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1.
Rev Environ Contam Toxicol ; 251: 1-24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31011831

RESUMO

Heavy metal pollution in surface water is a global environmental problem. This study analyzed the trends, health risks, and sources of eight dissolved heavy metal species in river and lake water across five continents (Africa, Asia, Europe, North America, and South America; Oceania was excluded owing to a lack of data) for the period 1970-2017. We wanted to assess the effects of various implemented countermeasures to pollution and to determine those that could be adopted worldwide. Collectively, the water system showed increasing trends for Cd, Cr, Cu, Ni, Mn, and Fe and decreasing trends for Pb and Zn. The mean dissolved concentrations of most heavy metals were highest in Asia and lowest in Europe. Most heavy metals had low non-carcinogenic risks over this period. The cancer risks associated with Pb were lower than the hazardous level on all five continents over the five decades, whereas the cancer risks related to Cr exceeded the hazardous level in the 1970s, 2000s, and 2010s, and in Africa, Asia, and North America over the entire period. Mining and manufacturing were consistently found to be critical sources of metal pollution from 1970 to 2017. However, the heavy metal sources differed significantly by continent, with waste discharge and rock weathering dominant in Africa; mining and manufacturing, along with rock weathering, are dominant in Asia and South America; fertilizer and pesticide use, along with rock weathering, are dominant in North America; and mining and manufacturing, waste discharge, and rock weathering are dominant in Europe. Global trends in the metal loadings in water and in relevant pollution-control measures suggest that countermeasures in Europe have successfully controlled heavy metal pollution. The successful measures include implementing rigorous standards for metal emissions, limiting the metal concentrations in products, and rigorously treating metal-contaminated waste. Therefore, the measures implemented in Europe should be extended worldwide to treat heavy metal pollution in water.


Assuntos
Exposição Ambiental/análise , Metais Pesados/análise , Poluentes Químicos da Água/análise , Poluição Química da Água/estatística & dados numéricos , Exposição Ambiental/estatística & dados numéricos , Monitoramento Ambiental , Humanos , Lagos , Rios
2.
Ann Bot ; 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31585004

RESUMO

BACKGROUND AND AIMS: Most primary auxin response genes are classified into three families: AUX/IAAs, GH3s, and SAURs. Few studies have been conducted on Arabidopsis SAURs, possibly due to genetic redundancy among different subfamily members. Data mining on Arabidopsis transcriptional profiles indicates that the SAUR41 subfamily members of SMALL AUXIN UP RNA genes are, strikingly, induced by an inhibitory phytohormone, abscisic acid. We aimed to reveal the physiological roles of Arabidopsis SAUR41 subfamily genes containing SAUR40, SAUR41, SAUR71, and SAUR72. METHODS: Transcriptional responses of Arabidopsis SAUR41s to phytohormones were determined by quantitative real-time PCR. Knock out of SAUR41s was carried out with the CRISPR/Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR Associated Protein 9) genome editing technique. The saur41/40/71/72 quadruple mutants, the SAUR41 overexpression lines and the wild-type were subjected to ultra-structure observation, transcriptome analysis, and physiological characterization. KEY RESULTS: Transcription of Arabidopsis SAUR41 subfamily genes is activated by ABA but not by gibberellic acids and brassinosteroids. Quadruple mutations in saur41/40/71/72 led to reduced cell expansion/elongation in cotyledons and hypocotyls, opposite to the SAUR41 overexpression; however, irregular arrangement of cell size and shape was observed in both cases. The quadruple mutants had increased transcription of calcium homeostasis/signaling genes in seedling shoots, and the SAUR41 overexpression lines had decreased transcription of iron homeostasis genes in roots and increased ABA biosynthesis in shoots. Notably, both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress during seedling establishment, whereas specific expression of SAUR41 under the ABA-responsive RD29A (Responsive to Desiccation 29A) promoter in the quadruple mutants rescued the inhibitory effect of salt stress. CONCLUSIONS: The SAUR41 subfamily genes of Arabidopsis are ABA-inducible to modulate cell expansion, ion homeostasis and salt tolerance. Our work may provide new candidate genes for improvement of plant abiotic stress tolerance.

3.
J Virol ; 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619552

RESUMO

Refolding of the HIV-1 gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) juxtaposes viral and cellular membranes for fusion. The CHR-derived peptide T20 is the only clinically approved viral fusion inhibitor and has potent anti-HIV activity; however, its mechanism of action is not fully understood. In this study, we surprisingly found that T20 disrupted the α-helical conformation of the NHR-derived peptide N54 through its C-terminal tryptophan-rich motif (TRM), and that synthetic short-peptides containing the TRM sequence, TRM8 and TRM12, disrupted the N54 helix in a dose-dependent manner. Interestingly, TRM8 efficiently interfered with the secondary structures of three overlapped NHR peptides (N44, N38, and N28) and interacted with N28 that mainly contains the deep NHR pocket-forming sequence in a high affinity, suggesting that TRM targeted the NHR pocket site to mediate the disruption. Different from the TRM8, the short-peptide corresponding to the pocket-binding domain (PBD) of the CHR helix had no such disruptive effect and the CHR peptide C34 could form a stable 6-HB with the NHR helix; however, addition of the TRM to the C-terminus of C34 resulted in a peptide (C46) that destroyed the NHR helix. Although the TRM peptides alone had no anti-HIV activity and could not block the formation of 6-HB conformation, substitution of the TRM for the PBD in C34 resulted in a mutant inhibitor (C34TRM) with high binding and inhibitory capacities. Combined, the present data inform a new mode of action of T20 and the structure-function relationship of gp41.IMPORTANCE The HIV-1 Env glycoprotein mediates membrane fusion and is conformationally labile. Despite extensive efforts, the structural property of the native fusion protein gp41 is largely unknown and the mechanism of action of the gp41-derived fusion inhibitor T20 remains elusive. Here, we report that T20 and its C-terminal tryptophan-rich motif (TRM) can efficiently impair the conformation of the gp41 N-terminal heptad repeat (NHR) coiled coil, which is through the interaction with the deep NHR pocket site. The TRM sequence has been verified to possess ability to substitute the pocket-binding domain of C34, a fusion inhibitor peptide with high anti-HIV potency. Therefore, our studies have not only facilitated to understand the mechanism of action of T20 and develop novel HIV-1 fusion inhibitors but also provided new insights into the structural property of the pre-fusion state gp41.

4.
Int J Phytoremediation ; 21(3): 240-245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30656962

RESUMO

Bioaccumulation of heavy metals in aquatic plants is significantly affected by hydrological regime and therefore the accumulation and translocation of cadmium in five organs-panicle, leaf, stem, root, and bud-of an emergent plant (Miscanthus sacchariflorus) were compared between the submerged environment and non-submerged environment. In the submerged condition, the cadmium concentration was higher in the panicle and leaf than in the stem, root, and bud. Cadmium concentration in the root exhibited a positive regression with cadmium concentration in the sediment. However, cadmium concentration in the panicle, leaf, stem, and bud exhibited no significant regression with cadmium concentration in the sediment. In the non-submerged environment, the cadmium concentration was higher in the below-ground organs than in the aboveground organs. The mean bioaccumulation coefficient in the 24 investigated plots in the submerged environment was higher than that in the 20 and 40 mg kg-1 cadmium treatments in the non-submerged environment. The mean translocation factor in the submerged environment was nine times higher than that in non-submerged environment. These results indicate that submergence enhanced cadmium bioaccumulation in the aboveground organs and that this plant can be used to remove heavy metals from polluted rivers and lakes.


Assuntos
Metais Pesados , Poluentes do Solo , Biodegradação Ambiental , Cádmio , Poaceae
5.
AIDS ; 33(1): 1-11, 2019 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-30096076

RESUMO

OBJECTIVE: The peptide drug T20 (enfuvirtide), derived from the C-terminal heptad repeat region of HIV-1 gp41, is the only membrane fusion inhibitor available for treatment of viral infection; however, its mechanism of action remains elusive and its structural basis is lacking. DESIGN: We focused on determining the crystal structure of T20 in complex with N39, a target mimic peptide derived from the N-terminal heptad repeat region of gp41. On the basis of the structural information, the mechanisms of action of T20 and its resistance were further characterized. METHODS: A panel of peptides was synthesized. The T20/N39 complex was assembled for crystallization studies. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), native polyacrylamide gel electrophoresis (N-PAGE), and mutational analysis were applied to analyze the structural and functional properties. RESULTS: A crystal structure of six-helical bundle (6-HB) structure formed by T20 and N39 was determined with a resolution limit of 2.3 Å, which revealed the critical intrahelical and interhelical interactions underlying the mechanism of action of T20 and its resistance mutations. Although the structural properties in the C-terminal tryptophan-rich motif (TRM) of T20 and the fusion peptide proximal region (FPPR) of N39 could not be finely defined by the structure, the data from biophysical and mutational analyses verified the essential roles of the TRM and FPPR motifs for the binding and inhibitory activities of T20. CONCLUSION: For the first time, our studies provide a structural basis of T20, which help our understanding on the mechanisms of HIV-1 fusion and its inhibition.

6.
J Pharm Biomed Anal ; 160: 397-403, 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30125733

RESUMO

Sitafloxacin (STFX) is a new generation of broad-spectrum oral fluoroquinolones. STFX has significantly enhanced antibacterial activity than most similar drugs. Clinically, this drug is mainly used to treat respiratory and urinary tract infections and other serious bacterial infections. In this study, the interaction between sitafloxacin and human serum albumin (HSA) was investigated by spectroscopic methods and molecular simulations. Fluorescence quenching experiments showed that the interaction mechanism between STFX and HSA was static quenching, which was confirmed by time-resolved fluorescence. Thermodynamic parameters and docking results indicated that hydrophobic and electrostatic forces played a key role in this mechanism. Probe experiments and molecular docking results indicated that the major binding of STFX was at site I. 3D fluorescence showed that the insertion of STFX had minimal impact on the microenvironment. Analysis of the protein secondary structure showed that the insertion of STFX had little effect on the secondary structure of the protein. Hydrophobicity experiments showed the slight decrease in the overall hydrophobicity index of the system. Molecular dynamics simulations further validated the stability of the HSA-STFX complex. This study are useful for further drug development, in vivo toxicity studies, and can provide guidance for the clinical application of STFX to study its pharmacokinetic properties.

7.
J Biol Chem ; 293(33): 12703-12718, 2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-29929981

RESUMO

Host cell infection with HIV-1 requires fusion of viral and cell membranes. Sifuvirtide (SFT) is a peptide-based HIV-1 fusion inhibitor approved for phase III clinical trials in China. Here, we focused on characterizing HIV-1 variants highly resistant to SFT to gain insight into the molecular resistance mechanism. Three primary substitutions (V38A, A47I, and Q52R) located at the inhibitor-binding site of HIV-1's envelope protein (Env) and one secondary substitution (N126K) located at the C-terminal heptad repeat region of the viral protein gp41, which is part of the envelope, conferred high SFT resistance and cross-resistance to the anti-HIV-1 drug T20 and the template peptide C34. Interestingly, SFT's resistance profile could be dramatically improved with an M-T hook structure-modified SFT (MTSFT) and with short-peptide inhibitors that mainly target the gp41 pocket (2P23 and its lipid derivative LP-19). We found that the V38A and Q52R substitutions reduce the binding stabilities of SFT, C34, and MTSFT, but they had no effect on the binding of 2P23 and LP-19; in sharp contrast, the A47I substitution enhanced fusion inhibitor binding. Furthermore, the primary resistance substitutions impaired Env-mediated membrane fusion and cell entry and changed the conformation of the gp41 core structure. Importantly, whereas the V38A and Q52R substitutions disrupted the N-terminal helix of gp41, a single A47I substitution greatly enhanced its thermostability. Taken together, our results provide crucial structural insights into the mechanism of HIV-1 resistance to gp41-dependent fusion inhibitors, which may inform the development of additional anti-HIV drugs.

8.
Artigo em Inglês | MEDLINE | ID: mdl-29744779

RESUMO

Bioaccumulation of five heavy metals (Cd, Cu, Mn, Pb, and Zn) in six plant organs (panicle, leaf, stem, root, rhizome, and bud) of the emergent and perennial plant species, Miscanthus sacchariflorus, were investigated to estimate the plant's potential for accumulating heavy metals in the wetlands of Dongting Lake. We found the highest Cd concentrations in the panicles and leaves; while the highest Cu and Mn were observed in the roots, the highest Pb in the panicles, and the highest Zn in the panicles and buds. In contrast, the lowest Cd concentrations were detected in the stem, roots, and buds; the lowest Cu concentrations in the leaves and stems; the lowest Mn concentrations in the panicles, rhizomes, and buds; the lowest Pb concentrations in the stems; and the lowest Zn concentrations in the leaves, stems, and rhizomes. Mean Cu concentration in the plant showed a positive regression coefficient with plot elevation, soil organic matter content, and soil Cu concentration, whereas it showed a negative regression coefficient with soil moisture and electrolyte leakage. Mean Mn concentration showed positive and negative regression coefficients with soil organic matter and soil moisture, respectively. Mean Pb concentration exhibited positive regression coefficient with plot elevation and soil total P concentration, and Zn concentration showed a positive regression coefficient with soil available P and total P concentrations. However, there was no significant regression coefficient between mean Cd concentration in the plant and the investigated environmental parameters. Stems and roots were the main organs involved in heavy metal accumulation from the environment. The mean quantities of heavy metals accumulated in the plant tissues were 2.2 mg Cd, 86.7 mg Cu, 290.3 mg Mn, 15.9 mg Pb, and 307 mg Zn per square meter. In the Dongting Lake wetlands, 0.7 × 103 kg Cd, 22.9 × 103 kg Cu, 77.5 × 103 kg Mn, 3.1 × 103 kg Pb, and 95.9 × 103 kg Zn per year were accumulated by aboveground organs and removed from the lake through harvesting for paper manufacture.

9.
J Biol Chem ; 293(14): 5323-5334, 2018 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-29425101

RESUMO

Enfuvirtide (T20) is the only viral fusion inhibitor approved for clinical use, but it has relatively weak anti-HIV activity and easily induces drug resistance. In succession to T20, T1249 has been designed as a 39-mer peptide composed of amino acid sequences derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV); however, its development has been suspended due to formulation difficulties. We recently developed a T20-based lipopeptide (LP-40) showing greatly improved pharmaceutical properties. Here, we generated a T1249-based lipopeptide, termed LP-46, by replacing its C-terminal tryptophan-rich sequence with fatty acid. As compared with T20, T1249, and LP-40, the truncated LP-46 (31-mer) had dramatically increased activities in inhibiting a large panel of HIV-1 subtypes, with IC50 values approaching low picomolar concentrations. Also, LP-46 was an exceptionally potent inhibitor against HIV-2, SIV, and T20-resistant variants, and it displayed obvious synergistic effects with LP-40. Furthermore, we showed that LP-46 had increased helical stability and binding affinity with the target site. The crystal structure of LP-46 in complex with a target surrogate revealed its critical binding motifs underlying the mechanism of action. Interestingly, it was found that the introduced pocket-binding domain in LP-46 did not interact with the gp41 pocket as expected; instead, it adopted a mode similar to that of LP-40. Therefore, our studies have provided an exceptionally potent and broad fusion inhibitor for developing new anti-HIV drugs, which can also serve as a tool to exploit the mechanisms of viral fusion and inhibition.

10.
Environ Sci Pollut Res Int ; 25(8): 8002-8011, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29305802

RESUMO

The growth performance of Salix triandroides cuttings at three water cadmium (Cd) concentrations (0, 20, and 40 mg L-1) and three water levels (- 40 cm, water level 40 cm below the soil surface; 0 cm, water level even with the soil surface; and 40 cm, water level 40 cm above soil surface) was investigated to evaluate its potential in phytoextraction strategies. Compared to cuttings in the - 40 or 0 cm water levels, cuttings in the 40 cm water level showed significantly lower biomass, height, and adventitious root length and significantly fewer leaves and adventitious roots. However, these growth and morphological parameters were not different among the three water Cd concentrations. Water level decreased stomatal conduction and transpiration rate but showed no significant effects on chlorophyll concentration or photosynthetic rate. Chlorophyll concentration and stomatal conductance were higher at 40 mg L-1 Cd treatment than at 0 or 20 mg L-1 Cd treatment; yet, photosynthetic rate and transpiration rate were not different. Cd concentration in the leaves and stems increased as the water level increased, but the highest Cd concentration in the roots occurred in the 0 cm water level. As water Cd concentration increased, Cd concentration in the leaves, stems, and roots increased in all three water levels, except in stems in the - 40 cm water level. Under Cd stress, cuttings in the - 40 or 0 cm water levels were characterized by a higher bioaccumulation coefficient, but a lower translocation factor, than those in the 40 cm water level. However, the bioaccumulation coefficient increased with increasing water Cd concentration in all three water levels, as did the translocation factor in the 40 cm water level. The tolerance index for the cuttings was the same among all water levels and water Cd concentrations. The results clearly indicated that the low water level increased plant growth and Cd accumulation in underground parts, while the high water level decreased plant growth but increased Cd accumulation in aboveground parts.


Assuntos
Cádmio/toxicidade , Salix/efeitos dos fármacos , Poluentes do Solo/toxicidade , Eliminação de Resíduos Líquidos/métodos , Água/química , Água/metabolismo , Biodegradação Ambiental , Biomassa , Cádmio/química , Cádmio/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Salix/crescimento & desenvolvimento , Salix/metabolismo , Solo/química , Poluentes do Solo/química , Poluentes do Solo/metabolismo
11.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29321334

RESUMO

SC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.


Assuntos
Farmacorresistência Viral/genética , Proteína gp41 do Envelope de HIV , Inibidores da Fusão de HIV/farmacologia , HIV-1 , Mutação de Sentido Incorreto , Peptídeos/farmacologia , Substituição de Aminoácidos , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Peptídeos/química , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
12.
J BUON ; 22(5): 1314-1321, 2017 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29135119

RESUMO

PURPOSE: To investigate the expression and relevant clinical and pathological significance of AT-rich interactive domaincontaining protein 1A (ARID1A) mRNA in endometriosis-associated ovarian cancer. METHODS: The clinical and pathological data of 63 patients with ovarian clear cell carcinoma (OCCC) and of 43 patients with ovarian endometrioid adenocarcinoma (OEAC) were collected. The expression of ARID1A-encoded protein, baf250a, in ovarian cancer tissues was detected using immunohistochemistry. The ARID1A mRNA expression was detected via RNAscope hybridization in situ, and its correlation with the clinical and pathological features of patients was analyzed. RESULTS: The age at the onset of OEAC patients accompanied with endometriosis (CM-EAC) was lower than that of those not accompanied with endometriosis (NCM-EAC) (p<0.001). For patients with OCCC, the lymph node metastasis (LNM) rate of CM-CCC patients was significantly lower compared to NCM-CCC (p=0.02) and FIGO stage was earlier (stage I and II) (p=0.013). The expression of baf250a in OCCC group was significantly lower than that in the EAC group (p=0.033). In the OCCC group, baf250a was significantly related to early FIGO staging (stage I and II) (p=0.026), while its expression was not significantly associated with FIGO staging of EAC, age, tumor size, occurrence site and LND. The mRNA expression of ARID1A was positively correlated with the expression of baf250a (in OCCC group, rp=0.936, p<0.01; in OEAC group, rp=0.325, p=0.035). Analysis of prognosis showed that baf250a was an important prognostic factor rather than an independent prognostic factor, affecting the overall survival (OS) of patients with OCCC, while patients with low ARID1A mRNA expression had a longer-term OS. CONCLUSION: The decreased gene and protein expression levels of ARID1A are related to the occurrence and development of endometriosis-associated ovarian cancer, especially OCCC. The detection of ARID1A mRNA expression may be used to predict the OS of OCCC.

13.
J Virol ; 91(18)2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28659478

RESUMO

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.


Assuntos
Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV/efeitos dos fármacos , Lipopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Cristalografia por Raios X , Enfuvirtida , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/isolamento & purificação , Lipopeptídeos/química , Lipopeptídeos/isolamento & purificação , Ligação Proteica
14.
J Virol ; 91(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28356533

RESUMO

Peptides derived from the C-terminal heptad repeat (CHR) region of the human immunodeficiency virus type 1 (HIV-1) fusogenic protein gp41 are potent viral entry inhibitors, and currently, enfuvirtide (T-20) is the only one approved for clinical use; however, emerging drug resistance largely limits its efficacy. In this study, we generated a novel lipopeptide inhibitor, named LP-19, by integrating multiple design strategies, including an N-terminal M-T hook structure, an HIV-2 sequence, intrahelical salt bridges, and a membrane-anchoring lipid tail. LP-19 showed stable binding affinity and highly potent, broad, and long-lasting antiviral activity. In in vitro studies, LP-19 efficiently inhibited HIV-1-, HIV-2-, and simian immunodeficiency virus (SIV)-mediated cell fusion, viral entry, and infection, and it was highly active against diverse subtypes of primary HIV-1 isolates and inhibitor-resistant mutants. Ex vivo studies demonstrated that LP-19 exhibited dramatically increased anti-HIV activity and an extended half-life in rhesus macaques. In short-term monotherapy, LP-19 reduced viral loads to undetectable levels in acutely and chronically simian-human immunodeficiency virus (SHIV)-infected monkeys. Therefore, this study offers an ideal HIV-1/2 fusion inhibitor for clinical development and emphasizes the importance of the viral fusion step as a drug target.IMPORTANCE The peptide drug T-20 is the only viral fusion inhibitor in the clinic, which is used for combination therapy of HIV-1 infection; however, it requires a high dosage and easily induces drug resistance, calling for a new drug with significantly improved pharmaceutical profiles. Here, we have developed a short-lipopeptide-based fusion inhibitor, termed LP-19, which mainly targets the conserved gp41 pocket site and shows highly potent inhibitory activity against HIV-1, HIV-2, and even SIV isolates. LP-19 exhibits dramatically increased antiviral activity and an extended half-life in rhesus macaques, and it has potent therapeutic efficacy in SHIV-infected monkeys, highlighting its high potential as a new viral fusion inhibitor for clinical use.


Assuntos
Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Lipopeptídeos/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Desenho de Drogas , Descoberta de Drogas , Enfuvirtida , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/farmacologia , Proteína gp41 do Envelope de HIV/uso terapêutico , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/isolamento & purificação , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-2/fisiologia , Meia-Vida , Humanos , Lipopeptídeos/química , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/uso terapêutico , Macaca mulatta , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
15.
J Virol ; 91(1)2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27795437

RESUMO

Human immunodeficiency virus type 2 (HIV-2) has already spread to different regions worldwide, and currently about 1 to 2 million people have been infected, calling for new antiviral agents that are effective on both HIV-1 and HIV-2 isolates. T20 (enfuvirtide), a 36-mer peptide derived from the C-terminal heptad repeat region (CHR) of gp41, is the only clinically approved HIV-1 fusion inhibitor, but it easily induces drug resistance and is not active on HIV-2. In this study, we first demonstrated that the M-T hook structure was also vital to enhancing the binding stability and inhibitory activity of diverse CHR-based peptide inhibitors. We then designed a novel short peptide (23-mer), termed 2P23, by introducing the M-T hook structure, HIV-2 sequences, and salt bridge-forming residues. Promisingly, 2P23 was a highly stable helical peptide with high binding to the surrogate targets derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV). Consistent with this, 2P23 exhibited potent activity in inhibiting diverse subtypes of HIV-1 isolates, T20-resistant HIV-1 mutants, and a panel of primary HIV-2 isolates, HIV-2 mutants, and SIV isolates. Therefore, we conclude that 2P23 has high potential to be further developed for clinical use, and it is also an ideal tool for exploring the mechanisms of HIV-1/2- and SIV-mediated membrane fusion. IMPORTANCE: The peptide drug T20 is the only approved HIV-1 fusion inhibitor, but it is not active on HIV-2 isolates, which have currently infected 1 to 2 million people and continue to spread worldwide. Recent studies have demonstrated that the M-T hook structure can greatly enhance the binding and antiviral activities of gp41 CHR-derived inhibitors, especially for short peptides that are otherwise inactive. By combining the hook structure, HIV-2 sequence, and salt bridge-based strategies, the short peptide 2P23 has been successfully designed. 2P23 exhibits prominent advantages over many other peptide fusion inhibitors, including its potent and broad activity on HIV-1, HIV-2, and even SIV isolates, its stability as a helical, oligomeric peptide, and its high binding to diverse targets. The small size of 2P23 would benefit its synthesis and significantly reduce production cost. Therefore, 2P23 is an ideal candidate for further development, and it also provides a novel tool for studying HIV-1/2- and SIV-mediated cell fusion.


Assuntos
Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Peptídeos/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Sítios de Ligação , Desenho de Drogas , Farmacorresistência Viral/efeitos dos fármacos , Enfuvirtida , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/síntese química , HIV-1/química , HIV-1/metabolismo , HIV-2/química , HIV-2/metabolismo , Humanos , Fragmentos de Peptídeos/farmacologia , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/metabolismo , Relação Estrutura-Atividade , Internalização do Vírus/efeitos dos fármacos
16.
Guang Pu Xue Yu Guang Pu Fen Xi ; 37(1): 189-93, 2017 01.
Artigo em Chinês | MEDLINE | ID: mdl-30196585

RESUMO

The modeling and predicting of vegetation Leaf area index (LAI) is an important component of land surface model and assimilation of remote sensing data. The MODIS LAI product (i.e. MOD15A2) is one of the most widely used LAI data sources. However, the time series of MODIS LAI contains some data of low quality. For example, because of the influence of the cloud, aerosol, etc., the MODIS LAI presents the characteristics of the discontinuous in time and space. In fact, the time series of MODIS LAI include both linear and nonlinear components, which cannot be accurately modeled and predicted by either linear method or nonlinear method alone. In this paper, the original LAI time series data were first smoothed with Savitzky-Golay (SG) filtration and linear interpolation; SARIMA, BP neural network and a hybrid method of SARIMA-BP neural network were then used for modeling and predicting MODIS LAI time series. The SARIMA-BP neural network combined both SARIMA and BP neural network, which could model the linear and the nonlinear component of MODIS LAI time series respectively. That is, the final result of SARIMA-BP neural network was the sum of results of the two methods. Experiments showed that the time series of MODIS LAI that were smoothed with the SG filtration and linear interpolation were more smooth than original time series, with a determination coefficient up to 0.981, closer to 1 than that of SARIMA (0.941) and BP neural network (0.884); the correlation coefficient between SARIMA-BP neural network and the observation is 0.991, higher than that of between SARIMA (0.971) or BP neural network (0.942) SARIMA and the observation. Thus, it can be concluded that, the proposed SARIMA-BP neural network method can better adapt to the LAI time series, and it outperforms the SARIMA and BP neural network methods.


Assuntos
Redes Neurais (Computação) , Aerossóis , Folhas de Planta
17.
Biochem Biophys Res Commun ; 480(1): 69-74, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27717824

RESUMO

Myeloid differentiation primary-response protein-88 (MyD88) is one of adaptor proteins mediating Toll-like receptors (TLRs) signaling. Activation of MyD88 results in the activation of nuclear factor kappa B (NFκB) and the increase of inflammatory responses. Evidences have demonstrated that TLRs signaling contributes to cerebral ischemia/reperfusion (I/R) injury. However, the role of MyD88 in this mechanism of action is disputed and needs to be clarified. In the present study, in a mouse model of cerebral I/R, we examined the activities of NFκB and interferon factor-3 (IRF3), and the inflammatory responses in ischemic brain tissue using ELISA, Western blots, and real-time PCR. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. Our results showed that NFκB activity increased in ischemic brains, but IRF3 was not activated after cerebral I/R, in wild-type (WT) mice. MyD88 deficit inhibited the activation of NFκB, and the expression of interleukin-1ß (IL-1ß), IL-6, Beclin-1 (BECN1), pellino-1, and cyclooxygenase-2 (COX-2) increased by cerebral I/R compared with WT mice. Interestingly, the expression of interferon Beta 1 (INFB1) and vascular endothelial growth factor (VEGF) increased in MyD88 KO mice. Unexpectedly, although the neurological function improved in the MyD88 knockout (KO) mice, the deficit of MyD88 failed to reduce cerebral infarct size compared to WT mice. We concluded that MyD88-dependent signaling contributes to the inflammatory responses induced by cerebral I/R. MyD88 deficit may inhibit the increased inflammatory response and increase neuroprotective signaling.


Assuntos
Isquemia Encefálica/fisiopatologia , Encefalite/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Proteína Beclina-1/metabolismo , Western Blotting , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Isquemia Encefálica/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/metabolismo
18.
Yi Chuan ; 38(8): 72017 Apr, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27531614

RESUMO

IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Vetores Genéticos/genética , Edição de RNA/genética , RNA Guia/genética , Fatores de Transcrição/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Mutação/genética
19.
Comput Math Methods Med ; 2016: 4941235, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27313656

RESUMO

Support vector machines are powerful tools used to solve the small sample and nonlinear classification problems, but their ultimate classification performance depends heavily upon the selection of appropriate kernel and penalty parameters. In this study, we propose using a particle swarm optimization algorithm to optimize the selection of both the kernel and penalty parameters in order to improve the classification performance of support vector machines. The performance of the optimized classifier was evaluated with motor imagery EEG signals in terms of both classification and prediction. Results show that the optimized classifier can significantly improve the classification accuracy of motor imagery EEG signals.


Assuntos
Eletroencefalografia , Imaginação/fisiologia , Processamento de Sinais Assistido por Computador , Máquina de Vetores de Suporte , Adulto , Algoritmos , Simulação por Computador , Pé/fisiologia , Mãos/fisiologia , Voluntários Saudáveis , Humanos , Modelos Estatísticos , Destreza Motora , Movimento , Análise de Componente Principal , Reprodutibilidade dos Testes , Transdução de Sinais
20.
Free Radic Biol Med ; 77: 152-67, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25308698

RESUMO

Candida albicans is one of the most important opportunistic pathogens, causing both mucosal candidiasis and life-threatening systemic infections. To survive in the host immune defense system, this pathogen uses an elaborate signaling network to recognize and respond to oxidative stress, which is essential for its pathogenicity. However, the exact mechanisms that this fungus employs to integrate the oxidative stress response (OSR) with functions of various organelles remain uncharacterized. Our previous work implicated a connection between the calcium signaling system and the OSR. In this study, we find that the vacuolar transient receptor potential (TRP) channel Yvc1, one of the calcium signaling members, plays a critical role in cell tolerance to oxidative stress. We further provide evidence that this channel is required not only for activation of Cap1-related transcription of OSR genes but also for maintaining the stability of both the mitochondria and the vacuole in a potassium- and calcium-dependent manner. Element assays reveal that this TRP channel affects calcium influx and potassium transport from the vacuole to the mitochondria. Therefore, the TRP channel governs the novel interaction among the OSR, the vacuole, and the mitochondria by mediating ion transport in this pathogen under oxidative stress.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/fisiologia , Estresse Oxidativo , Canais de Receptores Transientes de Potencial/fisiologia , Vacúolos/fisiologia , Animais , Apoptose , Transporte Biológico , Sinalização do Cálcio , Candida albicans/efeitos dos fármacos , Candida albicans/ultraestrutura , Catalase/metabolismo , Células Cultivadas , Feminino , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Macrófagos/microbiologia , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Oxidantes/farmacologia , Superóxido Dismutase/metabolismo
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