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1.
Annu Rev Biochem ; 88: 383-408, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30939043

RESUMO

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.


Assuntos
Desenvolvimento de Medicamentos/métodos , Proteoma/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Ligantes , Espectrometria de Massas , Ligação Proteica , Proteoma/química , Proteômica
2.
Cell ; 173(6): 1481-1494.e13, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29706543

RESUMO

Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of the biochemical processes determining cellular events. Using a proteome-wide implementation of the cellular thermal shift assay (CETSA) to study specific cell-cycle phases, we uncover changes of interaction states for more than 750 proteins during the cell cycle. Notably, many protein complexes are modulated in specific cell-cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and disintegration of the nuclear envelope. Surprisingly, only small differences in the interaction states were seen between the G1 and the G2 phase, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle and establishes proteome-wide CETSA as a new strategy to study modulation of protein-interaction states in intact cells.


Assuntos
Ciclo Celular , Mapeamento de Interação de Proteínas , Divisão Celular , Cromatina/química , Análise por Conglomerados , Replicação do DNA , Fase G1 , Fase G2 , Humanos , Células K562 , Membrana Nuclear , Proteoma , Proteômica/métodos
3.
Acta Pharmacol Sin ; 45(2): 391-404, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37803139

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers in the world. The therapeutic outlook for HCC patients has significantly improved with the advent and development of systematic and targeted therapies such as sorafenib and lenvatinib; however, the rise of drug resistance and the high mortality rate necessitate the continuous discovery of effective targeting agents. To discover novel anti-HCC compounds, we first constructed a deep learning-based chemical representation model to screen more than 6 million compounds in the ZINC15 drug-like library. We successfully identified LGOd1 as a novel anticancer agent with a characteristic levoglucosenone (LGO) scaffold. The mechanistic studies revealed that LGOd1 treatment leads to HCC cell death by interfering with cellular copper homeostasis, which is similar to a recently reported copper-dependent cell death named cuproptosis. While the prototypical cuproptosis is brought on by copper ionophore-induced copper overload, mechanistic studies indicated that LGOd1 does not act as a copper ionophore, but most likely by interacting with the copper chaperone protein CCS, thus LGOd1 represents a potentially new class of compounds with unique cuproptosis-inducing property. In summary, our findings highlight the critical role of bioavailable copper in the regulation of cell death and represent a novel route of cuproptosis induction.


Assuntos
Carcinoma Hepatocelular , Aprendizado Profundo , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Cobre , Neoplasias Hepáticas/tratamento farmacológico , Ionóforos , Apoptose
4.
Med Res Rev ; 41(6): 2893-2926, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33533067

RESUMO

Small-molecule drugs modulate biological processes and disease states through engagement of target proteins in cells. Assessing drug-target engagement on a proteome-wide scale is of utmost importance in better understanding the molecular mechanisms of action of observed beneficial and adverse effects, as well as in developing next generation tool compounds and drugs with better efficacies and specificities. However, systematic assessment of drug-target engagement has been an arduous task. With the continuous development of mass spectrometry-based proteomics instruments and techniques, various chemical proteomics approaches for drug target deconvolution (i.e., the identification of molecular target for drugs) have emerged. Among these, the label-free target deconvolution approaches that do not involve the chemical modification of compounds of interest, have gained increased attention in the community. Here we provide an overview of the basic principles and recent biological applications of the most important label-free methods including the cellular thermal shift assay, pulse proteolysis, chemical denaturant and protein precipitation, stability of proteins from rates of oxidation, drug affinity responsive target stability, limited proteolysis, and solvent-induced protein precipitation. The state-of-the-art technical implications and future outlook for the label-free approaches are also discussed.


Assuntos
Proteoma , Proteômica , Sistemas de Liberação de Medicamentos , Humanos , Oxirredução , Proteoma/metabolismo , Proteômica/métodos , Solventes
5.
Immunology ; 152(2): 344-355, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28581024

RESUMO

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T-cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T-cell epitopes identified using the interferon-γ ELISPOT assay with 15-mer overlapping peptides. C57BL/6 mice were sensitized with bone-marrow-derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H-2b restricted epitopes (Bt576-90 and Bt5106-115 ) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5-pulsed BMDC and challenged with intranasal Blo t 5 Bt576-90 and Bt5106-115 , peptide-specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin-5 and interleukin-13. Intradermal administration of synthetic peptides encoding the identified T-cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T-cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T-cell response in a murine model of allergic airway inflammation.


Assuntos
Alérgenos/imunologia , Antiasmáticos/imunologia , Asma/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Ácaros/imunologia , Peptídeos/imunologia , Pneumonia/prevenção & controle , Vacinas de DNA/imunologia , Alérgenos/administração & dosagem , Alérgenos/genética , Animais , Antiasmáticos/administração & dosagem , Asma/imunologia , Asma/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , ELISPOT , Mapeamento de Epitopos , Imunização , Injeções Intradérmicas , Interferon gama/imunologia , Interferon gama/metabolismo , Testes de Liberação de Interferon-gama , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Peptídeos/genética , Pneumonia/imunologia , Pneumonia/metabolismo , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/metabolismo , Eosinofilia Pulmonar/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética
6.
J Virol ; 87(23): 12510-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24027334

RESUMO

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ(-/-) and IFN-γR(-/-) compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ(-/-) mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ(-/-) and IFN-γR(-/-) mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Interferon gama/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Feminino , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Interferon gama/deficiência , Interferon gama/genética , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Especificidade da Espécie , Receptor de Interferon gama
7.
J Prosthodont ; 23(3): 252-5, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24118216

RESUMO

The success of an ocular prosthesis depends largely on the correct orientation of the iris disk. Various methods have been put forth to achieve this. This article emphasizes one such simplified method, wherein a customized scale has been used to orient the iris disk mediolaterally, superoinferiorly, and anteroposteriorly in an ocular prosthesis. A scleral wax pattern was fabricated. The customized scale was used to measure the dimension and orientation of the natural iris. These measurements were then transferred to the scleral wax pattern with the customized scale. An iris disk was fabricated using black crayon on the scleral wax pattern according to the measurements. The scleral wax pattern, including the iris disk, was then placed in the eye socket to verify its dimension and orientation. A prefabricated iris disk was modified according to the measured dimensions and transferred to the final scleral wax pattern. The transfer of these dimensions to the definitive prosthesis was achieved successfully, ultimately improving the patient's social and psychological well being.


Assuntos
Olho Artificial , Iris , Desenho de Prótese/instrumentação , Adulto , Humanos , Masculino , Propriedades de Superfície , Ceras/química
8.
Sci Rep ; 14(1): 1878, 2024 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-38253642

RESUMO

Mass spectrometry-coupled cellular thermal shift assay (MS-CETSA), a biophysical principle-based technique that measures the thermal stability of proteins at the proteome level inside the cell, has contributed significantly to the understanding of drug mechanisms of action and the dissection of protein interaction dynamics in different cellular states. One of the barriers to the wide applications of MS-CETSA is that MS-CETSA experiments must be performed on the specific cell lines of interest, which is typically time-consuming and costly in terms of labeling reagents and mass spectrometry time. In this study, we aim to predict CETSA features in various cell lines by introducing a computational framework called CycleDNN based on deep neural network technology. For a given set of n cell lines, CycleDNN comprises n auto-encoders. Each auto-encoder includes an encoder to convert CETSA features from one cell line into latent features in a latent space [Formula: see text]. It also features a decoder that transforms the latent features back into CETSA features for another cell line. In such a way, the proposed CycleDNN creates a cyclic prediction of CETSA features across different cell lines. The prediction loss, cycle-consistency loss, and latent space regularization loss are used to guide the model training. Experimental results on a public CETSA dataset demonstrate the effectiveness of our proposed approach. Furthermore, we confirm the validity of the predicted MS-CETSA data from our proposed CycleDNN through validation in protein-protein interaction prediction.


Assuntos
Aprendizado Profundo , Biofísica , Linhagem Celular , Dissecação , Espectrometria de Massas
9.
Cell Rep ; 43(10): 114784, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39365699

RESUMO

Cellular phenotypes of apoptosis, as well as the activation of apoptosis caspase cascades, are well described. However, sequences and locations of early biochemical effector events after apoptosis initiation are still only partly understood. Here, we use integrated modulation of protein interaction states-cellular thermal shift assay (IMPRINTS-CETSA) to dissect the cellular biochemistry of early stages of apoptosis at the systems level. Using 5 families of cancer drugs and a new CETSA-based method to monitor the cleavage of caspase targets, we discover the initial biochemistry of the effector stage of apoptosis for all the studied drugs being focused on the peripheral nuclear region rather than the cytosol. Despite very different candidate apoptosis-inducing mechanisms of the drug families, as revealed by the CETSA data, they converge into related biochemical modulations in the peripheral nuclear region. This implies a higher control of the localization of the caspase cascades than previously anticipated and highlights the nuclear periphery as a critical vulnerability for cancer therapies.


Assuntos
Apoptose , Núcleo Celular , Proteoma , Apoptose/efeitos dos fármacos , Humanos , Proteoma/metabolismo , Núcleo Celular/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Células HeLa
10.
Cell Chem Biol ; 31(4): 743-759.e8, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38593807

RESUMO

Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine. Enzymatic assays showed that these compounds can inhibit FLN proteolytic activity. Their interaction with FLN was explored systematically by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with lowered FLN expression demonstrated statistically significant increases in susceptibility toward MK-4815, MMV000848, and several quinolines. Importantly, the hydrophobic pocket of FLN appears amenable to inhibition and the structures reported here can guide the development of novel drugs against malaria.


Assuntos
Antimaláricos , Malária , Metilaminas , Quinolinas , Humanos , Antimaláricos/química , Malária/tratamento farmacológico , Fenóis/uso terapêutico , Quinolinas/farmacologia , Quinolinas/metabolismo , Desenvolvimento de Medicamentos
11.
J Virol ; 86(5): 2817-25, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22205730

RESUMO

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.


Assuntos
Antígenos Virais/imunologia , Fatores de Transcrição Forkhead/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
J Immunol ; 187(11): 6011-21, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22043017

RESUMO

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Antígenos CD/imunologia , Antígenos Virais/imunologia , Separação Celular , Células Dendríticas/virologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Cadeias alfa de Integrinas/imunologia , Pulmão/imunologia , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Annu Int Conf IEEE Eng Med Biol Soc ; 2022: 1659-1662, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-36085889

RESUMO

The Cellular Thermal Shift Assay (CETSA) is a biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins. This technology has revolutionized cell-based target engagement studies and has been used as guidance for drug design. Although many ap-plications of CETSA data have been explored, the correlations between CETSA data and protein-protein interactions (PPI) have barely been touched. In this study, we conduct the first exploration study applying CETSA data for PPI prediction. We use a machine learning method, Decision Tree, to predict PPI scores using proteins' CETSA features. It shows promising results that the predicted PPI scores closely match the ground-truth PPI scores. Furthermore, for a small number of protein pairs, whose PPI score predictions mismatch the ground truth, we use iterative clustering strategy to gradually reduce the number of these pairs. At the end of iterative clustering, the remaining protein pairs may have some unusual properties and are of scientific value for further biological investigation. Our study has demonstrated that PPI is a brand-new application of CETSA data. At the same time, it also manifests that CETSA data can be used as a new data source for PPI exploration study.


Assuntos
Bioensaio , Projetos de Pesquisa , Biofísica , Análise por Conglomerados , Domínios Proteicos
14.
Annu Int Conf IEEE Eng Med Biol Soc ; 2022: 1647-1650, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-36085941

RESUMO

Cellular Thermal Shift Assay (CETSA) has been widely used in drug discovery, cancer cell biology, immunology, etc. One of the barriers for CETSA applications is that CETSA experiments have to be conducted on various cell lines, which is extremely time-consuming and costly. In this study, we make an effort to explore the translation of CETSA features cross cell lines, i.e., known CETSA feature of a given protein in one cell line, can we automatically predict the CETSA feature of this protein in another cell line, and vice versa? Inspired by pix2pix and CycleGAN, which perform well on image-to-image translation cross various domains in computer vision, we propose a novel deep neural network model called CycleDNN for CETSA feature translation cross cell lines. Given cell lines A and B, the proposed CycleDNN consists of two auto-encoders, the first one encodes the CETSA feature from cell line A into Z in the latent space [Formula: see text], then decodes Z into the CETSA feature in cell line B., Similarly, the second one translates the CETSA feature from cell line B to cell line A through the latent space [Formula: see text]. In such a way, the two auto-encoders form a cyclic feature translation between cell lines. The reconstructed loss, cycle-consistency loss, and latent vector regularization loss are used to guide the training of the model. The experimental results on a public CETSA dataset demonstrate the effectiveness of the proposed approach.


Assuntos
Descoberta de Drogas , Redes Neurais de Computação , Linhagem Celular , Descoberta de Drogas/métodos , Proteínas , Projetos de Pesquisa
15.
Cell Chem Biol ; 29(4): 572-585.e8, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-34265272

RESUMO

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.


Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Proteoma , Proteômica , RNA
16.
J Virol ; 84(7): 3201-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20071572

RESUMO

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD(50)) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.


Assuntos
Baculoviridae/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Feminino , Células HCT116 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunoglobulina G/sangue , Mucosa Intestinal/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Spodoptera , Vacinação , Vacinas Sintéticas/imunologia
17.
Dent Update ; 38(6): 414-6, 418, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21905355

RESUMO

UNLABELLED: The fabrication of ear prosthesis is considered by many prosthetists to be one of the more difficult replacements in maxillofacial reconstruction. The severe undercuts and pronounced convolutions of the ear's surface present a challenge in simulating a natural proportioned prosthesis. The mould for the ear is generally made by creating a three surface die to reproduce the unique configuration adequately and to allow retrieval of the finished prosthesis without damage. This article presents an outlined procedure in the basic fabrication of a prosthetic ear by a conventional technique where the wax pattern is fabricated from the impression of an individual with a similarly proportioned ear. CLINICAL RELEVANCE: Fabricating an auricular prosthesis may be part of the work of a maxillofacial department.


Assuntos
Orelha Externa , Próteses e Implantes , Desenho de Prótese , Resinas Acrílicas/química , Adulto , Materiais Biocompatíveis/química , Cartilagem da Orelha , Humanos , Masculino , Satisfação do Paciente , Pigmentação em Prótese , Propriedades de Superfície
18.
J Virol ; 83(6): 2553-62, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19109379

RESUMO

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Antivirais/uso terapêutico , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Células CHO , Fusão Celular , Cricetinae , Cricetulus , Mapeamento de Epitopos , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Ligação Proteica , Análise de Sobrevida
19.
Curr Opin Chem Biol ; 54: 54-62, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31838273

RESUMO

The Cellular Thermal Shift Assay (CETSA) has recently emerged as a promising method to directly monitor functional modulations of protein interaction states in intact cells and tissue samples. Recent data support that the mass spectrometry-coupled proteome-wide implementation of CETSA (MS-CETSA) generates stringent information on a wide range of different interaction classes and is uniquely well suited to study the modulation of protein interaction states in cellular processes and during drug action. To expand the mechanistic insight of CETSA shifts, and to complement information from CETSA experiments, we outline how the integration of MS-CETSA with other proteomics techniques can provide a new platform for detailed, comprehensive, and interactive studies of the functional modulations of proteomes in situ.


Assuntos
Proteínas/química , Proteínas/metabolismo , Proteômica/métodos , Humanos , Metabolômica/métodos , Mapas de Interação de Proteínas , Espectrometria de Massas em Tandem
20.
Nat Protoc ; 15(6): 1881-1921, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341577

RESUMO

Despite decades of research, little is known about the cellular targets and the mode of action of the vast majority of antimalarial drugs. We recently demonstrated that the cellular thermal shift assay (CETSA) protocol in its two variants: the melt curve and the isothermal dose-response, represents a comprehensive strategy for the identification of antimalarial drug targets. CETSA enables proteome-wide target screening for unmodified antimalarial compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug-protein interactions. The experimental workflow involves treatment of P. falciparum-infected erythrocytes with a compound of interest, heat exposure to denature proteins, soluble protein isolation, enzymatic digestion, peptide labeling with tandem mass tags, offline fractionation, and liquid chromatography-tandem mass spectrometry analysis. Methodological optimizations necessary for the analysis of this intracellular parasite are discussed, including enrichment of parasitized cells and hemoglobin depletion strategies to overcome high hemoglobin abundance in the host red blood cells. We outline an effective data processing workflow using the mineCETSA R package, which enables prioritization of drug-target candidates for follow-up studies. The entire protocol can be completed within 2 weeks.


Assuntos
Antimaláricos/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Descoberta de Drogas/métodos , Eritrócitos/parasitologia , Humanos , Malária Falciparum/metabolismo , Terapia de Alvo Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Plasmodium falciparum/metabolismo , Proteoma/metabolismo
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