aimed to summarize the prevalence of TB and HIV co-infection worldwide, using meta-analysis based on systematic review of published articles.
Methods
We searched PubMed, Embase, and Web of Science for studies of the prevalence of TB/HIV co-infection. We also searched bibliographic indices, scanned reference lists, and corresponded with authors. We summarized the estimates using meta-analysis and explored potential sources of heterogeneity in the estimates by metaregression analysis.
Results
We identified 47 eligible studies with a total population of 272,466. Estimates of TB/HIV co-infection prevalence ranged from 2.93% to 72.34%; the random effects pooled prevalence of TB/HIV co-infection was 23.51% (95% CI 20.91–26.11). We noted substantial heterogeneity (Cochran’s χ2 = 10945.31, p<0.0001; I2 = 99.58%, 95% CI 99.55–99.61). Prevalence of TB/HIV co-infection was 31.25%(95%CI 19.30–43.17) in African countries, 17.21%(95%CI 9.97–24.46) in Asian countries, 20.11%(95%CI 13.82–26.39) in European countries, 25.06%(95%CI 19.28–30.84) in Latin America countries and 14.84%(95%CI 10.44–19.24) in the USA. Prevalence of TB/HIV co-infection was higher in studies in which TB diagnosed by chest radiography and HIV diagnosis based on blood analyses than in those which used other diagnostic methods, and in countries with higher prevalence HIV in the general population than in countries with lower general prevalence.
Conclusions
Our analyses suggest that it is necessary to attach importance to HIV/TB co-infection, especially screening of TB/HIV co-infection using methods with high sensitivity, specificity and predictive values in the countries with high HIV/AIDS prevalence in the general population.
doi:10.1371/journal.pone.0064915
PMCID: PMC3669088  PMID: 23741419
19.  Activation of Type 1 Cannabinoid Receptor (CB1R) Promotes Neurogenesis in Murine Subventricular Zone Cell Cultures 
PLoS ONE  2013;8(5):e63529.
The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+]i) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.
doi:10.1371/journal.pone.0063529
PMCID: PMC3660454  PMID: 23704915
20.  TGF-β Superfamily Gene Expression and Induction of the Runx1 Transcription Factor in Adult Neurogenic Regions after Brain Injury 
PLoS ONE  2013;8(3):e59250.
Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-β (TGF-β) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-β, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-β Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.
doi:10.1371/journal.pone.0059250
PMCID: PMC3605457  PMID: 23555640
21.  Fast, Potent Pharmacological Expansion of Endogenous Hes3+/Sox2+ Cells in the Adult Mouse and Rat Hippocampus 
PLoS ONE  2012;7(12):e51630.
The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).
doi:10.1371/journal.pone.0051630
PMCID: PMC3518467  PMID: 23251599
22.  Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells 
PLoS ONE  2012;7(11):e49700.
Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.
doi:10.1371/journal.pone.0049700
PMCID: PMC3507916  PMID: 23209593
23.  The bHLH Repressor Deadpan Regulates the Self-renewal and Specification of Drosophila Larval Neural Stem Cells Independently of Notch 
PLoS ONE  2012;7(10):e46724.
Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using the Drosophila larval NSCs called neuroblasts (NBs) as a model, we demonstrate that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. Our results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. Our data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification.
doi:10.1371/journal.pone.0046724
PMCID: PMC3466283  PMID: 23056424
24.  Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase (SHP) 2 and p38 Regulate the Expression of Chemokine CXCL8 in Human Astrocytes 
PLoS ONE  2012;7(9):e45596.
CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.
doi:10.1371/journal.pone.0045596
PMCID: PMC3448633  PMID: 23029125
25.  Astrocyte Senescence as a Component of Alzheimer’s Disease 
PLoS ONE  2012;7(9):e45069.
Aging is the main risk factor for Alzheimer’s disease (AD); however, the aspects of the aging process that predispose the brain to the development of AD are largely unknown. Astrocytes perform a myriad of functions in the central nervous system to maintain homeostasis and support neuronal function. In vitro, human astrocytes are highly sensitive to oxidative stress and trigger a senescence program when faced with multiple types of stress. In order to determine whether senescent astrocytes appear in vivo, brain tissue from aged individuals and patients with AD was examined for the presence of senescent astrocytes using p16INK4a and matrix metalloproteinase-1 (MMP-1) expression as markers of senescence. Compared with fetal tissue samples (n = 4), a significant increase in p16INK4a-positive astrocytes was observed in subjects aged 35 to 50 years (n = 6; P = 0.02) and 78 to 90 years (n = 11; P<10−6). In addition, the frontal cortex of AD patients (n = 15) harbored a significantly greater burden of p16INK4a-positive astrocytes compared with non-AD adult control subjects of similar ages (n = 25; P = 0.02) and fetal controls (n = 4; P<10−7). Consistent with the senescent nature of the p16INK4a-positive astrocytes, increased metalloproteinase MMP-1 correlated with p16INK4a. In vitro, beta-amyloid 1–42 (Aβ1–42) triggered senescence, driving the expression of p16INK4a and senescence-associated beta-galactosidase. In addition, we found that senescent astrocytes produce a number of inflammatory cytokines including interleukin-6 (IL-6), which seems to be regulated by p38MAPK. We propose that an accumulation of p16INK4a-positive senescent astrocytes may link increased age and increased risk for sporadic AD.
doi:10.1371/journal.pone.0045069
PMCID: PMC3440417  PMID: 22984612

Results 1-25 (54)