Publications by authors named "Meijuan Tian"

12 Publications

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Evolution-Guided Structural and Functional Analyses of the HERC Family Reveal an Ancient Marine Origin and Determinants of Antiviral Activity.

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

Western University, Schulich School of Medicine and Dentistry, Department of Microbiology and Immunology, London, Ontario, Canada

In humans, homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses, including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution, and function of HERC5, we performed phylogenetic, structural, and functional analyses of the entire human small-HERC family, which includes HERC3, HERC4, HERC5, and HERC6. We demonstrated that the family emerged >595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs is highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity toward HIV-1 and that HERC5 provides the strongest inhibition. Notably, coelacanth HERC5 inhibited simian immunodeficiency virus (SIV), but not HIV-1, particle production, suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection, particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution, and biological importance of the human restriction factor HERC5 and the other HERC family members. Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origins, evolution, and functions of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, two of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates.
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http://dx.doi.org/10.1128/JVI.00528-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002735PMC
July 2018

Development of an anti-HIV vaccine eliciting broadly neutralizing antibodies.

AIDS Res Ther 2017 Sep 12;14(1):50. Epub 2017 Sep 12.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada.

The extreme HIV diversity posts a great challenge on development of an effective anti-HIV vaccine. To solve this problem, it is crucial to discover an appropriate immunogens and strategies that are able to prevent the transmission of the diverse viruses that are circulating in the world. Even though there have been a number of broadly neutralizing anti-HIV antibodies (bNAbs) been discovered in recent years, induction of such antibodies to date has only been observed in HIV-1 infection. Here, in this mini review, we review the progress in development of HIV vaccine in eliciting broad immune response, especially production of bNAbs, discuss possible strategies, such as polyvalent sequential vaccination, that facilitates B cell maturation leading to bNAb response.
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http://dx.doi.org/10.1186/s12981-017-0178-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594608PMC
September 2017

An in vitro Model to Mimic Selection of Replication-Competent HIV-1 Intersubtype Recombination in Dual or Superinfected Patients.

J Mol Biol 2017 07 1;429(14):2246-2264. Epub 2017 May 1.

Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Department of Microbiology and Immunology, Western University, London, Ontario N6A 5C1, Canada. Electronic address:

The low frequency of HIV-1 recombinants within entire viral populations in both individual patients and culture-based infection models impedes investigation of the underlying factors contributing to either the occurrence of recombinants or the survival of recombinants once they are formed. So far, most of the related studies have no consideration of recombinants' functionality. Here, we established a functional recombinant production (FRP) system to produce pure and functional HIV-1 intersubtype Env recombinants and utilized 454 pyrosequencing to investigate the distribution of over 4000 functional and non-functional recombination breakpoints from either the FRP system or dual infection cultures. The results revealed that most of the breakpoints converged in gp41 (62%) and C1 (25.3%) domains of gp120, which has strong correlation with the similarity between the two recombining sequences. Yet, the breakpoints also appeared in C2 (5.2%) and C5 (4.6%) domains not correlated with the recombining sequence similarity. Interestingly, none of the intersubtype gp120 recombinants recombined between C1 and gp41 regions either from the FRP system or from the dual infection culture, and very few from the HIV epidemic were functional. The present study suggests that the selection of functional Env recombinants is one of the reasons for the predominance of C1 and gp41 Env recombinants in the HIV epidemic, and it provides an in vitro model to mimic the selection of replication-competent HIV-1 intersubtype recombination in dual or superinfected patients.
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http://dx.doi.org/10.1016/j.jmb.2017.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202033PMC
July 2017

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections.

AIDS Res Ther 2016 11 25;13(1):41. Epub 2016 Nov 25.

Division of Infectious Diseases, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106 USA.

Background: New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques.

Results: Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans.

Conclusion: Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.
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http://dx.doi.org/10.1186/s12981-016-0125-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124249PMC
November 2016

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

Retrovirology 2016 Nov 28;13(1):82. Epub 2016 Nov 28.

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, 1400 Western Road, London, ON, N6G 2V4, Canada.

Background: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity.

Results: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes.

Conclusion: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.
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http://dx.doi.org/10.1186/s12977-016-0317-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126836PMC
November 2016

Pathogenic infection of Rhesus macaques by an evolving SIV-HIV derived from CCR5-using envelope genes of acute HIV-1 infections.

Virology 2016 12 7;499:298-312. Epub 2016 Oct 7.

Division of Infectious Diseases and HIV Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada. Electronic address:

For studies on vaccines and therapies for HIV disease, SIV-HIV chimeric viruses harboring the HIV-1 env gene (SHIVenv) remain the best virus in non-human primate models. However, there are still very few SHIVenv viruses that can cause AIDS in non-CD8-depleted animals. In the present study, a recently created CCR5-using SHIVenv_B3 virus with env gene derived from acute/early HIV-1 infections (AHI) successfully established pathogenic infection in macaques. Through a series of investigations on the evolution, mutational profile, and phenotype of the virus and the resultant humoral immune response in infected rhesus macaques, we found that the E32K mutation in the Env C1 domain was associated with macaque pathogenesis, and that the electrostatic interactions in Env may favor E32K at the gp120 N terminus and "lock" the binding to heptad repeat 1 of gp41 in the trimer and produce a SHIVenv with increased fitness and pathogenesis during macaque infections.
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http://dx.doi.org/10.1016/j.virol.2016.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899904PMC
December 2016

Functional bottlenecks for generation of HIV-1 intersubtype Env recombinants.

Retrovirology 2015 May 23;12:44. Epub 2015 May 23.

Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH, 44106, USA.

Background: Intersubtype recombination is a powerful driving force for HIV evolution, impacting both HIV-1 diversity within an infected individual and within the global epidemic. This study examines if viral protein function/fitness is the major constraint shaping selection of recombination hotspots in replication-competent HIV-1 progeny. A better understanding of the interplay between viral protein structure-function and recombination may provide insights into both vaccine design and drug development.

Results: In vitro HIV-1 dual infections were used to recombine subtypes A and D isolates and examine breakpoints in the Env glycoproteins. The entire env genes of 21 A/D recombinants with breakpoints in gp120 were non-functional when cloned into the laboratory strain, NL4-3. Likewise, cloning of A/D gp120 coding regions also produced dead viruses with non-functional Envs. 4/9 replication-competent viruses with functional Env's were obtained when just the V1-V5 regions of these same A/D recombinants (i.e. same A/D breakpoints as above) were cloned into NL4-3.

Conclusion: These findings on functional A/D Env recombinants combined with structural models of Env suggest a conserved interplay between the C1 domain with C5 domain of gp120 and extracellular domain of gp41. Models also reveal a co-evolution within C1, C5, and ecto-gp41 domains which might explain the paucity of intersubtype recombination in the gp120 V1-V5 regions, despite their hypervariability. At least HIV-1 A/D intersubtype recombination in gp120 may result in a C1 from one subtype incompatible with a C5/gp41 from another subtype.
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http://dx.doi.org/10.1186/s12977-015-0170-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445978PMC
May 2015

SiRNA-induced mutation in HIV-1 polypurine tract region and its influence on viral fitness.

PLoS One 2015 10;10(4):e0122953. Epub 2015 Apr 10.

Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America; Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, United States of America.

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3' and central polypurine tracts (3' and cPPTs). The 5' and 3' termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3'PPT sequence. Introducing either or both mutations into the 3'PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3'PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3'PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3'PPT is capable of replication, this nucleotide substitution shifts the 3'-terminal cleavage site in the 3'PPT by one nucleotide (nt) and significantly reduces viral fitness.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122953PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393142PMC
March 2016

Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome?

J AIDS Immune Res 2015;1(1). Epub 2015 Mar 2.

Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, 10900, Euclid Ave, Cleveland, Ohio 44106, USA; Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio 44106, USA.

Although the process of reverse transcription is well elucidated, it remains unclear if viral core disruption provides a more cellular or viral milieu for HIV-1 reverse transcription. We have devised a method to require mixing of viral cores or core constituents to produce infectious progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each other and when together can complete viral reverse transcription. Only the heterodiploid virus containing both the nfl and cplt_R/U5/gag/Δpol sgRNAs can complete reverse transcription and propagate infectious virus upon de novo infection. Dual exposure of U87.CD4.CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/Δpol virus particles did not result in productive virus infection. On the other hand, in early endosomes, the HIV-1 sgRNAs released from viral cores can retain function and complete the reverse transcription and result in productive infection. These findings confirm the assumptions that, in natural infection, HIV-1 cores, and likely other retrovirus cores, remain largely intact and do not mix/fuse in the cytoplasm during the reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA (produced through transfection) could not help the complementary sgRNA in the viral core to complement the reverse transcription process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882926PMC
March 2015

Deletion of C7L and K1L genes leads to significantly decreased virulence of recombinant vaccinia virus TianTan.

PLoS One 2013 1;8(7):e68115. Epub 2013 Jul 1.

Division of Research on Virology and Immunology, State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention-NCAIDS, China CDC, Beijing, China.

The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068115PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698190PMC
February 2014

Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

PLoS One 2013 12;8(4):e60557. Epub 2013 Apr 12.

State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention, Beijing, China.

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060557PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625194PMC
October 2013

Proteomic identification of angiomotin by ProteomeLab PF-2D and correlation with clinical outcome in human clear cell renal cell carcinoma.

Int J Oncol 2013 Jun 10;42(6):2078-86. Epub 2013 Apr 10.

Medical Oncology Department, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710061, P.R. China.

Identification of new therapeutic and prognostic biomarkers for clear cell renal cell carcinoma (ccRCC) is urgently required since most patients are in advanced stages of ccRCC at initial diagnosis and the recurrence rate is high. Differentially expressed proteins between the ccRCC cell line RLC-310 and the normal renal cell line HK-2 were identified by a comparative proteomic approach based on a protein fractionation two-dimensional (PF-2D) liquid-phase fractionation system and capillary liquid chromatography electrospray ionization mass spectrometry/mass spectrometry (LC-ESI-MS/MS). Differentially expressed proteins (n=196) were identified. Of the 13 differentially expressed proteins newly discovered in ccRCC, angiomotin (Amot) was the focus of this study since its role in ccRCC progression has been obscure. The overexpression of Amot in ccRCC tissues was confirmed by comparing Amot expression in 18 primary ccRCC tissues and adjacent normal renal tissues (ANRT) using western blot analysis. Quantitative RT-PCR using 127 ccRCC tissues revealed that high levels of Amot transcripts were associated with poor differentiation, venous invasion and decreased survival (p<0.0001, <0.05 and <0.05). Multivariate analysis indicated that Amot transcript was an independent prognostic factor for the survival of ccRCC patients (p<0.05). These data suggest that Amot may serve as a novel prognostic factor of ccRCC.
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http://dx.doi.org/10.3892/ijo.2013.1889DOI Listing
June 2013