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1.
bioRxiv ; 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32743589

RESUMO

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome 1, 2 . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides 3-6 . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve 7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

2.
Am J Gastroenterol ; 115(7): 1129-1132, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32618665

RESUMO

INTRODUCTION: High rates of concurrent gastrointestinal manifestations have been noted in patients with corona virus disease 2019 (COVID-19); however, the association between these digestive manifestations and need for hospitalization has not been established. METHODS: This is a retrospective review of consecutive patients diagnosed with COVID-19. A total of 207 patients were identified; 34.5% of patients noted concurrent gastrointestinal symptoms, with 90% of gastrointestinal symptoms being mild. RESULTS: In a multivariate regression model controlled for demographics and disease severity, an increased risk of hospitalization was noted in patients with any digestive symptom (adjusted odds ratio 4.84, 95% confidence interval: 1.68-13.94). DISCUSSION: The presence of digestive symptoms in COVID-19 is associated with a need for hospitalization.


Assuntos
Infecções por Coronavirus/complicações , Gastroenteropatias/etiologia , Pneumonia Viral/complicações , Adulto , Idoso , Betacoronavirus , Doenças do Sistema Digestório/etiologia , Doenças do Sistema Digestório/virologia , Feminino , Gastroenteropatias/virologia , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Estudos Retrospectivos
3.
medRxiv ; 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32511634

RESUMO

BACKGROUND: High rates of concurrent gastrointestinal manifestations have been noted in patients with COVID- 19, however the association between these digestive manifestations and need for hospitalization has not been established. METHODS: Following expedited approval from our Institutional Review Board, we analyzed retrospectively collected data from consecutive patients with confirmed COVID-19 based on a positive polymerase chain reaction testing at our institution from March 03, 2020 to April 7, 2020. Baseline demographic, clinical, laboratory and patient-reported symptom data were collected at presentation in the emergency room. Multivariable logistic regression analyses were performed to evaluate the association between hospitalization and presence of gastrointestinal symptoms. RESULTS: During this study period, we identified 207 consecutive patients with confirmed COVID-19. 34.5% noted concurrent gastrointestinal symptoms; of which 90% of gastrointestinal symptoms were mild. In a multivariate regression model controlled for demographics and disease severity, an increased risk for hospitalization was noted in patients with any gastrointestinal symptom (adjusted OR 4.84 95% CI: 1.68-13.94]. Diarrhea was associated with a seven-fold higher likelihood for hospitalization (adjusted OR=7.58, 95% CI: 2.49-20.02, P <0.001) and nausea or vomiting had a four times higher odds (adjusted OR 4.39, 95% CI: 1.61-11.4, P = 0.005). CONCLUSION: We demonstrate that a significant portion of COVID19 patients have concurrent mild gastrointestinal symptoms and that the presence of these digestive symptoms is associated with a need for hospitalization. With the current focus on streamlining triaging efforts, first responders and frontline providers should consider assessing for digestive symptoms in their initial clinical evaluation and decision-making.

4.
RNA ; 26(8): 937-959, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32398273

RESUMO

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nt as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 "SARS-related-conserved" regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 "SARS-CoV-2-conserved-structured" regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5' UTR, frameshifting stimulation element, and 3' UTR. Lastly, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 "SARS-CoV-2-conserved-unstructured" genomic regions may be most easily accessible by hybridization in primer-based diagnostic strategies.


Assuntos
Betacoronavirus/genética , RNA Viral/química , RNA Viral/genética , Sequência de Bases , Betacoronavirus/classificação , Evolução Molecular , Genoma Viral , Conformação de Ácido Nucleico , Alinhamento de Sequência , Termodinâmica
6.
Dig Dis Sci ; 65(3): 897-905, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32020359

RESUMO

Chronic liver disease is a major cause of morbidity and mortality worldwide. Even though effective treatments are now available for most chronic viral hepatitis, treatment options for other causes of chronic liver disease remain inadequate. Recent research has revealed a previously unappreciated role that the human intestinal microbiome plays in mediating the development and progression of chronic liver diseases. The recent remarkable success of fecal microbiota transplantation (FMT) in treating Clostridioides difficile demonstrates that the intestinal microbiota can be manipulated to obtain favorable therapeutic benefits and that FMT may become an important component of a total therapeutic approach to effectively treat hepatic disorders.


Assuntos
Transplante de Microbiota Fecal/tendências , Microbioma Gastrointestinal/fisiologia , Hepatopatias/microbiologia , Hepatopatias/terapia , Doença Crônica , Transplante de Microbiota Fecal/métodos , Previsões , Humanos , Hepatopatias/patologia
7.
Sci Transl Med ; 12(527)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969487

RESUMO

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.

8.
Gastroenterology ; 157(4): 1055-1066.e11, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31251928

RESUMO

BACKGROUND & AIMS: Trends of mortality associated with extrahepatic complications of chronic liver disease might be changing. We studied trends in mortality from extrahepatic complications of viral hepatitis, alcoholic liver disease (ALD), and nonalcoholic fatty liver disease in the United States. METHODS: We performed a population-based study using US Census and the National Center for Health Statistics mortality records from 2007 through 2017. We identified trends in age-standardized mortality using Joinpoint trend analysis with estimates of annual percent change. RESULTS: The liver-related mortality among patients with hepatitis C virus (HCV) infection increased from 2007 through 2013 and then decreased once patients began receiving treatment with direct-acting antiviral (DAA) agents, from 2014 through 2017. Among patients with HCV infection, the age-standardized mortality for extrahepatic cancers was 2.6%, for cardiovascular disease was 1.9%, and for diabetes was 3.3%. Among individuals with hepatitis B virus infection, liver-related mortality decreased steadily from 2007 through 2017. During the study, age-standardized mortality from hepatitis B virus-related extrahepatic complications increased by an average of 2.0% each year. Although liver-related mortality from ALD continued to increase, mortality from extrahepatic complications of ALD did not change significantly during the 11-year study. Among patients with nonalcoholic fatty liver disease, the cause of death was most frequently cardiovascular disease, which increased gradually over the study period, whereas liver-related mortality increased rapidly. CONCLUSIONS: In an analysis of US Census and the National Center for Health Statistics mortality records, we found that after widespread use of DAA agents for treatment of viral hepatitis, cause-specific mortality from extrahepatic cancers increased, whereas mortality from cardiovascular disease or diabetes increased only among patients with HCV infection. These findings indicate the need to reassess risk and risk factors for extrahepatic cancer, cardiovascular disease, and diabetes in individuals successfully treated for HCV infection with DAA agents.


Assuntos
Causas de Morte/tendências , Hepatite B Crônica/mortalidade , Hepatite C Crônica/mortalidade , Hepatopatias Alcoólicas/mortalidade , Hepatopatia Gordurosa não Alcoólica/mortalidade , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Censos , Bases de Dados Factuais , Atestado de Óbito , Feminino , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Humanos , Hepatopatias Alcoólicas/diagnóstico , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Prevalência , Fatores de Proteção , Medição de Risco , Fatores de Risco , Fatores de Tempo , Estados Unidos/epidemiologia , Adulto Jovem
9.
Nat Commun ; 10(1): 794, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770827

RESUMO

Prolonged exposure of CD8+ T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8+ T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα+ CD8+ T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα+ cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8+ T cell-killing in vivo. SIRPα+ CD8+ T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8+ T cells during chronic infection expands the cytotoxic subset of SIRPα+ CD8+ T cells.


Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores Imunológicos/imunologia , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Feminino , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia
10.
Curr Hepatol Rep ; 15(4): 237-244, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27917363

RESUMO

Although currently available therapies for chronic hepatitis B virus infection can suppress viremia and provide long-term benefits for patients, they do not lead to a functional cure for most patients. Advances in our understanding of the virus-host interaction and the recent remarkable success of immunotherapy in cancer offer new and promising strategies for developing immune modulators that may become important components of a total therapeutic approach to hepatitis B, some of which are now in clinical development. Among the immunomodulatory agents currently being investigated to combat chronic HBV are toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, and engineered T cells. The efficacy of some immune modulatory therapies is compromised by high viral antigen levels. Cutting edge strategies, including RNA interference and CRISPR/Cas9, are now being studied that may ultimately be shown to have the capacity to lower viral antigen levels sufficiently to substantially increase the efficacy of these agents. The current advances in therapies for chronic hepatitis B are leading us toward the possibility of a functional cure.

11.
ACS Cent Sci ; 2(7): 456-66, 2016 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-27504492

RESUMO

Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.

14.
Dig Dis Sci ; 60(12): 3552-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26467703

RESUMO

BACKGROUND: Despite the availability of safe and effective direct-acting antiviral drugs (DAAs), the vast majority of patients with chronic hepatitis C (HCV) in the USA remain untreated, in part due to lack of access to specialist providers. AIMS: To determine the effectiveness of DAA-based treatment in medically underserved areas in California, in a healthcare model dependent on task-shifting--wherein a visiting hepatologist assesses patients for treatment eligibility, but subsequent routine follow-up evaluation of patients prescribed treatment is devolved to a part-time licensed vocational nurse under remote supervision of the hepatologist. METHODS: We retrospectively determined rates of sustained virologic response 12 weeks after treatment completion (SVR-12), adverse events, and treatment discontinuations in patients who received sofosbuvir-based DAA regimens between December 2013 and November 2014. RESULTS: Despite limited specialist provider involvement in medically underserved areas, all but two of 58 patients completed treatment, and 88 % of patients achieved the curative endpoint of undetectable HCV RNA 12 weeks after completing treatment (sustained virologic response, SVR-12). Almost 80 % of patients with cirrhosis and 85 % of patients with prior treatment experience achieved SVR-12. CONCLUSIONS: Treatment effectiveness with sofosbuvir-based regimens in medically underserved areas utilizing task-shifting from a specialist to a mid-level provider is comparable to those achieved in pivotal clinical trials for these regimens, and to "real-world" experiences of tertiary care centers in the USA.


Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Área Carente de Assistência Médica , Idoso , Idoso de 80 Anos ou mais , Antivirais/administração & dosagem , California , Feminino , Acesso aos Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Ribavirina/administração & dosagem , Ribavirina/uso terapêutico , Simeprevir/administração & dosagem , Simeprevir/uso terapêutico , Sofosbuvir/administração & dosagem , Sofosbuvir/uso terapêutico
15.
Gastroenterology ; 148(3): 616-25, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25479136

RESUMO

BACKGROUND & AIMS: Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle. METHODS: We used a combination of quartz crystal microbalance, circular dichroism, molecular genetics, and immunofluorescence to study specific binding of PI(4,5)P2 by the HCV NS5A protein. We evaluated the effects of PI(4,5)P2 on the function of NS5A by expressing wild-type or mutant forms of Bart79I or FL-J6/JFH-5'C19Rluc2AUbi21 RNA in Huh7 cells. We also studied the effects of strategies designed to inhibit PI(4,5)P2 on HCV replication in these cells. RESULTS: The N-terminal amphipathic helix of NS5A bound specifically to PI(4,5)P2, inducing a conformational change that stabilized the interaction between NS5A and TBC1D20, which is required for HCV replication. A pair of positively charged residues within the amphipathic helix (the basic amino acid PI(4,5)P2 pincer domain) was required for PI(4,5)P2 binding and replication of the HCV-RNA genome. A similar motif was found to be conserved across all HCV isolates, as well as amphipathic helices of many pathogens and apolipoproteins. CONCLUSIONS: PI(4,5)P2 binds to HCV NS5A to promote replication of the viral RNA genome in hepatocytes. Strategies to disrupt this interaction might be developed to inhibit replication of HCV and other viruses.


Assuntos
Genoma Viral , Hepacivirus/genética , Hepatócitos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sobrevivência Celular , Dicroísmo Circular , Hepacivirus/metabolismo , Humanos , Microscopia de Fluorescência , Estrutura Secundária de Proteína , Técnicas de Microbalança de Cristal de Quartzo , Análise de Sequência de RNA , Proteínas rab1 de Ligação ao GTP/metabolismo
16.
J Virol ; 86(2): 1250-4, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22072754

RESUMO

MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5'-untranslated region (UTR) of HCV. We used selective 2'-hydroxyl acylation analyzed by primer extension to resolve the HCV 5'-UTR's RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of 'tail' miR-122 nucleotide interactions.


Assuntos
Hepacivirus/química , Hepatite C/virologia , MicroRNAs/metabolismo , RNA Viral/química , Regiões 5' não Traduzidas , Sequência de Bases , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/genética , Hepatite C/metabolismo , Humanos , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral
17.
Nucleic Acids Res ; 39(22): e151, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21965531

RESUMO

SHAPE (Selective 2'-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ~900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest.


Assuntos
RNA/química , Software , Acilação , Algoritmos , Sequência de Bases , Primers do DNA , Eletroforese Capilar , Hepacivirus/genética , Ligantes , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/química , Reprodutibilidade dos Testes , Proteínas do Core Viral/genética
18.
Cancer Sci ; 101(12): 2637-43, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20849469

RESUMO

Development of cancer therapeutics requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Three-dimensional (3-D) in vitro models have therefore been investigated for drug screening. In this study, we have developed a novel in vitro model in which multicellular aggregates, or spheroids, were incorporated into 3-D porous scaffolds. Drug resistance assays showed that spheroid-seeded scaffolds have much higher drug resistance than monolayer cultures, spheroids on flat substrates, or scaffolds seeded with dispersed cells. Furthermore, spheroid-seeded scaffolds demonstrated higher lactate production leading to acidosis, and higher expression of angiogenic factors. These data suggest that the spheroid-seeded 3-D scaffolds might serve as a useful in vitro system for screening cancer therapeutics.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Esferoides Celulares/efeitos dos fármacos , Tecidos Suporte , Células Tumorais Cultivadas/efeitos dos fármacos , Humanos , Esferoides Celulares/citologia , Células Tumorais Cultivadas/citologia
19.
J Biomed Mater Res B Appl Biomater ; 92(2): 499-507, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19957351

RESUMO

As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects. Thus, additional steps are required to ensure complete isolation of cells from the 3D scaffold. The diphenylamine assay isolates cellular DNA, degrades the polymeric matrix materials, and reacts with the DNA to yield a colorimetric response. Thus, we report here a practical modification of the diphenylamine assay and show that the assay quantifies cells in 3D polyester scaffolds reliably and reproducibly as long as the necessary amount of the acidic working reagent is present. Our study also demonstrates that the sensitivity of the assay can be optimized by controlling the dimensions of the sampling volume. Overall, the DPA assay offers an attractive solution for challenges associated with 3D cell quantification.


Assuntos
Implantes Absorvíveis , Contagem de Células/métodos , Difenilamina , Tecidos Suporte , Materiais Biocompatíveis , Linhagem Celular , DNA/biossíntese , DNA/isolamento & purificação , Fluorometria , Humanos , Indicadores e Reagentes , Ácido Láctico , Poliésteres , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Desenho de Prótese , Padrões de Referência
20.
J Control Release ; 133(3): 178-84, 2009 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-18992290

RESUMO

We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC(50) values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf.


Assuntos
Citotoxinas/administração & dosagem , Engenharia de Proteínas/métodos , Transferrina/metabolismo , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/química , Substituição de Aminoácidos , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Citotoxinas/química , Citotoxinas/farmacologia , Toxina Diftérica/administração & dosagem , Toxina Diftérica/química , Toxina Diftérica/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ácido Edético/química , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Ferro/química , Cinética , Mutação , Receptores da Transferrina/química , Receptores da Transferrina/metabolismo , Ressonância de Plasmônio de Superfície , Transferrina/química , Transferrina/genética
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