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1.  Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival 
Infection and Immunity  2014;82(7):2840-2850.
We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants.
doi:10.1128/IAI.01765-14
PMCID: PMC4097617  PMID: 24752516
2.  Intracellular Poly(I:C) Initiated Gastric Adenocarcinoma Cell Apoptosis and Subsequently Ameliorated NK Cell Functions 
Natural killer (NK) cells are granular lymphocytic cells that exert essential functions in viral infection defense and tumor immune surveillance. However, the functions of NK cells were impaired in cancer patients. Polycytidylic acid [poly(I:C)] has been used as an immune adjuvant to improve innate and adaptive immune responses. In this study, intracellular poly(I:C) could trigger gastric adenocarcinoma cells apoptosis quickly. Meanwhile, the sensitivity of poly(I:C)-treated gastric adenocarcinoma cells to NK cell cytolysis was increased, concomitant with the elevated expression of MICA/B and Fas. Furthermore, the cytolytic activity of NK cells against tumor cells was augmented significantly by the supernatant from poly(I:C)-transfected tumor cells compared with NK cells treated by the supernatant from untreated tumor cells, as well as the proliferation and migration abilities of NK cells. In this process, the activating receptors and cytolysis-associated molecules of NK cells were up-regulated. Further investigation showed that type I interferon (IFN) produced by poly(I:C)-transfected gastric adenocarcinoma cells played an important role in this process. Our findings demonstrated that intracellular poly(I:C) not only triggered gastric adenocarcinoma cell apoptosis, but also enhanced NK responses via inducing type I IFN production by gastric adenocarcinoma cells. These functions make poly(I:C) a promising therapeutic medicine for gastric adenocarcinoma.
doi:10.1089/jir.2012.0118
PMCID: PMC3887439  PMID: 24032591
3.  Engineering geminivirus resistance in Jatropha curcus 
Biotechnology for Biofuels  2014;7(1):149.
Background
Jatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge.
Results
We generated transgenic J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus.
Conclusions
In this study we developed transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-014-0149-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-014-0149-z
PMCID: PMC4210599  PMID: 25352912
Biodiesel; Indian cassava mosaic virus; Jatropha curcus; Virus resistance; Transgenic
4.  NOTCH1 signaling contributes to cell growth, anti-apoptosis and metastasis in salivary adenoid cystic carcinoma 
Oncotarget  2014;5(16):6885-6895.
Background: Numerous studies have reported both the tumor-suppressive and oncogenic roles of the Notch pathway, indicating that Notch activity regulates tumor biology in a complex, context-dependent manner. The aim of the present study was to identify the role of NOTCH1 in the cell growth and metastasis of SACC.
Methods: We analyzed the expression of NOTCH1 in clinical SACC samples using immunohistochemical staining. We silenced the expression of NOTCH1 and overexpressed activated NOTCH1 to elucidate the effects of NOTCH1 on proliferation, migration and invasion. NOTCH1 target genes were validated by real-time PCR.
Results: Our results showed that NOTCH1 was upregulated in SACC tissues when compared with normal tissues, and this upregulation was further enhanced in SACC tissues with metastasis and recurrence when compared with SACC tissues without metastasis. Overexpression of NOTCH1 in SACC cells promoted cell growth, migration and invasion, and knockdown of NOTCH1 inhibited cell proliferation in vitro and tumorigenicity in vivo by inducing cell apoptosis.
Conclusions:The results of this study suggest that NOTCH1 plays a key role in the cell growth, anti-apoptosis, and metastasis of SACC. NOTCH1 inhibitors might therefore have potential therapeutic applications in treating SACC patients by inhibiting cancer cell growth and metastasis.
PMCID: PMC4196170  PMID: 25149541
NOTCH1; salivary adenoid cystic carcinoma; proliferation; apoptosis; metastasis
5.  Converted neural cells: induced to a cure? 
Protein & cell  2012;3(2):91-97.
Many neurodegenerative disorders such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and others often occur as a result of progressive loss of structure or function of neurons. Recently, many groups were able to generate neural cells, either differentiated from induced pluripotent stem cells (iPSCs) or converted from somatic cells. Advances in converted neural cells have opened a new era to ease applications for modeling diseases and screening drugs. In addition, the converted neural cells also hold the promise for cell replacement therapy (Kikuchi et al., 2011; Krencik et al., 2011; Kriks et al., 2011; Nori et al., 2011; Rhee et al., 2011; Schwartz et al., 2012). Here we will mainly discuss most recent progress on using converted functional neural cells to treat neurological diseases and highlight potential clinical challenges and future perspectives.
doi:10.1007/s13238-012-2029-2
PMCID: PMC4104580  PMID: 22410787
converted neural cell; pluripotent stem cell; transdifferentiation; transplantation; neurodegenerative diseases
6.  “TET-on” pluripotency 
Cell Research  2013;23(7):863-865.
doi:10.1038/cr.2013.72
PMCID: PMC3698639  PMID: 23732523
7.  Surpassing the advanced comes from continuous accumulation 
Protein & Cell  2014;5(6):409-410.
doi:10.1007/s13238-014-0056-x
PMCID: PMC4026420  PMID: 24792691
8.  Surpassing the advanced comes from continuous accumulation 
Protein & Cell  2014;5(6):409-410.
doi:10.1007/s13238-014-0056-x
PMCID: PMC4026420  PMID: 24792691
9.  Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses 
PLoS ONE  2014;9(4):e95635.
Background
Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection.
Results
Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens.
Conclusions
Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.
doi:10.1371/journal.pone.0095635
PMCID: PMC3994070  PMID: 24752452
12.  Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes 
Protein & Cell  2014;5(1):59-68.
With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT+) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-013-0016-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-013-0016-x
PMCID: PMC3938846  PMID: 24474197
human cardiomyocyte; DNA methylation; microarray; heart development
13.  Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes 
Protein & Cell  2014;5(1):59-68.
With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT+) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-013-0016-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-013-0016-x
PMCID: PMC3938846  PMID: 24474197
human cardiomyocyte; DNA methylation; microarray; heart development
15.  Optimal Duration of Fluorouracil-Based Adjuvant Chemotherapy for Patients with Resectable Gastric Cancer 
PLoS ONE  2013;8(12):e83196.
Background
Although several clinical trials have suggested that postoperative adjuvant chemotherapy can improve survival of patients with gastric cancer, the optimal treatment duration has not been studied. This retrospective analysis evaluated the outcomes of patients with gastric cancer treated with six cycles of fluorouracil-based treatment compared with a cohort treated with four or eight cycles.
Methods
We retrospectively identified 237 patients with stage IB–IIIC gastric cancer who received four, six, or eight cycles of fluorouracil-based adjuvant chemotherapy administered every 3 weeks after radical gastrectomy. The endpoint was overall survival (OS). Factors associated with prognosis were also analyzed.
Results
The estimated 3-year OS rates for the four-, six-, and eight-cycle cohorts were 54.4%, 76.1%, and 68.9%, respectively; and the estimated 5-year OS rates were 41.2%, 74.0%, and 65.8%, respectively. Patients who received six cycles were more likely to have a better OS than those who received four cycles (P = 0.002). Eight cycles failed to show an additional survival benefit (P = 0.454). In the multivariate analysis, the number of chemotherapy cycles was associated with OS independent of clinical covariates (P<0.05). Subgroup analysis suggested that among patients in all age groups examined, male patients, and subgroups of fluorouracil plus oxaliplatin combined chemotherapy, stage III, poor differentiation, and gastrectomy with D2 lymphadenectomy, six cycles of adjuvant chemotherapy were associated with a statistically significant benefit of OS compared with four cycles (P<0.05).
Conclusions
Six cycles of adjuvant chemotherapy might lead to a favorable outcome for patients with gastric cancer, and two further cycles could not provide an additional clinical benefit.
doi:10.1371/journal.pone.0083196
PMCID: PMC3873471  PMID: 24386161
16.  Discovery and study of cutaneous leishmaniasis in Karamay of Xinjiang, West China 
Cutaneous leishmaniasis (CL) was discovered in the farms of the Karamay suburb, Xinjiang Uygur Autonomous Region in the 1990s. Between 1992 and 1994, a house-to-house survey revealed a prevalence of 1.0-1.6% in the residents. The clinical types of skin lesions included papule, plaque, ulcer and nodular prurigo. Observations verified that, in some cases, the skin lesions healed spontaneously in 10–14 months, whilst in other cases, they persisted for several years. Sporadic cases of CL have continued to appear at the dermatology clinic of the local hospital since 2000. Phlebotomus wui (Ph. wui), subgenus Larroussius was confirmed as the transmitting vector. The causative agent is Leishmania infantum sensu lato.
doi:10.1186/2049-9957-2-20
PMCID: PMC3856449  PMID: 24010525
17.  S-nitrosylation of Cdk5 
Prion  2012;6(4):364-370.
Aberrant activation of Cdk5 has been implicated in the process of neurodegenerative diseases such as Alzheimer's disease (AD). We recently reported that S-nitrosylation of Cdk5 (forming SNO-Cdk5) at specific cysteine residues results in excessive activation of Cdk5, contributing to mitochondrial dysfunction, synaptic damage, and neuronal cell death in models of AD. Furthermore, SNO-Cdk5 acts as a nascent S-nitrosylase, transnitrosylating the mitochondrial fission protein Drp1 and enhancing excessive mitochondrial fission in dendritic spines. However, a molecular mechanism that leads to the formation of SNO-Cdk5 in neuronal cells remained obscure. Here, we demonstrate that neuronal nitric oxide synthase (NOS1) interacts with Cdk5 and that the close proximity of the two proteins facilitates the formation of SNO-Cdk5. Interestingly, as a negative feedback mechanism, Cdk5 phosphorylates and suppresses NOS1 activity. Thus, together with our previous report, these findings delineate an S-nitrosylation pathway wherein Cdk5/NOS1 interaction enhances SNO-Cdk5 formation, mediating mitochondrial dysfunction and synaptic loss during the etiology of AD.
doi:10.4161/pri.21250
PMCID: PMC3609065  PMID: 22874667
nitrosative stress; Cyclin-dependent kinase 5; nitric oxide; neuronal NO synthase; transnitrosylation
18.  Microarray-Based Identification of Differentially Expressed Genes in Intracellular Brucella abortus within RAW264.7 Cells 
PLoS ONE  2013;8(8):e67014.
Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella’s virulence and potential for chronic infection include the ability to circumvent the host cell’s internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292) had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.
doi:10.1371/journal.pone.0067014
PMCID: PMC3737221  PMID: 23950864
19.  Gating neural development and aging via nuclear pores 
Cell Research  2012;22(8):1212-1214.
doi:10.1038/cr.2012.35
PMCID: PMC3411164  PMID: 22410792
20.  A reported death case of a novel bunyavirus in Shanghai, China 
Virology Journal  2013;10:187.
This paper describes the first case of infection with a recently described novel bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), in Shanghai, China. The case is originally from Chizhou City, Anhui province within an endemic area for SFTSV. We describe the etiology, epidemiological characteristics, clinical diagnosis and treatment of this fatal case. This case is unique because major cause of death was renal failure, whereas other reported cases have been due to hemorrhage. The investigation and response to this case provides meaningful insight for the early and rapid diagnosis, treatment, prevention and control of severe fever with thrombocytopenia syndrome virus in non-endemic regions in China and globally.
doi:10.1186/1743-422X-10-187
PMCID: PMC3689053  PMID: 23758684
Severe fever and thrombocytopenia syndrome; Bunyavirus; Genetic analysis
23.  Hepatitis C Virus Activates Bcl-2 and MMP-2 Expression through Multiple Cellular Signaling Pathways 
Journal of Virology  2012;86(23):12531-12543.
Hepatitis C virus (HCV) infection is associated with numerous liver diseases and causes serious global health problems, but the mechanisms underlying the pathogenesis of HCV infections remain largely unknown. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-2 (MMP-2), and B-cell lymphoma 2 (Bcl-2) are significantly stimulated in HCV-infected patients. We further show that HCV activates STAT3, MMP-2, Bcl-2, extracellular regulated protein kinase (ERK), and c-Jun N-terminal kinase (JNK) in infected Huh7.5.1 cells. Functional screening of HCV proteins revealed that nonstructural protein 4B (NS4B) is responsible for the activation of MMP-2 and Bcl-2 by stimulating STAT3 through repression of the suppressor of cytokine signaling 3 (SOCS3). Our results also demonstrate that multiple signaling cascades, including several members of the protein kinase C (PKC) family, JNK, ERK, and STAT3, play critical roles in the activation of MMP-2 and Bcl-2 mediated by NS4B. Further studies revealed that the C-terminal domain (CTD) of NS4B is sufficient for the activation of STAT3, JNK, ERK, MMP-2, and Bcl-2. We also show that amino acids 227 to 250 of NS4B are essential for regulation of STAT3, JNK, ERK, MMP-2, and Bcl-2, and among them, three residues (237L, 239S, and 245L) are crucial for this regulation. Thus, we reveal a novel mechanism underlying HCV pathogenesis in which multiple intracellular signaling cascades are cooperatively involved in the activation of two important cellular factors, MMP-2 and Bcl-2, in response to HCV infection.
doi:10.1128/JVI.01136-12
PMCID: PMC3497616  PMID: 22951829
24.  Progress and prospects in stem cell therapy 
Acta Pharmacologica Sinica  2013;34(6):741-746.
In the past few years, progress being made in stem cell studies has incontestably led to the hope of developing cell replacement based therapy for diseases deficient in effective treatment by conventional ways. The induced pluripotent stem cells (iPSCs) are of great interest of cell therapy research because of their unrestricted self-renewal and differentiation potentials. Proof of principle studies have successfully demonstrated that iPSCs technology would substantially benefit clinical studies in various areas, including neurological disorders, hematologic diseases, cardiac diseases, liver diseases and etc. On top of this, latest advances of gene editing technologies have vigorously endorsed the possibility of obtaining disease-free autologous cells from patient specific iPSCs. Here in this review, we summarize current progress of stem cell therapy research with special enthusiasm in iPSCs studies. In addition, we compare current gene editing technologies and discuss their potential implications in clinic application in the future.
doi:10.1038/aps.2013.77
PMCID: PMC3674518  PMID: 23736002
induced pluripotent stem cells (iPSCs); stem cell therapy; gene editing; neurological disorders; hematologic diseases; cardiac diseases; liver diseases
25.  Progressive degeneration of human neural stem cells caused by pathogenic LRRK2 
Nature  2012;491(7425):603-607.
Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging1-4. It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice5. Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.
doi:10.1038/nature11557
PMCID: PMC3504651  PMID: 23075850

Results 1-25 (46)