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ABSTRACT: Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides.
PLoS ONE 01/2013; 8(3):e59777. · 4.09 Impact Factor
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ABSTRACT: OBJECTIVES: We recently demonstrated that both 3-hydroxyphthalic anhydride (HP)- and maleic anhydride-modified chicken ovalbumin (OVA) could effectively inhibit HIV-1 infection. But because OVA may cause allergy in some human subjects, here we replaced OVA with human serum albumin (HSA) in designing a new anti-HIV-1 agent, HP-HSA, and then tested its anti-HIV-1 activity and cytotoxicity. METHODS: The in vitro anti-HIV-1 activities of HP-HSA were detected by measuring p24 production and luciferase activity. The cytotoxicities of HP-HSA on target cells and human vaginal and cervical epithelial cells and the effect of HP-HSA on human peripheral blood mononuclear cell (PBMC) proliferation were evaluated by XTT assay. The effect of HP-HSA on interferon-γ secretion by PBMCs was detected by enzyme-linked immunospot (ELISPOT) assay. RESULTS: We found that HP-HSA exhibited broad and potent antiviral activity against infection by the HIV-1 strains tested, including drug-resistant strains. HP-HSA displayed no or low cytotoxicity on human vaginal and cervical epithelial cells and the cells used for testing HIV-1 infectivity. In addition, HP-HSA had no significant effect on proliferation or interferon-γ secretion by normal or phytohaemagglutinin-stimulated human PBMCs. A time-of-addition assay indicated that HP-HSA was an HIV-1 entry inhibitor. CONCLUSIONS: Because of its broad and potent anti-HIV-1 activity, low cytotoxicity and low immunogenicity to humans, HP-HSA has great potential for further development as a microbicide to prevent the sexual transmission of HIV.
Journal of Antimicrobial Chemotherapy 12/2012; · 5.07 Impact Factor
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ABSTRACT: Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.
Biochimica et Biophysica Acta 07/2012; 1818(12):2950-7. · 4.66 Impact Factor
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ABSTRACT: The membrane proximal external region (MPER) of gp41 is extremely conserved among diverse HIV-1 variants, implying its important
role in viral infection. Interestingly, two of the most broadly neutralizing antibodies, 2F5 and 4E10, specifically recognize
this region. Our previous study demonstrated that the antigenicity and immunogenicity of 4E10 epitope are affected by remodeling
gp41 fusion core, suggesting that the MPER may be associated with gp41 core and involved in gp41-mediated membrane fusion.
Here we measured the binding activity of 4E10 epitope peptide (D4E10P) with various gp41 core-derived peptides and found that
the N-trimer region in a construct designated N-trimer-6HB interacted significantly with D4E10P. Using N-trimer-6HB to screen
a phage library, we identified a motif (WF) located in 4E10 epitope that may play a certain role in the interaction of gp41
MPER with the N-trimer in gp41 fusion core and, we thus speculated upon the potential involvement of MPER in the fusion process
between viral envelope and target cell membrane.
Chinese Science Bulletin 04/2012; 54(10):1707-1712. · 1.32 Impact Factor
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ABSTRACT: To prove that the peptidic HIV-1 fusion inhibitors containing the pocket-binding domain (PBD) mainly target the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR), we constructed pseudoviruses by replacement of Q64 in the gp41 pocket region with Ala (Q64A) or Leu (Q64L). These viruses were highly resistant to C34 and CP32M containing the PBD, while they were susceptible to T20 (enfuvirtide) lacking the PBD but containing the GIV-motif-binding domain (GBD) and lipid-binding domain (LBD). They were also sensitive to C52L, which contains the PBD, GBD, and LBD. Those mutations may disrupt the hydrophilic interaction between Q64 in the NHR and N113 in the peptides containing the PBD. This report provides insights into the mechanisms of drug resistance, with implications for the design of novel HIV fusion and entry inhibitors.
Journal of Virology 01/2012; 86(1):589-93. · 5.40 Impact Factor
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ABSTRACT: Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library and identified a novel gp41-binding protein, CD74 (the MHC class II-associated invariant chain). Here, we report possible biological effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could bind directly to host CD74 in HIV-1-infected cells, and the peptide 6358 derived from gp41 loop region (aa 597-611) could effectively block the gp41-CD74 interaction. As a result of this binding, recombinant soluble gp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1 infection in vitro. Conversely, the enhancing effect could be suppressed by the recombinant CD74 extracellular domain. These results reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.
Journal of Biological Chemistry 12/2011; 286(52):44869-77. · 4.77 Impact Factor
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ABSTRACT: Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many
host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library
and identified a novel gp41 binding protein, CD74 (the MHC class II-associated invariant chain). Here we report possible biological
effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could directly bind to host CD74 in HIV-1-infected
cells and the peptide 6358 derived from gp41 loop region (aa597-611) could effectively block gp41-CD74 interaction. As a result
of this binding, rsgp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1
infection in vitro. Conversely, the enhancing effect could be suppressed by recombinant CD74 extracellular domain. These results
reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.
Journal of Biological Chemistry 10/2011; · 4.77 Impact Factor
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ABSTRACT: T20 (enfuvirtide, Fuzeon) is the first generation HIV-1 fusion inhibitor approved for salvage therapy of HIV-1-infected patients refractory to current antiretroviral drugs. However, its application is limited by the high cost of peptide synthesis, rapid proteolysis, and poor efficacy against emerging drug-resistant strains. Here we reported the design of a novel chimera protein-based fusion inhibitor targeting gp41, TLT35, that uses a flexible 35-mer linker to couple T20 and T1144, the first and next generation HIV-1 fusion inhibitors, respectively. TLT35, which was expressed in Escherichia coli with good yield, showed low nm activity against HIV-1-mediated cell-cell fusion and infection by laboratory-adapted HIV-1 strains (X4 or R5), including T20-resistant variants and primary HIV-1 isolates of clades A to G and group O (R5 or X4R5). TLT35 was stable in human sera and in peripheral blood mononuclear cell culture and was more resistant to proteolysis than either T20 or T1144 alone. Circular dichroism spectra showed that TLT35 folded into a thermally stable conformation with high α-helical content and T(m) value in aqueous solution. It formed a highly stable complex with gp41 N-terminal heptad repeat peptide and blocked formation of the gp41 six-helix-bundle core. These merits combined with an anticipated low production cost for expression of TLT35 in E. coli make this novel protein-based fusion inhibitor a promising candidate for further development as an anti-HIV-1 microbicide or therapeutic for the prevention and treatment of HIV-1 infection.
Journal of Biological Chemistry 06/2011; 286(32):28425-34. · 4.77 Impact Factor
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ABSTRACT: Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.
Journal of Biological Chemistry 02/2011; 286(5):3277-87. · 4.77 Impact Factor
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ABSTRACT: The development of a safe, effective, and affordable microbicide to prevent the sexual transmission of HIV combination is urgently needed. Our previous studies demonstrated that 3-hydroxyphthalic anhydride-modified chicken ovalbumin (HP-OVA) exhibited potent antiviral activity against a broad spectrum of HIV, simian immunodeficiency virus, and herpes simplex virus, making it a promising candidate as a component of combination microbicide. We intended to evaluate potential the synergistic anti-HIV-1 effect of HP-OVA in combination with antiretroviral drug (ARV)-based microbicide candidates.
The antiviral activity of HP-OVA and the ARVs, including HIV-1 entry inhibitors (T20, C52L, NB64, NBD556, AMD3100, and Maraviroc) and reverse transcriptase inhibitors (Tenofovir, UC781, and TMC120), tested alone or in combination, against HIV-1 X4 and R5 viruses, including some drug-resistant strains, was determined in MT-2 and peripheral blood mononuclear cells using p24 assay. The immune responses induced by HP-OVA that was applied in the vaginas of rats were detected by enzyme-linked immunosorbent assay.
When each of these ARV-based microbicide candidates was combined with HP-OVA, synergistic activity was observed against infection by both X4 and R5 strains, and the degree of synergy differed in each case. HP-OVA was highly effective against several ARV-resistant HIV-1 strains, suggesting that combining HP-OVA with these ARV-based microbicide candidates might work cooperatively against both drug-sensitive and -resistant HIV-1 strains. Human body fluids and human proteins had little or no effects on HP-OVA-mediated inhibitory activity against HIV-1 infection. HP-OVA formulated in the universal gel maintained its antiviral activity for at least 1 month and only induced weak immune responses after its multiple applications in the vaginas of rats.
Synergistic and complementary effects against infection by a broad spectrum of HIV-1 strains were observed by combining HP-OVA with the ARV-based microbicide candidates. These findings provide a sound scientific platform for the development of a safe, effective, and affordable combination microbicide to prevent the sexual transmission of HIV and other sexually transmissible viruses.
JAIDS Journal of Acquired Immune Deficiency Syndromes 01/2011; 56(5):384-92. · 4.43 Impact Factor
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ABSTRACT: The membrane-proximal external region (MPER) of the HIV-1 gp41 consists of epitopes for the broadly cross-neutralizing monoclonal antibodies 2F5 and 4E10. However, antigens containing the linear sequence of these epitopes are unable to elicit potent and broad neutralizing antibody responses in vaccinated hosts, possibly because of inappropriate conformation of these epitopes. Here we designed a recombinant antigen, designated NCM, which comprises the N- and C-terminal heptad repeats that can form a six-helix bundle (6HB) core and the MPER domain of gp41. Two mutations (T569A and I675V) previously reported to expose the neutralization epitopes were introduced into NCM to generate mutants named NCM(TA), NCM(IV), and NCM(TAIV). Our results showed that NCM and its mutants could react with antibodies specific for 6HB and MPER of gp41, suggesting that these antigens are in the form of a trimer of heterodimer (i.e., 6HB) with three exposed MPER tails. Antigen with double mutations, NCM(TAIV), elicited much stronger antibody response in rabbits than immunogens with single mutation, NCM(TA) and NCM(IV), or no mutation, NCM. The purified MPER-specific antibodies induced by NCM(TAIV) exhibited broad neutralizing activity, while the purified 6HB-specific antibodies showed no detectable neutralizing activity. Our recombinant antigen design supported by an investigation of its underlying molecular mechanisms provides a strong scientific platform for the discovery of a gp41 MPER-based AIDS vaccine.
PLoS ONE 01/2011; 6(3):e18233. · 4.09 Impact Factor
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ABSTRACT: Sifuvirtide, a novel fusion inhibitor against human immunodeficiency type I (HIV-1) which is more potent than enfuvirtide
(T20) in cell culture is currently under clinical investigation for the treatment of HIV-1 infection. We now report that
in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide
has led to several specific mutations in the gp41 region which had not been previously reported. Many of these substitutions
were confined to the N-terminal heptad repeat (NHR) region at positions 37, 38, 41, and 43, either singly or in combination.
A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat (CHR) region was also found. Site-directed
mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the
resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some
of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses.
Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide.
Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic
utility of sifuvirtide.
Journal of Biological Chemistry 11/2010; · 4.77 Impact Factor
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ABSTRACT: This paper studies a common traffic problem in China: a large number of bus lines share the same stop. In rush hours, a long queue of buses waiting to get into the stop, which causes a rise of bus delay and a drop of traffic capacity nearby. First, a cellular automaton model is proposed to simulate such traffic scenarios. Second, to re-designing such busy bus stops to enhance efficiency, some principles are proposed and evaluated by simulations. Finally, a specific design example is given.
Intelligent Transportation Systems (ITSC), 2010 13th International IEEE Conference on; 10/2010
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4(+) cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.
Journal of Virology 09/2010; 84(18):9359-68. · 5.40 Impact Factor
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ABSTRACT: Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.
Journal of Biological Chemistry 08/2010; 285(33):25506-15. · 4.77 Impact Factor
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ABSTRACT: This paper studies the following driving scenario that is common in China and many Asian developing countries: a bus has to temporarily occupy the bicycle lane in order to stop and pick up passengers, where the mixed traffic flow is composed of motorized vehicles and bicycles. A special cellular automation model is proposed to examine the governing factors that influence the delay of a bus near a bus stop. Vehicle inflow rate, bicycle inflow rate and bus inflow rate are specially addressed. Finally, some useful suggestions are presented.
Intelligent Vehicles Symposium (IV), 2010 IEEE; 07/2010
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IEEE Intell. Transport. Syst. Mag. 01/2010; 2:19-24.
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Lin Li,
Pengyuan Qiao,
Jie Yang, Lu Lu,
Suiyi Tan,
Hong Lu,
Xiujuan Zhang,
Xi Chen,
Shuguang Wu,
Shibo Jiang,
Shuwen Liu
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ABSTRACT: Background: Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, β-lactoglobulin (β-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission. Results: Maleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than β-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.
Retrovirology. 01/2010; 7.
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Lin Li,
Pengyuan Qiao,
Jie Yang, Lu Lu,
Suiyi Tan,
Hong Lu,
Xiujuan Zhang,
Xi Chen,
Shuguang Wu,
Shibo Jiang,
Shuwen Liu
[show abstract]
[hide abstract]
ABSTRACT: Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, beta-lactoglobulin (beta-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.
Maleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than beta-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.
ML-OVA is a potent HIV fusion/entry inhibitor with the potential to be developed as an effective, safe and inexpensive anti-HIV microbicide.
Retrovirology 01/2010; 7:37. · 6.47 Impact Factor
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ABSTRACT: From 8th to 24th in August 2008, a set of Traffic Demand Management (TDM) policies were applied in Beijing during the 2008 Olympic Games. These TDM strategies include improving performance of the existing transportation network, staggering work schedules and limiting the use of public vehicles and private vehicles. This paper studies the performance targets, the implementation details and the effects of these TDM measures. Some useful conclusions about how to improve the quality of transport services and how to satisfy the need of international community and local public during important activities are drawn from this paper. We are happy to witness that fluent traffic services were provided for Beijing Summer Olympics 2008 via the aid of these transport services.
Intelligent Transportation Systems, 2009. ITSC '09. 12th International IEEE Conference on; 11/2009
